Antibodies and chimeric antigen receptors targeting glypican-3 (GPC3) and methods of use thereof

Abstract

Provided are anti-glypican-3 (GPC3) antibodies or antigen-binding fragments thereof, and chimeric antigen receptors (CARs) that bind to glypican-3 (GPC3) and contain anti-GPC3 antibodies in the extracellular domain, transmembrane domain, and intracellular signaling domain. Immune effector cells transduced with the disclosed CAR constructs can be used for cancer immunotherapy.

Description

[Technical Field]

[0001] cross reference This application claims the benefit of priority from International Patent Application PCT / CN2020 / 076937, filed on February 27, 2020, the contents of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing This application incorporates by reference the Sequence Listing entitled "14651-019-228_SEQ_LISTING", created on February 23, 2021, having a size of 226,307 bytes, which was submitted herewith in text form.

[0003] The present disclosure relates to the fields of antibodies, chimeric antigen receptors and modified immune cells that target GPC3 and methods of use thereof. [Background technology]

[0004] Glypican-3 (GPC3), a 580-amino acid, 65-kDa protein, is a heparan sulfate proteoglycan anchored to the cell membrane by glycosylphosphatidylinositol. Various important proteins, including fibroblast growth factors and Wnts, bind to two heparan sulfate chains. The function of the heparan chains remains unclear, but they are thought to regulate the association of the bound protein with its corresponding receptor. GPC3 is expressed in hepatocellular carcinoma (HCC), ovarian clear cell carcinoma (OCCC), melanoma, lung squamous cell carcinoma, hepatoblastoma, nephroblastoma (Wilms' tumor), and yolk sac tumor, as well as certain gastric cancers, such as those producing α-fetoprotein. Although the precise function of secreted and membrane-anchored GPC3 in these cancers is not fully understood, it has been demonstrated that it is involved in neoplastic transformation, for example, in HCC (Shirakawa H., et al., Cancer Sci. 2009; 100: 1403-1407). Notably, this protein is almost absent in any other cancer forms. Based on these characteristics, GPC3 may be an ideal target for cancer immunotherapy. There is a need in the art for novel anti-GPC3 antibodies and immunotherapies such as GPC3 CAR-T therapy. Summary of the Invention [Means for solving the problem]

[0005] In one aspect, the present specification provides an antibody or antigen-binding fragment thereof that binds to glypican-3 (GPC3), comprising an HCDR1 selected from the group consisting of SEQ ID NOs: 1 to 10, an HCDR2 selected from the group consisting of SEQ ID NOs: 11 to 20, and an HCDR3 selected from the group consisting of SEQ ID NOs: 21 to 30; and an LCDR1 selected from the group consisting of SEQ ID NOs: 31 to 40, an LCDR2 selected from the group consisting of SEQ ID NOs: 41 to 50, and an LCDR3 selected from the group consisting of SEQ ID NOs: 51 to 60.

[0006] In some embodiments, in the antibodies or antigen-binding fragments provided herein, (i) HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, HCDR2 comprises the amino acid sequence of SEQ ID NO: 11, HCDR3 comprises the amino acid sequence of SEQ ID NO: 21, LCDR1 comprises the amino acid sequence of SEQ ID NO: 31, LCDR2 comprises the amino acid sequence of SEQ ID NO: 41, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 51; or (ii) HCDR1 comprises the amino acid sequence of SEQ ID NO: 2, HCDR2 comprises the amino acid sequence of SEQ ID NO: 12, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 2 2, wherein LCDR1 comprises the amino acid sequence of SEQ ID NO: 32, LCDR2 comprises the amino acid sequence of SEQ ID NO: 42, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 52, or (iii) HCDR1 comprises the amino acid sequence of SEQ ID NO: 3, HCDR2 comprises the amino acid sequence of SEQ ID NO: 13, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 23, wherein LCDR1 comprises the amino acid sequence of SEQ ID NO: 33, LCDR2 comprises the amino acid sequence of SEQ ID NO: 43, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 53, or (iv) HCDR HCDR1 comprises the amino acid sequence of SEQ ID NO:4, HCDR2 comprises the amino acid sequence of SEQ ID NO:14, HCDR3 comprises the amino acid sequence of SEQ ID NO:24, LCDR1 comprises the amino acid sequence of SEQ ID NO:34, LCDR2 comprises the amino acid sequence of SEQ ID NO:44, and LCDR3 comprises the amino acid sequence of SEQ ID NO:54; or (v) HCDR1 comprises the amino acid sequence of SEQ ID NO:5, HCDR2 comprises the amino acid sequence of SEQ ID NO:15, HCDR3 comprises the amino acid sequence of SEQ ID NO:25, LCDR1 comprises the amino acid sequence of SEQ ID NO:35, and LCDR2 comprises the amino acid sequence of SEQ ID NO: (vi) HCDR1 comprises the amino acid sequence of SEQ ID NO: 6, HCDR2 comprises the amino acid sequence of SEQ ID NO: 16, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 26, LCDR1 comprises the amino acid sequence of SEQ ID NO: 36, LCDR2 comprises the amino acid sequence of SEQ ID NO: 46, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 56, or (vii) HCDR1 comprises the amino acid sequence of SEQ ID NO: 7 and HCDR2 comprises the amino acid sequence of SEQ ID NO: 17;HCDR3 comprises the amino acid sequence of SEQ ID NO:27, LCDR1 comprises the amino acid sequence of SEQ ID NO:37, LCDR2 comprises the amino acid sequence of SEQ ID NO:47, and LCDR3 comprises the amino acid sequence of SEQ ID NO:57, or (viii) HCDR1 comprises the amino acid sequence of SEQ ID NO:8, HCDR2 comprises the amino acid sequence of SEQ ID NO:18, HCDR3 comprises the amino acid sequence of SEQ ID NO:28, LCDR1 comprises the amino acid sequence of SEQ ID NO:38, LCDR2 comprises the amino acid sequence of SEQ ID NO:48, and LCDR3 comprises the amino acid sequence of SEQ ID NO:58, or (ix) HCDR1 comprises the amino acid sequence of SEQ ID NO:9 wherein HCDR2 comprises the amino acid sequence of SEQ ID NO: 19, HCDR3 comprises the amino acid sequence of SEQ ID NO: 29, LCDR1 comprises the amino acid sequence of SEQ ID NO: 39, LCDR2 comprises the amino acid sequence of SEQ ID NO: 49, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 59, or (x) HCDR1 comprises the amino acid sequence of SEQ ID NO: 10, HCDR2 comprises the amino acid sequence of SEQ ID NO: 20, HCDR3 comprises the amino acid sequence of SEQ ID NO: 30, LCDR1 comprises the amino acid sequence of SEQ ID NO: 40, LCDR2 comprises the amino acid sequence of SEQ ID NO: 50, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 60.

[0007] In some embodiments, the antibodies or antigen-binding fragments provided herein comprise: (i) a VH domain comprising an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 61 and a VL domain comprising an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 71; or (ii) a VH domain comprising an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 62 and a VL domain comprising an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 72; or (iii) a VH domain comprising an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 63 and a VL domain comprising an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 73; or (iv) a VH domain comprising an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 64 and a VL domain comprising an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 74; or (v) an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 65. (vi) a VH domain comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 66 and a VL domain comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 76; or (vii) a VH domain comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 67 and a VL domain comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 77; or (viii) a VH domain comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 68 and a VL domain comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 78; or (ix) a VH domain comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 69 and a VL domain comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 79.or (x) a VH domain comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 70 and a VL domain comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 80, or (xi) a VH domain comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 117 and a VL domain comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 123, or (xii) a VH domain comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 118 and a VL domain comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 123, or (xiii) a VH domain comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 119 and a VL domain comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 123, or (xiv) a VH domain comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 120 and and a VL domain comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 123, or (xv) a VH domain comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 121 and a VL domain comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 123, or (xvi) a VH domain comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 122 and a VL domain comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 123, or (xvii) a VH domain comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 117 and a VL domain comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 124, or (xviii) a VH domain comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 118 and a VL domain comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 124,or (xix) a VH domain comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 119 and a VL domain comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 124, or (xx) a VH domain comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 120 and a VL domain comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 124, or (xxi) a VH domain comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 121 and a VL domain comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 124, or (xxii) a VH domain comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 122 and a VL domain comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 124, or (xxiii) a VH domain comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 117. (xxiv) a VH domain comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 118 and a VL domain comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 125, or (xxv) a VH domain comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 119 and a VL domain comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 125, or (xxvi) a VH domain comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 120 and a VL domain comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 125, or (xxvii) a VH domain comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 121 and a VL domain comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 125,or (xxviii) a VH domain comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 122 and a VL domain comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 125.

[0008] In some embodiments, the antibody or antigen-binding fragment provided herein comprises (i) a VH domain comprising the amino acid sequence of SEQ ID NO: 61 and a VL domain comprising the amino acid sequence of SEQ ID NO: 71, or (ii) a VH domain comprising the amino acid sequence of SEQ ID NO: 62 and a VL domain comprising the amino acid sequence of SEQ ID NO: 72, or (iii) a VH domain comprising the amino acid sequence of SEQ ID NO: 63 and a VL domain comprising the amino acid sequence of SEQ ID NO: 73, or (iv) a VH domain comprising the amino acid sequence of SEQ ID NO: 64 and a VL domain comprising the amino acid sequence of SEQ ID NO: 74, or (v) a VH domain comprising the amino acid sequence of SEQ ID NO: 65 and a VL domain comprising the amino acid sequence of SEQ ID NO: 75, or (vi) a VH domain comprising the amino acid sequence of SEQ ID NO: 66 and a VL domain comprising the amino acid sequence of SEQ ID NO: 76, or (vii) a VH domain comprising the amino acid sequence of SEQ ID NO: 67 and a VL domain comprising the amino acid sequence of SEQ ID NO: 77, or (viii) a VH domain comprising the amino acid sequence of SEQ ID NO: 68 and a VL domain comprising the amino acid sequence of SEQ ID NO: 78, or (ix) the amino acid sequence of SEQ ID NO: 69 and a VL domain comprising the amino acid sequence of SEQ ID NO: 79, or (x) a VH domain comprising the amino acid sequence of SEQ ID NO: 70 and a VL domain comprising the amino acid sequence of SEQ ID NO: 80, or (xi) a VH domain comprising the amino acid sequence of SEQ ID NO: 117 and a VL domain comprising the amino acid sequence of SEQ ID NO: 123, or (xii) a VH domain comprising the amino acid sequence of SEQ ID NO: 118 and a VL domain comprising the amino acid sequence of SEQ ID NO: 123, or (xiii) a VH domain comprising the amino acid sequence of SEQ ID NO: 119 and a VL domain comprising the amino acid sequence of SEQ ID NO: 123. (xiv) a VH domain comprising the amino acid sequence of SEQ ID NO: 120 and a VL domain comprising the amino acid sequence of SEQ ID NO: 123, or (xv) a VH domain comprising the amino acid sequence of SEQ ID NO: 121 and a VL domain comprising the amino acid sequence of SEQ ID NO: 123, or (xvi) a VH domain comprising the amino acid sequence of SEQ ID NO: 122 and a VL domain comprising the amino acid sequence of SEQ ID NO: 123, or (xvii) a VH domain comprising the amino acid sequence of SEQ ID NO: 117 and a VL domain comprising the amino acid sequence of SEQ ID NO: 124,or (xviii) a VH domain comprising the amino acid sequence of SEQ ID NO: 118 and a VL domain comprising the amino acid sequence of SEQ ID NO: 124, or (xix) a VH domain comprising the amino acid sequence of SEQ ID NO: 119 and a VL domain comprising the amino acid sequence of SEQ ID NO: 124, or (xx) a VH domain comprising the amino acid sequence of SEQ ID NO: 120 and a VL domain comprising the amino acid sequence of SEQ ID NO: 124, or (xxi) a VH domain comprising the amino acid sequence of SEQ ID NO: 121 and a VL domain comprising the amino acid sequence of SEQ ID NO: 124, or (xxii) a VH domain comprising the amino acid sequence of SEQ ID NO: 122 and a VL domain comprising the amino acid sequence of SEQ ID NO: 124, or (xxiii) a VH domain comprising the amino acid sequence of SEQ ID NO: 117 and and a VL domain comprising the amino acid sequence of SEQ ID NO: 125, or (xxiv) a VH domain comprising the amino acid sequence of SEQ ID NO: 118 and a VL domain comprising the amino acid sequence of SEQ ID NO: 125, or (xxv) a VH domain comprising the amino acid sequence of SEQ ID NO: 119 and a VL domain comprising the amino acid sequence of SEQ ID NO: 125, or (xxvi) a VH domain comprising the amino acid sequence of SEQ ID NO: 120 and a VL domain comprising the amino acid sequence of SEQ ID NO: 125, or (xxvii) a VH domain comprising the amino acid sequence of SEQ ID NO: 121 and a VL domain comprising the amino acid sequence of SEQ ID NO: 125, or (xxviii) a VH domain comprising the amino acid sequence of SEQ ID NO: 122 and a VL domain comprising the amino acid sequence of SEQ ID NO: 125.

[0009] In some embodiments, the antibody or antigen-binding fragment is a Fab fragment, a Fab' fragment, a F(ab)'2 fragment, a single-chain variable fragment (scFv), or a disulfide-stabilized variable fragment (dsFv).

[0010] In some embodiments, the antibodies or antigen-binding fragments provided herein comprise an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 81-90, 128, and 129.

[0011] In some embodiments, the antibody is an IgG. In some embodiments, the antibody is a humanized antibody.

[0012] In another aspect, provided herein is a nucleic acid molecule encoding an antibody or antigen-binding fragment provided herein. In yet another aspect, provided herein is a vector comprising a nucleic acid molecule provided herein.

[0013] In yet another aspect, provided herein is a host cell transformed with a vector provided herein.

[0014] In yet another aspect, provided herein is a composition comprising a therapeutically effective amount of an antibody or antigen-binding fragment, nucleic acid molecule, or vector provided herein and a pharmaceutically acceptable carrier.

[0015] In yet another aspect, provided herein is a method of treating a patient having a cancer that expresses GPC3, comprising administering to the patient a composition provided herein, wherein optionally the cancer is selected from the group consisting of hepatocellular carcinoma (HCC), melanoma, ovarian clear cell carcinoma (OCCC), yolk sac tumor (YST), neuroblastoma, hepatoblastoma, nephroblastoma (Wilms' tumor), lung squamous cell carcinoma, lung adenocarcinoma, large cell lung carcinoma, small cell lung carcinoma, testicular nonseminomatous germ cell tumor, liposarcoma, cervical intraepithelial neoplasia, adrenal adenoma, schwannoma, embryonal tumor, gastric cancer, colorectal cancer, thyroid cancer, and esophageal cancer.

[0016] In yet another aspect, provided herein is a method for detecting GPC3 in a tissue sample, comprising contacting the tissue sample with an antibody or antigen-binding fragment provided herein; and detecting binding of the antibody to the tissue sample, wherein increased binding of the antibody to the tissue sample compared to binding of the antibody to a control sample detects GPC3 in the tissue sample.

[0017] In some embodiments, the antibody is directly labeled with a detectable marker.

[0018] In some embodiments, the method further comprises contacting the tissue sample with a secondary antibody that specifically binds to the antibody and detecting binding of the secondary antibody, wherein increased binding of the secondary antibody to the tissue sample compared to binding of the secondary antibody to a control sample detects GPC3 in the tissue sample. In some embodiments, the tissue sample comprises cells of hepatocellular carcinoma (HCC), melanoma, ovarian clear cell carcinoma (OCCC), yolk sac tumor (YST), neuroblastoma, hepatoblastoma, nephroblastoma (Wilm's tumor), lung squamous cell carcinoma, lung adenocarcinoma, large cell lung carcinoma, small cell lung carcinoma, testicular nonseminomatous germ cell tumor, liposarcoma, cervical intraepithelial neoplasia, adrenal adenoma, schwannoma, embryonal tumor, gastric cancer, colorectal cancer, thyroid cancer, and / or esophageal cancer.

[0019] In yet another aspect, provided herein is a chimeric antigen receptor (CAR), comprising a polypeptide comprising: (a) an extracellular antigen-binding domain comprising an antibody or antigen-binding fragment provided herein; (b) a transmembrane domain; and (c) an intracellular signaling domain.

[0020] In some embodiments, the antibody or antigen-binding fragment is an scFv.

[0021] In some embodiments, the CARs provided herein further comprise a signal peptide located at the N-terminus of the polypeptide. In some embodiments, the signal peptide is derived from a molecule selected from the group consisting of CD8α, GM-CSF receptor α, and IgG1 heavy chain.

[0022] In some embodiments, the CAR further comprises a hinge domain located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain. In some embodiments, the hinge domain is derived from CD8α.

[0023] In some embodiments, the transmembrane domain is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1.

[0024] In some embodiments, the intracellular signaling domain comprises a costimulatory signaling domain, hi some embodiments, the costimulatory signaling domain is derived from a costimulatory molecule selected from the group consisting of CD27, CD28, CD137, OX40, CD30, CD40, CD3, HVEM, ICOS, Myd88, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand for CD83, and combinations thereof.

[0025] In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell. In some embodiments, the primary intracellular signaling domain is derived from CD3ζ.

[0026] In some embodiments, the CAR provided herein has the amino acid sequence of: (i) SEQ ID NO: 91, or (ii) SEQ ID NO: 92, or (iii) SEQ ID NO: 93, or (iv) SEQ ID NO: 94, or (v) SEQ ID NO: 95, or (vi) SEQ ID NO: 96, or (vii) SEQ ID NO: 97, or (viii) SEQ ID NO: 98, or (ix) SEQ ID NO: 99, or (x) SEQ ID NO: 100, or (xi) SEQ ID NO: 107, or (x (ii) the amino acid sequence of SEQ ID NO: 108, or (xiii) the amino acid sequence of SEQ ID NO: 109, or (xiv) the amino acid sequence of SEQ ID NO: 110, or (xv) the amino acid sequence of SEQ ID NO: 111, or (xvi) the amino acid sequence of SEQ ID NO: 112, or (xvii) the amino acid sequence of SEQ ID NO: 113, or (xviii) the amino acid sequence of SEQ ID NO: 114, or (xix) the amino acid sequence of SEQ ID NO: 115, or (xx) the amino acid sequence of SEQ ID NO: 116, or (xxi) the amino acid sequence of SEQ ID NO: 130, or (xxii) the amino acid sequence of SEQ ID NO: 131.

[0027] In another aspect, provided herein is a polypeptide comprising a CAR provided herein and at least one of the p40 subunit of IL-12 and CCL-19. In some embodiments, p40 is human p40 comprising the amino acid sequence of SEQ ID NO: 135, and CCL-19 is human CCL-19 comprising the amino acid sequence of SEQ ID NO: 136. In some embodiments, p40 and CCL-19 are linked by a first self-cleaving peptide. In some embodiments, the first self-cleaving peptide is a 2A self-cleaving peptide T2A fragment comprising the amino acid sequence of SEQ ID NO: 139. In some embodiments, the p40 subunit of IL-12 and CCL-19 are present in a domain comprising the amino acid sequence of SEQ ID NO: 134. In some embodiments, the CAR is linked to the domain by a second self-cleaving peptide. In some embodiments, the second self-cleaving peptide is a 2A self-cleaving peptide P2A fragment comprising the amino acid sequence of SEQ ID NO: 138. In a specific embodiment, provided herein is a polypeptide comprising the amino acid sequence of SEQ ID NO: 133.

[0028] In yet another aspect, provided herein is a nucleic acid molecule encoding a CAR or polypeptide provided herein. In yet another aspect, provided herein is a vector comprising the nucleic acid molecule. In some embodiments, the vector is a viral vector or a non-viral vector.

[0029] In yet another aspect, provided herein is a host cell transformed with a vector provided herein. In some embodiments, the host cell is an immune effector cell selected from the group consisting of a T cell, a NK cell, a peripheral blood mononuclear cell (PBMC), a hematopoietic stem cell, a pluripotent stem cell, an embryonic stem cell, and a combination thereof. In a specific embodiment, the immune effector cell is a T cell.

[0030] In yet another aspect, provided herein is an immune effector cell, the immune effector cell expressing a CAR provided herein, exogenously introduced p40, and exogenously introduced CCL-19.

[0031] In yet another aspect, provided herein is a composition comprising a therapeutically effective amount of a host cell or immune effector cell provided herein and a pharmaceutically acceptable carrier.

[0032] In yet another aspect, provided herein is a method of treating a patient having a cancer that expresses GPC3, comprising administering to the patient a composition provided herein, wherein the cancer is optionally selected from the group consisting of hepatocellular carcinoma (HCC), melanoma, ovarian clear cell carcinoma (OCCC), yolk sac tumor (YST), neuroblastoma, hepatoblastoma, nephroblastoma (Wilms' tumor), lung squamous cell carcinoma, lung adenocarcinoma, large cell lung carcinoma, small cell lung carcinoma, testicular nonseminomatous germ cell tumor, liposarcoma, cervical intraepithelial neoplasia, adrenal adenoma, schwannoma, embryonal tumor, gastric cancer, colorectal cancer, thyroid cancer, and esophageal cancer.

[0033] In some embodiments, the host cells or immune effector cells are obtained from a patient, while in other embodiments, the host cells or immune effector cells are obtained from a healthy donor.

[0034] In some embodiments, the antibody or antigen-binding fragment or CAR provided herein binds to GPC3 comprising at least one amino acid sequence selected from GenBank Accession Nos. NM_001164617 and NP_001158089, NM_004484 and NP_004475, NM_001164618 and NP_001158090, and NM_001164619 and NP_001158091. [Brief explanation of the drawings]

[0035] [Figure 1] The expression levels of LIC19301 to LIC19310 CAR-T cells are shown.

[0036] [Figure 2] Figure 1 shows the in vitro cytotoxicity of LIC19301-LIC19310 CAR-T cells against GPC3-positive cell lines at various E / T ratios. E / T refers to the ratio of effector cells to target cells. UnT refers to T cells that have not been electroporated with CAR.

[0037] [Figure 3] Figure 1 shows the in vitro cytotoxicity of LIC19301-LIC19310 CAR-T cells against GPC3-negative cell lines at various E / T ratios. E / T refers to the ratio of effector cells to target cells. UnT refers to T cells that have not been electroporated with CAR.

[0038] [Figure 4] Cytokine secretion of LIC19301-LIC19310 CAR-T cells after 24 hours of co-culture with Huh7 or SK-HEP1. UnT refers to T cells not electroporated with CAR.

[0039] [Figure 5] Figure 1 shows the antitumor activity of LIC19309 CAR-T cells following administration to NCG mice transplanted with Huh7 cells.

[0040] [Figure 6] Expression levels of humanized CAR-T cells are shown.

[0041] [Figure 7] Figure 1 shows the in vitro cytotoxicity of humanized CAR-T cells and LIC19309 CAR-T cells against GPC3-positive cell lines at various E / T ratios. E / T refers to the ratio of effector cells to target cells. UnT refers to T cells not transfected with CAR.

[0042] [Figure 8]Figure 1 shows the in vitro cytotoxicity of humanized CAR-T cells and LIC19309 CAR-T cells against GPC3-negative cell lines at various E / T ratios. E / T refers to the ratio of effector cells to target cells. UnT refers to T cells not transfected with CAR.

[0043] [Figure 9] Figure 1 shows IFN-γ secretion of humanized CAR-T cells and LIC19309 CAR-T cells before and after 20 hours of co-culture with HepG2, Huh7, or PLC / PRF / 5 cell lines at various E / T ratios. UnT refers to T cells not transfected with a CAR.

[0044] [Figure 10] Figure 1 shows TNF-α secretion of humanized CAR-T cells and LIC19309 CAR-T cells before and after 20 hours of co-culture with HepG2, Huh7, or PLC / PRF / 5 cell lines at various E / T ratios. UnT refers to T cells not transfected with a CAR.

[0045] [Figure 11] 1 shows the antitumor activity of humanized CAR-T cells following administration to NCG mice transplanted with Huh7 cells.

[0046] [Figure 12] The structures of H93 CAR and H93M CAR are shown. SP refers to the signal peptide. TM refers to the transmembrane domain. TAA conjugate refers to the antigen-binding domain or the conjugate of the tumor-associated antigen (i.e., anti-GPC3 scFv).

[0047] [Figure 13] Figure 1 shows the positive rates of H93 CAR-T cells and H93M CAR-T cells expressing humanized anti-GPC3 scFv CAR. UnT refers to T cells not transduced with a CAR.

[0048] [Figure 14]Shows IL-23 (Figure 14A) and CCL-19 (Figure 14B) protein production in CAR-T cells cultured under normal conditions (RPMI-1640 + 300 IU / mL IL-2).

[0049] [Figure 15] Figure 15 shows the expansion of CAR-T cells in a re-challenge assay. Cells were stimulated overnight with PLC / PRF / 5 cells at a 1:1 E / T ratio every two days. At the end of each round, the fold expansion (Figure 15A) and viability (Figure 15B) of T cells were recorded.

[0050] [Figure 16] Shown are in vitro cytotoxicity (Figure 16A), TNF-α release levels (Figure 16B), and IFN-γ release levels (Figure 16C) of CAR-T cells against GPC3-positive cell lines at an E / T ratio of 1:1 after treatment with various stimulation rounds in a re-challenge assay.

[0051] [Figure 17] Positive rates of CAR-T cells after treatment with different stimulation rounds in a re-challenge assay are shown.

[0052] [Figure 18] PD-1 (Figures 18A and 18B) and LAG3 (Figures 18C and 18D) expression of CAR-T cells 24 hours after each stimulation round in a re-challenge assay is shown.

[0053] [Figure 19] Figure 19 shows a cell migration assay of CAR-T cells performed using a 96-well transwell chamber. Various CAR-T cells were co-cultured with PLC / PRF / 5 cells at a 1:1 E / T ratio for 30 hours, and the cell culture supernatant was collected and added to the lower chamber. After 4, 6, and 8 hours, the number of T cells migrated from the upper chamber to the lower chamber (Figure 19A) and the concentration of CCL-19 in the co-culture supernatant (Figure 19B) were quantified.

[0054] [Figure 20]Figure 20 shows the antitumor effect of CAR-T cells in an NCG mouse xenograft model. NCG mice were subcutaneously inoculated with Huh7 cells and treated with CAR-T cells at 0.2M and 0.6M doses (iv) (n = 4 mice per group). Tumor volume (Figures 20A and 20B) and CAR copy number in genomic DNA of peripheral blood of NCG mice (Figures 20C and 20D) were assessed.

[0055] [Figure 21] Shown are weight changes in mice treated with CAR-T cells at 0.2M (Figure 21A) and 0.6M (Figure 21B) dosages (n=4 mice per group) in an NCG mouse xenograft model.

[0056] [Figure 22] IL-23 protein (Figures 22A and 22B) and IFN-γ protein (Figures 22C and 22D) levels in the peripheral blood of NCG mice after treatment with CAR-T cells at a 0.2M or 0.6M dosage (n=3 mice per group).

[0057] [Figure 23] This figure shows T cell infiltration in tumor tissues from mice treated with 0.2M UnT, H93 CAR-T cells, or H93M CAR-T cells at the endpoint of an animal study in an NCG mouse xenograft model (n=3). Tumor tissues were formalin-fixed, paraffin-embedded, and used for immunohistochemistry (IHC) to detect T cell infiltration stained with 3,3'-diaminobenzidine (DAB). Images were obtained using 3D HISTECH (DRNJIER). The right column shows the magnified area of ​​the black box.

[0058] [Figure 24]This figure shows the recruitment of mouse macrophages and mouse dendritic cells in tumor tissue after treatment with 0.2M UnT, H93 CAR-T cells, or H93M CAR-T cells in an NCG mouse xenograft model. At the endpoint of the animal study, tumor tissue was formalin-fixed, paraffin-embedded, and used for immunohistochemistry (IHC). CD68-bearing macrophages (left panel) and CD11c-bearing dendritic cells (right panel) were stained with 3,3'-diaminobenzidine (DAB). Images were obtained using 3D HISTECH (DRNJIER). DETAILED DESCRIPTION OF THE INVENTION

[0059] The present disclosure is based, in part, on novel antibodies and chimeric antigen receptors that bind to GPC3 or modified cells containing same and their improved properties.

[0060] 5.1.Definition The techniques and procedures described or referenced herein include those that are generally well understood and / or commonly employed by those of skill in the art using conventional methodologies, such as the widely used methodologies described in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd ed. 2001); Current Protocols in Molecular Biology (Ausubel et al. eds., 2003); Therapeutic Monoclonal Antibodies: From Bench to Clinic (An ed. 2009); Monoclonal Antibodies: Methods and Protocols (Albitar ed. 2010); and Antibody Engineering Vols 1 and 2 (Kontermann and Duebel eds., 2nd ed. 2010). Unless otherwise defined herein, scientific and technical terms used in this description have the meanings commonly understood by those of skill in the art. For the purposes of interpreting this specification, the following explanations of terms shall apply, and whenever appropriate, terms used in the singular shall include the plural and vice versa. In the event that any explanation of a term provided contradicts any material incorporated herein by reference, the explanation of the term set forth below shall prevail.

[0061] The terms "antibody," "immunoglobulin," or "Ig" are used interchangeably herein and are used in the broadest sense, specifically encompassing, for example, monoclonal antibodies (including agonist, antagonist, neutralizing, full-length, or intact monoclonal antibodies), antibody compositions with polyepitopic or monoepitopic specificity, polyclonal or monovalent antibodies, multivalent antibodies, multispecific antibodies formed from at least two intact antibodies (e.g., bispecific antibodies, so long as they exhibit the desired biological activity), single-chain antibodies, and fragments thereof (e.g., domain antibodies) as described below. Antibodies may be human, humanized, chimeric, and / or affinity-matured antibodies, as well as antibodies from other species, such as mouse, rabbit, llama, etc. The term "antibody" is intended to include polypeptide products of B cells that are capable of binding to a specific molecular antigen and are within the immunoglobulin class of polypeptides composed of two identical pairs of polypeptide chains, each pair having one heavy chain (about 50-70 kDa) and one light chain (about 25 kDa), with the amino-terminal portion of each chain containing a variable region of about 100 to about 130 amino acids or more and the carboxy-terminal portion of each chain containing a constant region. See, e.g., Antibody Engineering (Borrebaeck ed., 2nd ed. 1995); and Kuby, Immunology (3rd ed. 1997). Antibodies also include, but are not limited to, synthetic antibodies, recombinantly produced antibodies, antibodies including those derived from Camelidae species (e.g., llamas or alpacas) or humanized variants thereof, intrabodies, anti-idiotypic (anti-Id) antibodies, and functional fragments (e.g., antigen-binding fragments) of any of the above (which refers to a portion of an antibody heavy or light chain polypeptide that retains some or all of the binding activity of the antibody from which the fragment is derived). Non-limiting examples of functional fragments (e.g., antigen-binding fragments) include single-chain Fvs (scFvs) (including, e.g., monospecific, bispecific, etc.), Fab fragments, F(ab') fragments, F(ab') fragments, F(ab') fragments, disulfide-linked Fvs (dsFvs), Fd fragments, Fv fragments, diabodies, triabodies, tetrabodies, and minibodies.Specifically, the antibodies provided herein include immunoglobulin molecules and molecules containing an immunologically active portion of an immunoglobulin molecule, such as an antigen-binding domain or site that binds to an antigen (e.g., one or more CDRs of an antibody). For such antibody fragments, see, for example, Harlow and Lane, Antibodies: A Laboratory Manual (1989); Mol. Biology and Biotechnology: A Comprehensive Desk Reference (Myers ed., 1995); Huston et al., 1993, Cell Biophysics 22:189-224; Plueckthun and Skerra, 1989, Meth. Enzymol. 178:497-515; and Day, Advanced Immunochemistry (2nd ed. 1990). The antibodies provided herein can be of any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) of immunoglobulin molecule. The antibody can be an agonist antibody or an antagonist antibody. The antibody can be neither an agonist antibody nor an antagonist antibody.

[0062] An "antigen" is a structure to which an antibody can selectively bind. A target antigen can be a polypeptide, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound. In some embodiments, the target antigen is a polypeptide. In certain embodiments, the antigen is associated with a cell, e.g., present on or within a cell.

[0063] An "intact" antibody is one that contains an antigen-binding site as well as a CL and at least heavy chain constant regions, CH1, CH2, and CH3. The constant region includes a human constant region or an amino acid sequence variant thereof. In certain embodiments, an intact antibody has one or more effector functions.

[0064] A "single-chain Fv," also abbreviated as "sFv" or "scFv," is an antibody fragment comprising the VH and VL antibody domains linked into a single polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the sFv to form the desired structure for antigen binding. For a review of sFvs, see Plückthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0065] The term "heavy chain-only antibody" or "HCAb" refers to a functional antibody that contains a heavy chain but lacks the light chain typically found in four-chain antibodies. Camelids (such as camels, llamas, or alpacas) are known to produce HCAbs.

[0066] The terms "bind" or "binding" refer to interactions between molecules, including, for example, forming a complex. The interaction can be a non-covalent interaction, including, for example, hydrogen bonding, ionic bonding, hydrophobic interactions, and / or van der Waals interactions. A complex can also include the association of two or more molecules held together by covalent or non-covalent bonds, interactions, or forces. The strength of the overall non-covalent interactions between a single antigen-binding site on an antibody and a single epitope of a target molecule, such as an antigen, is the affinity of the antibody or functional fragment for that epitope. The association rate (k on ) to the dissociation rate (k off ) ratio (k off / k on ) is the dissociation constant K D which is inversely proportional to the affinity. D The lower the value, the higher the affinity of the antibody. D The value of k varies depending on the antibody-antigen complex. on and k off The dissociation constant K of the antibodies provided herein depends on bothD can be determined using any method provided herein or any other method known to those skilled in the art. The affinity at one binding site does not always reflect the true strength of the interaction between an antibody and an antigen. When a complex antigen containing multiple repeating antigenic determinants, such as a multivalent antigen, contacts an antibody containing multiple binding sites, the interaction of the antibody with the antigen at one site will increase the likelihood of reaction at a second site. The strength of such multiple interactions between a multivalent antibody and an antigen is called avidity.

[0067] In the context of the binding molecules described herein, terms such as "binds to," "specifically binding to," and similar terms are also used interchangeably herein to refer to binding molecules, such as polypeptides, of an antigen-binding domain that specifically binds to an antigen. Binding molecules or antigen-binding domains that bind or specifically bind to an antigen can be identified, for example, by immunoassays, Octet®, Biacore®, or other techniques known to those skilled in the art. In some embodiments, a binding molecule or antigen-binding domain binds to or specifically binds to an antigen if it binds to the antigen with higher affinity than any cross-reactive antigen, as determined using experimental techniques such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). Typically, a specific or selective response will be at least twice the background signal or noise and may be more than 10 times the background. For a discussion of binding specificity, see, e.g., Fundamental Immunology 332-36 (Paul ed., 2d ed. 1989). In certain embodiments, the extent to which a binding molecule or antigen-binding domain binds to a "non-target" protein is less than about 10% of the binding of the binding molecule or antigen-binding domain to its specific target antigen, as determined, for example, by fluorescence-activated cell sorting (FACS) analysis or RIA. Binding molecules or antigen-binding domains that bind to antigens include those that have the ability to bind to antigens with sufficient affinity to make them useful for targeting antigens, e.g., as therapeutic and / or diagnostic agents. In certain embodiments, binding molecules or antigen-binding domains that bind to an antigen have a dissociation constant (K) of 1 μM, 800 nM, 600 nM, 550 nM, 500 nM, 300 nM, 250 nM, 100 nM, 50 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, or 0.1 nM or less. D In certain embodiments, the binding molecule or antigen-binding domain binds to an epitope that is conserved among antigens of different species.

[0068] In certain embodiments, a binding molecule or antigen-binding domain can comprise a "chimeric" sequence, in which a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of one or more chains is identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see U.S. Pat. No. 4,816,567; and Morrison et al., 1984, Proc. Natl. Acad. Sci. USA 81:6851-55). Chimeric sequences can include humanized sequences.

[0069] In certain embodiments, a binding molecule or antigen-binding domain can comprise a portion of a non-human (e.g., camelid, murine, non-human primate) antibody in a "humanized" form comprising sequences from a human immunoglobulin (e.g., recipient antibody), in which native CDR residues are replaced by residues from a corresponding CDR of a non-human species (e.g., donor antibody) such as camelid, mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and capacity. In some instances, one or more FR region residues of the human immunoglobulin sequence are replaced by corresponding non-human residues. Furthermore, humanized antibodies can comprise residues that are not found in the recipient antibody or the donor antibody. Such modifications are made to further refine antibody performance. A humanized antibody heavy or light chain can comprise substantially all of at least one or more variable regions, in which all or substantially all of the CDRs correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. In certain embodiments, a humanized antibody will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-25 (1986); Riechmann et al., Nature 332:323-29 (1988); Presta, Curr. Op. Struct. Biol. 2:593-96 (1992); Carter et al., Proc. Natl. Acad. Sci. USA 89:4285-89 (1992); U.S. Patent Nos. 6,800,738; 6,719,971; 6,639,055; 6,407,213; and 6,054,297.

[0070] In certain embodiments, a binding molecule or antigen-binding domain can comprise a portion of a "fully human antibody" or "human antibody," where these terms are used interchangeably herein and refer to an antibody comprising a human variable region and, for example, a human constant region. The binding molecule can comprise an antibody sequence. In specific embodiments, these terms refer to an antibody comprising variable and constant regions of human origin. A "fully human" antibody can also encompass, in certain embodiments, an antibody that binds a polypeptide and that is encoded by a nucleic acid sequence that is a naturally occurring somatic variant of a human germline immunoglobulin nucleic acid sequence. The term "fully human antibody" includes antibodies having variable and constant regions that correspond to human germline immunoglobulin sequences as described by Kabat et al. (See Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242). A "human antibody" is one having an amino acid sequence that corresponds to that of an antibody produced by a human and / or an antibody produced using human antibody production techniques. This definition of a human antibody specifically excludes humanized antibodies, which contain non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol. 227:381 (1991); Marks et al., J. Mol. Biol. 222:581 (1991)) and yeast display libraries (Chao et al., Nature Protocols 1:755-68 (2006)).For the preparation of human monoclonal antibodies, the methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy 77 (1985); Boerner et al., J. Immunol. 147(1):86-95 (1991); and van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001) can also be used. Human antibodies can be prepared by administering antigen to transgenic animals, e.g., mice, that have been modified to produce such antibodies in response to antigen challenge but render their endogenous gene loci incompetent (see, e.g., Jakobovits, Curr. Opin. Biotechnol. 6(5):561-66 (1995); Brueggemann and Taussing, Curr. Opin. Biotechnol. 8(4):455-58 (1997); and U.S. Pat. Nos. 6,075,181 and 6,150,584, regarding XENOMOUSE™ technology). See also, e.g., Li et al., Proc. Natl. Acad. Sci. USA 103:3557-62 (2006), regarding human antibodies generated by human B-cell hybridoma technology.

[0071] In certain embodiments, the binding molecule or antigen-binding domain may comprise a portion of a "recombinant human antibody," which term includes human antibodies prepared, expressed, produced or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant combinatorial human antibody library, antibodies isolated from animals (e.g., mice or cows) transgenic and / or transchromosomal for human immunoglobulin genes (see, e.g., Taylor, LD et al., Nucl. Acids Res. 20:6287-6295 (1992)), or antibodies prepared, expressed, produced or isolated by any other means involving splicing of human immunoglobulin gene sequences with other DNA sequences. Such recombinant human antibodies may have variable and constant regions derived from human germline immunoglobulin sequences (see Kabat, E.A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242). However, in certain embodiments, such recombinant human antibodies have been subjected to in vitro mutagenesis (or, when animals transgenic for human Ig sequences are used, in vivo somatic mutagenesis) such that the amino acid sequences of the VH and VL regions of the recombinant antibodies, while derived from and closely related to human germline VH and VL sequences, are sequences that may not naturally exist within the human antibody germline repertoire in vivo.

[0072] In certain embodiments, a binding molecule or antigen-binding domain can comprise a portion of a "monoclonal antibody," which term, as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical except for naturally occurring mutations that may be present in small amounts, or well-known post-translational modifications such as amino acid isomerization or deamidation, methionine oxidation, or asparagine or glutamine deamidation, such that each monoclonal antibody typically recognizes a single epitope on the antigen. In a specific embodiment, a "monoclonal antibody," as used herein, is an antibody produced by a single hybridoma or other cell. The term "monoclonal" is not limited to any particular method of making the antibody. For example, monoclonal antibodies useful in the present disclosure can be prepared by the hybridoma methodology first described by Kohler et al., Nature 256:495 (1975), or can be made using recombinant DNA methods in bacteria or eukaryotic animal or plant cells (see, e.g., U.S. Pat. No. 4,816,567). "Monoclonal antibodies" can also be isolated from phage antibody libraries using the techniques described, for example, in Clackson et al., Nature 352:624-28 (1991) and Marks et al., J. Mol. Biol. 222:581-97 (1991). Other methods for preparing clonal cell lines and the monoclonal antibodies they express are well known in the art. See, e.g., Short Protocols in Molecular Biology (Ausubel et al. eds., 5th ed. 2002).

[0073] A typical four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. In the case of IgG, the four-chain unit is generally approximately 150,000 daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has a variable domain (VH) at its N-terminus, followed by three constant domains (CH) for each of the α and γ chains and four CH domains for the μ and ε isotypes. Each L chain has a variable domain (VL) at its N-terminus, followed by a constant domain (CL) at its other end. The VL is aligned with the VH, and the CL is aligned with the first constant domain (CH1) of the heavy chain. Particular amino acid residues are believed to form an interface between the light-chain and heavy-chain variable domains. The pairing of a VH and a VL together forms a single antigen-binding site. The structure and properties of various antibody classes are described, for example, in Basic and Clinical Immunology 71 (Stites et al. eds., 8th ed. 1994); and Immunobiology (Janeway et al. eds., 5th ed. 1995). th ed. 2001).

[0074] The term "Fab" or "Fab region" refers to the antibody region that binds to an antigen. Conventional IgGs usually contain two Fab regions, each in one of the two arms of the Y-shaped IgG structure. Each Fab region typically consists of one variable region and one constant region from each of the heavy and light chains. More specifically, the variable and constant regions of the heavy chain in the Fab region are the VH and CH1 regions, and the variable and constant regions of the light chain in the Fab region are the VL and CL regions. The VH, CH1, VL, and CL regions in the Fab region can be arranged in various ways to confer antigen-binding ability according to the present disclosure. For example, as with the Fab region of a conventional IgG, the VH and CH1 regions can be on one polypeptide, while the VL and CL regions can be on separate polypeptides. Alternatively, as described in more detail in the following section, the VH, CH1, VL, and CL regions can all be on the same polypeptide and oriented in various orders.

[0075] The terms "variable region," "variable domain," "V region," or "V domain" refer to a portion of an antibody light or heavy chain, generally located at the amino terminus of the light or heavy chain, approximately 120-130 amino acids in length for heavy chains and approximately 100-110 amino acids in length for light chains, that is used in the binding and specificity of each particular antibody to its particular antigen. The variable region of a heavy chain may be referred to as "VH." The variable region of a light chain may be referred to as "VL." The term "variable" refers to the fact that certain segments of variable regions exhibit extensive sequence differences among antibodies. The V region mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, variability is not evenly distributed across the 110-amino acid span of the variable region. Instead, V regions consist of less variable (e.g., relatively invariant) stretches of approximately 15-30 amino acids called framework regions (FRs) separated by short regions of higher variability (e.g., hypervariability) called "hypervariable regions," each approximately 9-12 amino acids in length. The variable regions of the heavy and light chains each contain four FRs, each adopting a primarily β-sheet structure connected by three hypervariable regions that form loops connecting, and in some cases forming part of, the β-sheet structure. The hypervariable regions of each chain are held together in close proximity by the FRs and, together with the hypervariable regions of the other chain, contribute to the formation of the antigen-binding site of antibodies (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest (5th ed. 1991)). The constant regions are not directly involved in binding of antibodies to antigens but exhibit various effector functions, such as antibody participation in antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The variable regions vary widely in sequence among different antibodies, hi specific embodiments, the variable regions are human variable regions.

[0076] The terms "variable region residue numbering according to Kabat" or "numbering of amino acid positions as in Kabat" and variations thereof refer to the numbering system used for the heavy or light chain variable regions of the antibody sequences in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to shortening of, or insertion into, a FR or CDR of the variable domain. For example, a heavy chain variable domain may contain a single amino acid insertion after residue 52 (residue 52a according to Kabat) and three inserted residues after residue 82 (e.g., residues 82a, 82b, and 82c according to Kabat). The Kabat numbering of residues may be determined for a given antibody by alignment of the antibody's sequence at regions of homology with the "standard" Kabat numbering sequence. The Kabat numbering system is generally used when referring to residues in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al., supra). The "EU numbering system" or "EU index" is generally used when referring to residues in the immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., supra). "EU index as in Kabat" refers to the numbering of residues in a human IgG1 EU antibody. Other numbering systems are described, for example, by AbM, Chothia, Contact, IMGT, and AHon.

[0077] The term "heavy chain," when used in reference to an antibody, refers to a polypeptide chain of approximately 50 to 70 kDa, the amino-terminal portion of which contains a variable region of approximately 120 to 130 amino acids or more, and the carboxy-terminal portion of which contains a constant region. The constant region can be one of five distinct types (e.g., isotypes) designated alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ) based on the amino acid sequence of the heavy chain constant region. Distinct heavy chains vary in size: α, δ, and γ contain approximately 450 amino acids, while μ and ε contain approximately 550 amino acids. When combined with light chains, these distinct types of heavy chains give rise to the five well-known antibody classes (e.g., isotypes), IgA, IgD, IgE, IgG, and IgM, respectively, which include the four subclasses of IgG, namely, IgG1, IgG2, IgG3, and IgG4.

[0078] The term "light chain," when used in reference to an antibody, refers to a polypeptide chain of about 25 kDa, the amino-terminal portion of which contains a variable region of about 100 to about 110 amino acids or more, and the carboxy-terminal portion of which contains a constant region. Light chains are approximately 211 to 217 amino acids in length. There are two distinct types, called kappa (κ) or lambda (λ), based on the amino acid sequence of the constant domain.

[0079] As used herein, the terms "hypervariable region," "HVR," "complementarity-determining region," and "CDR" are used interchangeably. "CDR" refers to one of the three hypervariable regions (H1, H2, or H3) in the non-framework region of an immunoglobulin (Ig or antibody) VH β-sheet framework or one of the three hypervariable regions (L1, L2, or L3) in the non-framework region of an antibody VL β-sheet framework. CDR1, CDR2, and CDR3 in a VH domain are also referred to as HCDR1, HCDR2, and HCDR3, respectively. CDR1, CDR2, and CDR3 in a VL domain are also referred to as LCDR1, LCDR2, and LCDR3, respectively. Thus, CDRs are variable region sequences interspersed within framework region sequences.

[0080] CDR regions are well known to those skilled in the art and are defined by well-known numbering systems. For example, Kabat complementarity determining regions (CDRs) are based on sequence variability and are the most commonly used (see, for example, Kabat et al., supra; Nick Deschacht et al., J Immunol 2010;184:5696-5704). Chothia instead refers to the position of structural loops (see, for example, Chothia and Lesk, J. Mol. Biol.196:901-17(1987)). The end of the Chothia CDR-H1 loop, when numbered using the Kabat numbering convention, varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places insertions at H35A and H35B; if neither 35A nor 35B are present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and the Chothia structural loops and are used in Oxford Molecular's AbM antibody modeling software (see, e.g., Antibody Engineering Vol. 2 (Kontermann and Duebel eds., 2nd ed. 2010)). The "contact" hypervariable regions are based on an analysis of available complex crystal structures. Another universal numbering system that has been developed and widely adopted is the ImMunoGeneTics (IMGT) Information System® (Lafranc et al., Dev. Comp. Immunol. 27(1):55-77 (2003)). IMGT is a comprehensive information system dedicated to immunoglobulins (IGs), T cell receptors (TCRs), and major histocompatibility complexes (MHCs) of humans and other vertebrates. Here, CDRs are referenced both in terms of amino acid sequence and location within the light or heavy chain.Because the "location" of CDRs within the structure of immunoglobulin variable domains is conserved across species and resides in structures called loops, CDR and framework residues are readily identified by using a numbering system that aligns variable domain sequences according to structural features. This information can be used to graft and replace CDR residues from one species of immunoglobulin onto an acceptor framework, typically from a human antibody. An additional numbering system (AHon) has been developed by Honegger and Plueckthun, J. Mol. Biol. 309:657-70 (2001). Correspondence between numbering systems, including, for example, Kabat numbering and the IMGT proprietary numbering system, is well known to those skilled in the art (see, e.g., Kabat, supra; Chothia and Lesk, supra; Martin, supra; Lefranc et al., supra). Residues from each of these hypervariable regions or CDRs are illustrated in Table 1 below. [Table 1]

[0081] The boundaries of a given CDR may vary depending on the scheme used for identification. Thus, unless otherwise specified, the terms "CDR" and "complementarity-determining region" of a given antibody or region thereof, such as a variable region, and individual CDRs of the antibody or region thereof (e.g., CDR-H1, CDR-H2) should be understood to encompass the complementarity-determining regions as defined by any of the known schemes described hereinabove. In some instances, schemes for identifying a particular CDR or CDRs are designated, such as CDRs as defined by the IMGT, Kabat, Chothia, or Contact method. In other cases, the specific amino acid sequence of the CDR is provided. It should be noted that CDR regions can also be defined by a combination of various numbering schemes, such as a combination of the Kabat and Chothia numbering schemes or a combination of the Kabat and IMGT numbering schemes. Thus, a term such as "CDR1 as shown in a particular VH" includes, but is not limited to, any CDR1 as defined by the exemplary CDR numbering schemes described above. One of skill in the art will appreciate that, given a variable region (eg, VH or VL), the CDRs within that region may be defined by different numbering systems or combinations thereof.

[0082] The hypervariable regions may comprise "extended hypervariable regions" as follows: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in VL and 26-35 or 26-35A (H1), 50-65 or 49-65 (H2) and 93-102, 94-102 or 95-102 (H3) in VH.

[0083] The term "constant region" or "constant domain" refers to the carboxy-terminal portions of the light and heavy chains, which are not directly involved in binding to an antigen by an antibody but exhibit various effector functions, such as interaction with Fc receptors. This term refers to the portion of an immunoglobulin molecule that has a more conserved amino acid sequence than the other portions of the immunoglobulin, the variable region, which contain the antigen-binding site. The constant region may contain the CH1, CH2, and CH3 regions of the heavy chain and the CL region of the light chain.

[0084] The term "framework" or "FR" refers to variable region residues that flank the CDRs. FR residues are present, for example, in chimeric, humanized, human, domain antibodies, diabodies, linear antibodies, and bispecific antibodies. FR residues are variable domain residues other than hypervariable region or CDR residues.

[0085] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including, for example, native-sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is often defined to stretch from the amino acid residue at Cys226 or Pro230 to its carboxyl-terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) can be removed, for example, during antibody production or purification, or by recombinantly modifying the nucleic acid encoding the antibody heavy chain. Thus, an intact antibody composition can include antibody populations in which all K447 residues have been removed, antibody populations in which none of the K447 residues have been removed, and antibody populations having a mixture of antibodies with and without the K447 residue. A "functional Fc region" comprises an "effector function" of a native-sequence Fc region. Exemplary "effector functions" include C1q binding; CDC; Fc receptor binding; ADCC; phagocytosis; down-regulation of cell surface receptors (e.g., B cell receptors); and the like. Such effector functions generally require the Fc region to be in combination with a binding region or domain (e.g., an antibody variable region or domain), which can be assessed using various assays known to those of skill in the art. A "variant Fc region" comprises an amino acid sequence that differs from a native-sequence Fc region by virtue of at least one amino acid modification (e.g., substitution, addition, or deletion). In certain embodiments, the variant Fc region comprises at least one amino acid substitution relative to the native-sequence Fc region or relative to the Fc region of the parent polypeptide, e.g., from about 1 to about 10 amino acid substitutions, or from about 1 to about 5 amino acid substitutions, relative to the native-sequence Fc region or to the Fc region of the parent polypeptide. A variant Fc region herein may have at least about 80% homology to a native sequence Fc region and / or the Fc region of a parent polypeptide, or at least about 90% homology thereto, for example, at least about 95% homology thereto.

[0086] As used herein, "epitope" is a term used in the art and refers to a localized region in an antigen to which a binding molecule (e.g., an antibody) can specifically bind. An epitope can be a linear epitope or a conformational, non-linear, or discontinuous epitope. In the case of a polypeptide antigen, for example, an epitope can be consecutive amino acids of the polypeptide (a "linear" epitope), or it can include amino acids from two or more noncontiguous regions of the polypeptide (a "conformational," "non-linear," or "discontinuous" epitope). Those skilled in the art will generally understand that a linear epitope may or may not depend on secondary, tertiary, or quaternary structure. For example, in some embodiments, a binding molecule binds to a group of amino acids, regardless of whether those amino acids are folded into the native three-dimensional protein structure. In other embodiments, the amino acid residues that make up the epitope must adopt a particular conformation (eg, bend, twist, turn, or fold) in order for the binding molecule to recognize and bind to the epitope.

[0087] "Percent (%) amino acid sequence identity" and "homology," in the context of peptide, polypeptide, or antibody sequences, are defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a particular peptide or polypeptide sequence, after aligning the sequences to achieve the maximum percent sequence identity, introducing gaps if necessary, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be accomplished in a variety of ways that are within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences under comparison.

[0088] The term "specificity" refers to the selective recognition of an antigen-binding protein (such as a CAR or antibody) for a specific epitope of an antigen. Natural antibodies, for example, are monospecific. The term "multispecificity," as used herein, means that an antigen-binding protein (such as a CAR or antibody) has two or more antigen-binding sites, at least two of which bind to different antigens. "Bispecificity," as used herein, means that an antigen-binding protein (such as a CAR or antibody) has two different antigen-binding specificities. The term "monospecific" CAR, as used herein, means an antigen-binding protein (such as a CAR or antibody) with one or more binding sites that each bind to the same antigen.

[0089] The term "valent," as used herein, refers to the presence of a specified number of binding sites on an antigen-binding protein (such as a CAR or antibody). For example, a natural antibody or full-length antibody has two binding sites and is bivalent. Thus, the terms "trivalent," "tetravalent," "pentavalent," and "hexavalent" refer to the presence of two, three, four, five, and six binding sites on an antigen-binding protein (such as a CAR or antibody), respectively.

[0090] "Chimeric antigen receptor" or "CAR," as used herein, refers to a genetically engineered receptor that can be used to graft one or more antigen specificities onto immune effector cells, such as T cells. Some CARs are also known as "artificial T cell receptors," "chimeric T cell receptors," or "chimeric immune receptors." In some embodiments, a CAR comprises an extracellular antigen-binding domain specific for one or more antigens (such as tumor antigens), a transmembrane domain, and an intracellular signaling domain of a T cell and / or other receptor. "CAR-T cell" refers to a T cell that expresses a CAR.

[0091] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer can be linear or branched, it can contain modified amino acids, and it can be interrupted by non-amino acids. These terms also encompass amino acid polymers that have undergone natural or intervening modifications, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification. Also included within the definition are polypeptides containing one or more analogs of an amino acid, including, but not limited to, unnatural amino acids, as well as other modifications known in the art. Because the polypeptides of the present disclosure may be based on antibodies or other members of the immunoglobulin superfamily, it is understood that in certain embodiments, a "polypeptide" can exist as a single chain or as two or more associated chains.

[0092] As used interchangeably herein, "polynucleotide" or "nucleic acid" refers to a polymer of nucleotides of any length, including DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction. Polynucleotides can include modified nucleotides, such as methylated nucleotides, and their analogs. "Oligonucleotide," as used herein, refers to short, generally single-stranded, synthetic polynucleotides that generally, although not necessarily, do not exceed about 200 nucleotides in length. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The above description of polynucleotides is equally and fully applicable to oligonucleotides. Cells producing the binding molecules of the present disclosure can include parent hybridoma cells as well as bacterial and eukaryotic host cells into which nucleic acid encoding the antibody has been introduced. Unless otherwise specified, the left-hand end of any single-stranded polynucleotide sequence disclosed herein is the 5' end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The direction of addition of nascent RNA transcripts from 5' to 3' is referred to as the transcription direction; the region of the sequence on the DNA strand that is 5' to the 5' end of the RNA transcript and has the same sequence as the RNA transcript is referred to as the "upstream sequence"; the region of the sequence on the DNA strand that is 3' to the 3' end of the RNA transcript and has the same sequence as the RNA transcript is referred to as the "downstream sequence."

[0093] An "isolated nucleic acid" is a nucleic acid, e.g., RNA, DNA, or mixture of nucleic acids, that is substantially separated from other genomic DNA sequences and proteins or complexes, such as ribosomes and polymerases, that naturally accompany the native sequence. An "isolated" nucleic acid molecule is one that is separated from other nucleic acid molecules that are present in the nucleic acid molecule's natural source. Furthermore, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium if produced by recombinant techniques, or substantially free of chemical precursors or other chemicals if chemically synthesized. In specific embodiments, one or more nucleic acid molecules encoding an antibody as described herein are isolated or purified. This term encompasses a nucleic acid sequence that has been removed from its naturally occurring environment, including recombinant or cloned DNA isolates and chemically synthesized analogs or biologically synthesized analogs produced by heterologous systems. A substantially pure molecule can include a molecule in isolated form. Specifically, an "isolated" nucleic acid molecule encoding a CAR or antibody described herein is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is normally associated in the environment in which it is produced.

[0094] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence encoding a protein or RNA can also include introns, to the extent that the nucleotide sequence encoding the protein, depending on the version, may contain one or more introns.

[0095] The term "control sequence" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. Control sequences suitable for prokaryotes include, for example, a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0096] As used herein, the term "operably linked," and similar phrases (e.g., genetically fused), when used in reference to nucleic acids or amino acids, refers to an operable linkage in which nucleic acid or amino acid sequences, respectively, are placed in a functional relationship with each other. For example, operably linking a promoter, enhancer element, open reading frame, 5' and 3' UTRs, and terminator sequence results in the correct production of a nucleic acid molecule (e.g., RNA). In some embodiments, operably linked nucleic acid elements result in transcription of the open reading frame and ultimately production of a polypeptide (i.e., expression of the open reading frame). As another example, an operably linked peptide is one in which functional domains are positioned at an appropriate distance from each other to confer the intended function of each domain.

[0097] The term "vector" refers to a substance used to carry or contain nucleic acid sequences, including, for example, nucleic acid sequences encoding binding molecules (e.g., antibodies) as described herein, for introducing nucleic acid sequences into a host cell. Vectors applicable for use include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which may contain selection sequences or markers that can serve for stable integration into a host cell chromosome. In addition, a vector may contain one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes that can be included provide, for example, resistance to antibiotics or toxins, complement deficiencies of required nutrients, or supply important nutrients not present in the culture medium. Expression control sequences may include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like, which are well known in the art. When two or more nucleic acid molecules (e.g., both antibody heavy and light chains or antibody VH and VL) are to be coexpressed, both nucleic acid molecules can be inserted, for example, into a single expression vector or into separate expression vectors. For single vector expression, the encoding nucleic acids can be operably linked to one common expression control sequence, or can be linked to different expression control sequences, such as one inducible promoter and one constitutive promoter. Introduction of nucleic acid molecules into host cells can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA, immunoblotting for expression of gene products, or other suitable analytical methods for testing expression of the introduced nucleic acid sequence or its corresponding gene product. Those skilled in the art will understand that nucleic acid molecules will be expressed in amounts sufficient to produce the desired product, and will further understand that expression levels can be optimized to achieve sufficient expression using methods well known in the art.

[0098] The term "host," as used herein, refers to an animal, such as a mammal (e.g., a human).

[0099] The term "host cell," as used herein, refers to the particular subject cell that can be transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell. The progeny of such a cell may not be identical to the parent cell transfected with the nucleic acid molecule due to mutations or environmental influences that may occur in subsequent generations or due to integration of the nucleic acid molecule into the host cell genome.

[0100] As used herein, the term "autologous" is meant to refer to any material derived from the same individual that is subsequently reintroduced into the individual.

[0101] "Allogeneic" refers to a graft derived from a different individual of the same species.

[0102] The terms "transfected" or "transformed" or "transduced," as used herein, refer to the process of transferring or introducing exogenous nucleic acid into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed, or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0103] The term "pharmaceutically acceptable," as used herein, means approved by a federal or state regulatory agency for use in animals, and more particularly in humans, or listed in the United States Pharmacopoeia, the European Pharmacopoeia, or other generally recognized pharmacopoeias.

[0104] "Excipient" means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, solvent, or encapsulating material. Excipients include, for example, encapsulating materials or additives, such as absorption enhancers, antioxidants, binders, buffers, carriers, coating agents, colorants, diluents, disintegrants, emulsifiers, bulking agents, fillers, flavoring agents, humectants, lubricants, fragrances, preservatives, propellants, release agents, sterilizing agents, sweeteners, solubilizers, wetting agents, and mixtures thereof. The term "excipient" can also refer to a diluent, adjuvant (e.g., Freund's adjuvant (complete or incomplete)), or vehicle.

[0105] In some embodiments, the excipient is a pharmaceutically acceptable excipient. Examples of pharmaceutically acceptable excipients include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (e.g., less than about 10 amino acid residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™. Other examples of pharmaceutically acceptable excipients are described in Remington and Gennaro, Remington's Pharmaceutical Sciences (18th ed. 1990).

[0106] In one embodiment, each component is "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of the pharmaceutical formulation, suitable for use in contact with human and animal tissues or organs, without undue toxicity, irritation, allergic reaction, immunogenicity, or other problems or complications, and commensurate with a reasonable benefit-risk ratio. See, e.g., Lippincott Williams & Wilkins: Philadelphia, PA, 2005; Handbook of Pharmaceutical Excipients, 6th ed.; Rowe et al., Eds.; The Pharmaceutical Press and the American Pharmaceutical Association: 2009; Handbook of Pharmaceutical Additives, 3rd ed.; Ash and Ash Eds.; Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, 2nd ed.; Gibson Ed.; CRC Press LLC: Boca Raton, FL, 2009. In some embodiments, a pharmaceutically acceptable excipient is nontoxic to cells or mammals exposed thereto at the dosages and concentrations employed. In some embodiments, the pharmaceutically acceptable excipient is an aqueous pH buffered solution.

[0107] In some embodiments, the excipient is a sterile liquid, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is an exemplary excipient when the composition (e.g., pharmaceutical composition) is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid excipients, particularly for injectable solutions. Excipients can also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, nonfat dry milk, glycerol, propylene, glycol, water, ethanol, and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition can take the form of a solution, suspension, emulsion, tablet, pill, capsule, powder, sustained-release formulation, or the like. Oral compositions can include standard excipients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc., including formulations.

[0108] Compositions, including pharmaceutical compounds, can contain a binding molecule (eg, an antibody), eg, in isolated or purified form, together with a suitable amount of excipients.

[0109] The term "effective amount" or "therapeutically effective amount," as used herein, refers to an amount of an antibody or therapeutic molecule, including drugs and antibodies or pharmaceutical compositions provided herein, sufficient to bring about a desired result.

[0110] The terms "subject" and "patient" may be used interchangeably. As used herein, in certain embodiments, a subject is a mammal, such as a non-primate or a primate (e.g., a human). In specific embodiments, a subject is a human. In one embodiment, a subject is a mammal, e.g., a human, who has been diagnosed with a disease or disorder. In another embodiment, a subject is a mammal, e.g., a human, who is at risk of developing a disease or disorder.

[0111] "Administering" or "administration" refers to the act of injecting or otherwise physically delivering a substance as it exists outside the body to a patient, such as by mucosal, intradermal, intravenous, intramuscular delivery, and / or any other physical delivery method described herein or known in the art.

[0112] As used herein, the terms "treat," "treatment," and "treating" refer to the reduction or amelioration of the progression, severity, and / or duration of a disease or condition resulting from the administration of one or more therapies. Treatment may be determined by assessing whether there has been a decrease, alleviation, and / or mitigation of one or more symptoms associated with the underlying disorder, such that the patient experiences improvement even though the patient may still be suffering from the underlying disorder. The term "treatment" includes both maintenance and remission of the disease. The terms "maintain," "maintaining," and "maintenance" refer to the beneficial effect a subject derives from a therapy, but which does not necessarily result in a cure of the disease.

[0113] The terms "prevent," "preventing," and "prevention" refer to reducing the likelihood of a disease, disorder, condition, or one or more associated symptoms (e.g., diabetes or cancer) occurring (or recurring).

[0114] As used herein, "delaying" the onset of cancer means extending, preventing, slowing, retarding, stabilizing, and / or postponing the onset of the disease. This delay can be of varying lengths of time, depending on the course of the disease and / or individual being treated. As will be apparent to one of skill in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease. A method for "delaying" the onset of cancer is one that reduces the probability of disease onset within a given time frame and / or reduces the extent of disease within a given time frame, compared to the absence of the method. Such comparisons are typically based on clinical trials using a statistically significant number of individuals. The onset of cancer may be detectable using standard methods, including, but not limited to, computerized axial tomography (CAT scan), magnetic resonance imaging (MRI), abdominal ultrasound, coagulation studies, arteriography, or biopsy. Onset may also refer to the progression of cancer, which may be initially undetectable, and includes emergence, recurrence, and development.

[0115] As used herein, "GPC3-associated disease or disorder" refers to a disease or disorder involving cells or tissues in which GPC3 is expressed or overexpressed. In some embodiments, a GPC3-associated disease or disorder involves cells in which GPC3 is abnormally expressed. In other embodiments, a GPC3-associated disease or disorder involves cells in which at least one of the activities of GPC3 is deficient.

[0116] The terms "about" and "approximately" mean within 20%, within 15%, within 10%, within 9%, within 8%, within 7%, within 6%, within 5%, within 4%, within 3%, within 2%, within 1%, or less of a given value or range.

[0117] As used in this disclosure and the claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise.

[0118] Whenever an embodiment is described herein with the term "comprising," it is understood that otherwise similar embodiments described with the term "consisting of" and / or "consisting essentially of" are also provided. Whenever an embodiment is described herein with the phrase "consisting essentially of," it is also understood that otherwise similar embodiments described with the term "consisting of" are also provided.

[0119] The term "between," as used in phrases such as "between A and B" or "between A and B," refers to a range that includes both A and B.

[0120] The term "and / or," as used in phrases such as "A and / or B," is intended herein to include both A and B; A or B; A alone; and B alone. Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A alone; B alone; and C alone.

[0121] 5.2.GPC3 binding molecules 5.2.1. Antibodies that bind to GPC3 In one aspect, the present disclosure provides an antibody capable of binding to glypican-3 (GPC3). GPC3 is a member of the glypican family of heparan sulfate (HS) proteoglycans that are bound to cell surfaces by a glycosylphosphatidylinositol anchor (Filmus and Selleck, J Clin Invest 108:497-501, 2001). The GPC3 gene encodes an approximately 70 kD core protein, which can be cleaved by furin to yield an N-terminal 40 kD fragment and a C-terminal 30 kD fragment. Two HS chains are bound to the C-terminal portion of GPC3. GPC3 and other glypican family proteins play a role in regulating cell division and cell growth. GPC3 is highly expressed in HCC and some other human cancers, including melanoma, lung squamous cell carcinoma, and ovarian clear cell carcinoma (Ho and Kim, Eur J Cancer 47(3):333-338, 2011), but is not expressed in normal tissues. GPC3 is also known as SGB, DGSX, MXR7, SDYS, SGBS, OCI-5, SGBS1, and GTR2-2. There are four known isoforms of human GPC3 (isoforms 1 to 4). The nucleic acid and amino acid sequences of four isoforms of GPC3 are known, including GenBank accession numbers NM_001164617 and NP_001158089 (isoform 1); NM_004484 and NP_004475 (isoform 2); NM_001164618 and NP_001158090 (isoform 3); and NM_001164619 and NP_001158091 (isoform 4). In some embodiments, the anti-GPC3 antibodies provided herein can bind to any of the four isoforms. In some embodiments, the antibodies provided herein bind to human GPC3. In some embodiments, the anti-GPC3 antibodies provided herein modulate one or more GPC3 activities. In some embodiments, the anti-GPC3 antibodies provided herein are antagonistic antibodies.

[0122] In some embodiments, the anti-GPC3 antibodies provided herein have an antibody activity against GPC3 (e.g., human GPC3) of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g. 10 -8 M~10 -13 M, e.g., 10 -9 M~10 -13 Dissociation constant (K D ) Various methods of measuring binding affinity are known in the art, including, for example, by RIA performed on a Fab version of the antibody of interest and its antigen (Chen et al., 1999, J. Mol Biol 293:865-81); by Octet®, for example, using the Octet® Red 96 system, or by biolayer interferometry (BLI) or surface plasmon resonance (SPR) assays by Biacore®, for example, using a Biacore® TM-2000 or Biacore® TM-3000, any of which can be used for the purposes of this disclosure. The "on-rate" or "rate of association" or "association rate" or "k" may also be determined using the same biolayer interferometry (BLI) or surface plasmon resonance (SPR) techniques described above, for example, using an Octet® Red 96, Biacore® TM-2000, or Biacore® TM-3000 system.

[0123] In some embodiments, the anti-GPC3 antibodies provided herein are those described in Section 6 below. Accordingly, in some embodiments, the antibodies provided herein comprise one or more CDR sequences of any one of SEQ ID NOS: 61-80 and 117-125. The CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs follow the IMGT numbering system. In some embodiments, the CDRs follow the Kabat numbering system. In some embodiments, the CDRs follow the AbM numbering system. In other embodiments, the CDRs follow the Chothia numbering system. In other embodiments, the CDRs follow the Contact numbering system. In some embodiments, the anti-GPC3 antibodies are humanized antibodies. In some embodiments, the anti-GPC3 antibodies comprise an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0124] In some embodiments, the anti-GPC3 antibodies provided herein comprise HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 61. In some embodiments, the anti-GPC3 antibodies provided herein comprise HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 62. In some embodiments, the anti-GPC3 antibodies provided herein comprise HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 63. In some embodiments, the anti-GPC3 antibodies provided herein comprise HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 64. In some embodiments, the anti-GPC3 antibodies provided herein comprise HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 65. In some embodiments, the anti-GPC3 antibodies provided herein comprise HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 66. In some embodiments, the anti-GPC3 antibodies provided herein comprise HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 67. In some embodiments, the anti-GPC3 antibodies provided herein comprise HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 68. In some embodiments, the anti-GPC3 antibodies provided herein comprise HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 69. In some embodiments, the anti-GPC3 antibodies provided herein comprise HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 70. In some embodiments, the anti-GPC3 antibodies provided herein comprise HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 117. In some embodiments, the anti-GPC3 antibodies provided herein comprise HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 118. In some embodiments, the anti-GPC3 antibodies provided herein comprise HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 119. In some embodiments, the anti-GPC3 antibodies provided herein comprise HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 120.In some embodiments, the anti-GPC3 antibodies provided herein comprise HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 121. In some embodiments, the anti-GPC3 antibodies provided herein comprise HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 122. The CDR sequences may be determined according to any well-known numbering system or combination thereof. In some embodiments, the CDRs are numbered according to IMGT. In some embodiments, the CDRs are numbered according to Kabat. In some embodiments, the CDRs are numbered according to AbM. In other embodiments, the CDRs are numbered according to Chothia. In other embodiments, the CDRs are numbered according to Contact.

[0125] In some embodiments, the anti-GPC3 antibodies provided herein comprise LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO: 71. In some embodiments, the anti-GPC3 antibodies provided herein comprise LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO: 72. In some embodiments, the anti-GPC3 antibodies provided herein comprise LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO: 73. In some embodiments, the anti-GPC3 antibodies provided herein comprise LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO: 74. In some embodiments, the anti-GPC3 antibodies provided herein comprise LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO: 75. In some embodiments, the anti-GPC3 antibodies provided herein comprise LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO: 76. In some embodiments, the anti-GPC3 antibodies provided herein comprise LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO: 77. In some embodiments, the anti-GPC3 antibodies provided herein comprise LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO: 78. In some embodiments, the anti-GPC3 antibodies provided herein comprise LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO: 79. In some embodiments, the anti-GPC3 antibodies provided herein comprise LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO: 80. In some embodiments, the anti-GPC3 antibodies provided herein comprise LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO: 123. In some embodiments, the anti-GPC3 antibodies provided herein comprise LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO: 124. In some embodiments, the anti-GPC3 antibodies provided herein comprise LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO: 125. The CDR sequences can be determined according to well-known numbering systems or combinations thereof. In some embodiments, the CDRs are numbered according to the IMGT system. In some embodiments, the CDRs are according to Kabat numbering.In some embodiments, the CDRs are numbered according to AbM, in other embodiments, the CDRs are numbered according to Chothia, in other embodiments, the CDRs are numbered according to Contact.

[0126] In some embodiments, an antibody is provided that comprises HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 61, and LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO: 71. In some embodiments, an antibody is provided that comprises HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 62, and LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO: 72. In some embodiments, an antibody is provided that comprises HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 63, and LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO: 73. In some embodiments, an antibody is provided that comprises HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 64, and LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO: 74. In some embodiments, an antibody is provided that comprises HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 65, and LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO: 75. In some embodiments, an antibody is provided that comprises HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 66, and LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO: 76. In some embodiments, an antibody is provided that comprises HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 67, and LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO: 77. In some embodiments, an antibody is provided that comprises HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 68, and LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO: 78. In some embodiments, an antibody is provided that comprises HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 69, and LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO: 79. In some embodiments, an antibody is provided that comprises HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 70, and LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO: 80.In some embodiments, an antibody is provided that comprises HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 117, and LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO: 123. In some embodiments, an antibody is provided that comprises HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 118, and LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO: 123. In some embodiments, an antibody is provided that comprises HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 119, and LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO: 123. In some embodiments, an antibody is provided that comprises HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 120, and LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO: 123. In some embodiments, an antibody is provided that comprises HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 121, and LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO: 123. In some embodiments, an antibody is provided that comprises HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 122, and LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO: 123. In some embodiments, an antibody is provided that comprises HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 117, and LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO: 124. In some embodiments, an antibody is provided that comprises HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 118, and LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO: 124. In some embodiments, an antibody is provided that comprises HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 119, and LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO: 124. In some embodiments, an antibody is provided that comprises HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 120, and LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO: 124.In some embodiments, an antibody is provided that comprises HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 121, and LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO: 124. In some embodiments, an antibody is provided that comprises HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 122, and LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO: 124. In some embodiments, an antibody is provided that comprises HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 117, and LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO: 125. In some embodiments, an antibody is provided that comprises HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 118, and LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO: 125. In some embodiments, an antibody is provided that comprises HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 119, and LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO: 125. In some embodiments, an antibody is provided that comprises HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 120, and LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO: 125. In some embodiments, an antibody is provided that comprises HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 121, and LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO: 125. In some embodiments, an antibody is provided that comprises HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 122, and LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO: 125. CDR sequences may be determined according to well-known numbering systems or combinations thereof. In some embodiments, the CDRs are numbered according to IMGT numbering. In some embodiments, the CDRs follow Kabat numbering. In some embodiments, the CDRs follow AbM numbering. In other embodiments, the CDRs follow Chothia numbering. In other embodiments, the CDRs follow Contact numbering.

[0127] In other embodiments, the present specification provides a method for identifying a HCDR1 comprising: (i) an HCDR2 comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of SEQ ID NOs: 1 to 10; or (ii) an HCDR1 comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of SEQ ID NOs: 11 to 20. , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any one of SEQ ID NOs: 21 to 30; (iii) HCDR3 comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any one of SEQ ID NOs: 21 to 30; (i (v) LCDR1 comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of SEQ ID NOs: 31 to 40; (v) LCDR1 comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of SEQ ID NOs: 41 to 50; and / or (vi) an LCDR2 comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of SEQ ID NOs: 51-60. In some embodiments, the anti-GPC3 antibody is a humanized antibody. In some embodiments, the anti-GPC3 antibody comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0128] In some more specific embodiments, the antibodies or antigen-binding fragments provided herein comprise an HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 11, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 21, and an LCDR1 comprising the amino acid sequence of SEQ ID NO: 31, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 41, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 51.

[0129] In some more specific embodiments, the antibodies or antigen-binding fragments provided herein comprise an HCDR1 comprising the amino acid sequence of SEQ ID NO: 2, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 12, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 22, and an LCDR1 comprising the amino acid sequence of SEQ ID NO: 32, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 42, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 52.

[0130] In some more specific embodiments, the antibodies or antigen-binding fragments provided herein comprise an HCDR1 comprising the amino acid sequence of SEQ ID NO: 3, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 13, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 23, and an LCDR1 comprising the amino acid sequence of SEQ ID NO: 33, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 43, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 53.

[0131] In some more specific embodiments, the antibodies or antigen-binding fragments provided herein comprise an HCDR1 comprising the amino acid sequence of SEQ ID NO: 4, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 14, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 24, and an LCDR1 comprising the amino acid sequence of SEQ ID NO: 34, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 44, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 54.

[0132] In some more specific embodiments, the antibodies or antigen-binding fragments provided herein comprise an HCDR1 comprising the amino acid sequence of SEQ ID NO: 5, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 15, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 25, and an LCDR1 comprising the amino acid sequence of SEQ ID NO: 35, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 45, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 55.

[0133] In some more specific embodiments, the antibodies or antigen-binding fragments provided herein comprise an HCDR1 comprising the amino acid sequence of SEQ ID NO: 6, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 16, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 26, and an LCDR1 comprising the amino acid sequence of SEQ ID NO: 36, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 46, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 56.

[0134] In some more specific embodiments, the antibodies or antigen-binding fragments provided herein comprise an HCDR1 comprising the amino acid sequence of SEQ ID NO: 7, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 17, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 27, and an LCDR1 comprising the amino acid sequence of SEQ ID NO: 37, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 47, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 57.

[0135] In some more specific embodiments, the antibodies or antigen-binding fragments provided herein comprise an HCDR1 comprising the amino acid sequence of SEQ ID NO: 8, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 18, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 28, and an LCDR1 comprising the amino acid sequence of SEQ ID NO: 38, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 48, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 58.

[0136] In some more specific embodiments, the antibodies or antigen-binding fragments provided herein comprise an HCDR1 comprising the amino acid sequence of SEQ ID NO: 9, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 19, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 29, and an LCDR1 comprising the amino acid sequence of SEQ ID NO: 39, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 49, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 59.

[0137] In some more specific embodiments, the antibodies or antigen-binding fragments provided herein comprise an HCDR1 comprising the amino acid sequence of SEQ ID NO: 10, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 20, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 30, and an LCDR1 comprising the amino acid sequence of SEQ ID NO: 40, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 50, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 60.

[0138] In some embodiments, the antibody further comprises one or more framework regions of SEQ ID NOs: 61-80 and 117-125. In some embodiments, the antibody provided herein is a humanized antibody. In some embodiments, the humanized antibody can be made using the methods exemplified in Section 6 below or the methods described in the following sections. The framework regions described herein are determined based on the boundaries of the CDR numbering system. In other words, when CDRs are determined by, for example, Kabat, IMGT, or Chothia, then the framework regions are the amino acid residues surrounded by the CDRs in the variable region from N-terminus to C-terminus in the format FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. For example, FR1 is defined as the amino acid residues N-terminal to the CDR1 amino acid residues when defined, for example, by the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system; FR2 is defined as the amino acid residues between the CDR1 and CDR2 amino acid residues when defined, for example, by the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system; FR3 is defined as the amino acid residues between the CDR2 and CDR3 amino acid residues when defined, for example, by the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system; and FR4 is defined as the amino acid residues C-terminal to the CDR3 amino acid residues when defined, for example, by the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system.

[0139] In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a VH domain comprising SEQ ID NO: 61 and a VL domain comprising SEQ ID NO: 71. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a VH domain comprising SEQ ID NO: 62 and a VL domain comprising SEQ ID NO: 72. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a VH domain comprising SEQ ID NO: 63 and a VL domain comprising SEQ ID NO: 73. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a VH domain comprising SEQ ID NO: 64 and a VL domain comprising SEQ ID NO: 74. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a VH domain comprising SEQ ID NO: 65 and a VL domain comprising SEQ ID NO: 75. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a VH domain comprising SEQ ID NO: 66 and a VL domain comprising SEQ ID NO: 76. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a VH domain comprising SEQ ID NO: 67 and a VL domain comprising SEQ ID NO: 77. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a VH domain comprising SEQ ID NO: 68 and a VL domain comprising SEQ ID NO: 78. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a VH domain comprising SEQ ID NO: 69 and a VL domain comprising SEQ ID NO: 79. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a VH domain comprising SEQ ID NO: 70 and a VL domain comprising SEQ ID NO: 80. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a VH domain comprising SEQ ID NO: 117 and a VL domain comprising SEQ ID NO: 123. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a VH domain comprising SEQ ID NO: 118 and a VL domain comprising SEQ ID NO: 123. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a VH domain comprising SEQ ID NO: 119 and a VL domain comprising SEQ ID NO: 123.In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a VH domain comprising SEQ ID NO: 120 and a VL domain comprising SEQ ID NO: 123. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a VH domain comprising SEQ ID NO: 121 and a VL domain comprising SEQ ID NO: 123. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a VH domain comprising SEQ ID NO: 122 and a VL domain comprising SEQ ID NO: 123. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a VH domain comprising SEQ ID NO: 117 and a VL domain comprising SEQ ID NO: 124. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a VH domain comprising SEQ ID NO: 118 and a VL domain comprising SEQ ID NO: 124. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a VH domain comprising SEQ ID NO: 119 and a VL domain comprising SEQ ID NO: 124. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a VH domain comprising SEQ ID NO: 120 and a VL domain comprising SEQ ID NO: 124. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a VH domain comprising SEQ ID NO: 121 and a VL domain comprising SEQ ID NO: 124. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a VH domain comprising SEQ ID NO: 122 and a VL domain comprising SEQ ID NO: 124. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a VH domain comprising SEQ ID NO: 117 and a VL domain comprising SEQ ID NO: 125. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a VH domain comprising SEQ ID NO: 118 and a VL domain comprising SEQ ID NO: 125. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a VH domain comprising SEQ ID NO: 119 and a VL domain comprising SEQ ID NO: 125. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a VH domain comprising SEQ ID NO: 120 and a VL domain comprising SEQ ID NO: 125.In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a VH domain comprising SEQ ID NO: 121 and a VL domain comprising SEQ ID NO: 125. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a VH domain comprising SEQ ID NO: 122 and a VL domain comprising SEQ ID NO: 125. In some embodiments, the antibody is an scFv.

[0140] In certain embodiments, the antibodies or antigen-binding fragments thereof described herein comprise an amino acid sequence having a particular percent identity compared to any of the antibodies provided herein, e.g., those described in Section 6, below.

[0141] The determination of percent identity between two sequences (e.g., amino acid sequences or nucleic acid sequences) can be accomplished using a mathematical algorithm. A non-limiting example of a mathematical algorithm utilized for comparing two sequences is the algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990), modified as described in Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., J. Mol. Biol. 215:403 (1990). To obtain nucleotide sequences homologous to the nucleic acid molecules described herein, BLAST nucleotide searches can be performed, for example, with the NBLAST nucleotide program parameters set to score=100 and word length=12. To obtain amino acid sequences homologous to the protein molecules described herein, BLAST protein searches can be performed, for example, with the XBLAST program parameters set to score 50 and word length = 3. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described in Altschul et al., Nucleic Acids Res. 25:3389 3402 (1997). Alternatively, PSI BLAST can be used to perform an iterated search that detects distant relationships between molecules (ibid.). When using BLAST, Gapped BLAST, and PSI Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used (see, e.g., the National Center for Biotechnology Information (NCBI) on the World Wide Web at ncbi.nlm.nih.gov). Another non-limiting example of a mathematical algorithm used to compare sequences is the algorithm of Myers and Miller, CABIOS 4:11-17 (1998).Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are considered.

[0142] In some embodiments, the antibodies provided herein contain substitutions (e.g., conservative substitutions), insertions, or deletions compared to a reference sequence, but the anti-GPC3 antibodies comprising the sequence retain the ability to bind to GPC3. In some embodiments, a total of 1 to 10 amino acids in the reference amino acid sequence are substituted, inserted, and / or deleted. In some embodiments, the substitutions, insertions, or deletions are in regions other than the CDRs (i.e., in the FRs). Optionally, the anti-GPC3 antibodies provided herein contain post-translational modifications of the reference sequence.

[0143] In some embodiments, the antibody or antigen-binding fragment provided herein comprises a VH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 61, and a VL domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 71, wherein the antibody binds to GPC3. In some embodiments, the antibody or antigen-binding fragment provided herein comprises a VH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 62, and a VL domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 72, wherein the antibody binds to GPC3.In some embodiments, the antibody or antigen-binding fragment provided herein comprises a VH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 63, and a VL domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 73, wherein the antibody binds to GPC3. In some embodiments, the antibody or antigen-binding fragment provided herein comprises a VH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 64, and a VL domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 74, wherein the antibody binds to GPC3.In some embodiments, the antibody or antigen-binding fragment provided herein comprises a VH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 65, and a VL domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 75, wherein the antibody binds to GPC3. In some embodiments, the antibody or antigen-binding fragment provided herein comprises a VH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 66, and a VL domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 76, wherein the antibody binds to GPC3.In some embodiments, the antibody or antigen-binding fragment provided herein comprises a VH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 67, and a VL domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 77, wherein the antibody binds to GPC3. In some embodiments, the antibody or antigen-binding fragment provided herein comprises a VH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 68, and a VL domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 78, wherein the antibody binds to GPC3.In some embodiments, the antibody or antigen-binding fragment provided herein comprises a VH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 69, and a VL domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 79, wherein the antibody binds to GPC3. In some embodiments, the antibody or antigen-binding fragment provided herein comprises a VH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 70, and a VL domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 80, wherein the antibody binds to GPC3.In some embodiments, the antibody or antigen-binding fragment provided herein comprises a VH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 117, and a VL domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 123, wherein the antibody binds to GPC3. In some embodiments, the antibody or antigen-binding fragment provided herein comprises a VH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 118, and a VL domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 123, wherein the antibody binds to GPC3.In some embodiments, the antibody or antigen-binding fragment provided herein comprises a VH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 119, and a VL domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 123, wherein the antibody binds to GPC3. In some embodiments, the antibody or antigen-binding fragment provided herein comprises a VH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 120, and a VL domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 123, wherein the antibody binds to GPC3. In some embodiments, the antibodies or antigen-binding fragments provided herein have at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, or at least the same amino acid sequence as SEQ ID NO:121. and a VL domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 123, wherein the antibody binds to GPC3. In some embodiments, the antibody or antigen-binding fragment provided herein comprises a VH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 122, and a VL domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 123, wherein the antibody binds to GPC3. In some embodiments, the antibody or antigen-binding fragment provided herein comprises a VH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 117, and a VL domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 124, wherein the antibody binds to GPC3.In some embodiments, the antibody or antigen-binding fragment provided herein comprises a VH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 118, and a VL domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 124, wherein the antibody binds to GPC3. In some embodiments, the antibody or antigen-binding fragment provided herein comprises a VH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 119, and a VL domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 124, wherein the antibody binds to GPC3.In some embodiments, the antibody or antigen-binding fragment provided herein comprises a VH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 120, and a VL domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 124, wherein the antibody binds to GPC3. In some embodiments, the antibody or antigen-binding fragment provided herein comprises a VH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 121, and a VL domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 124, wherein the antibody binds to GPC3.In some embodiments, the antibody or antigen-binding fragment provided herein comprises a VH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 122, and a VL domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 124, wherein the antibody binds to GPC3. In some embodiments, the antibody or antigen-binding fragment provided herein comprises a VH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 117, and a VL domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 125, wherein the antibody binds to GPC3.In some embodiments, the antibody or antigen-binding fragment provided herein comprises a VH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 118, and a VL domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 125, wherein the antibody binds to GPC3. In some embodiments, the antibody or antigen-binding fragment provided herein comprises a VH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 119, and a VL domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 125, wherein the antibody binds to GPC3.In some embodiments, the antibody or antigen-binding fragment provided herein comprises a VH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 120, and a VL domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 125, wherein the antibody binds to GPC3. In some embodiments, the antibody or antigen-binding fragment provided herein comprises a VH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 121, and a VL domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 125, wherein the antibody binds to GPC3.In some embodiments, the antibody or antigen-binding fragment provided herein comprises a VH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 122, and a VL domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 125, wherein the antibody binds to GPC3. In some embodiments, the antibody is an scFv.

[0144] In some embodiments, functional epitope mapping, for example by combinatorial alanine scanning, can identify amino acids in the GPC3 protein required for interaction with the anti-GPC3 antibodies provided herein. In some embodiments, epitope identification can utilize the conformational structure and crystal structure of an anti-GPC3 antibody bound to GPC3. In some embodiments, the present disclosure provides antibodies that specifically bind to the same epitope as any of the anti-GPC3 antibodies provided herein. For example, in some embodiments, an antibody is provided that binds to the same epitope as an anti-GPC3 antibody comprising a VH domain comprising SEQ ID NO: 61 and a VL domain comprising SEQ ID NO: 71. In some embodiments, an antibody is provided that binds to the same epitope as an anti-GPC3 antibody comprising a VH domain comprising SEQ ID NO: 62 and a VL domain comprising SEQ ID NO: 72. In some embodiments, an antibody is provided that binds to the same epitope as an anti-GPC3 antibody comprising a VH domain comprising SEQ ID NO: 63 and a VL domain comprising SEQ ID NO: 73. In some embodiments, an antibody is provided that binds to the same epitope as an anti-GPC3 antibody comprising a VH domain comprising SEQ ID NO: 64 and a VL domain comprising SEQ ID NO: 74. In some embodiments, an antibody is provided that binds to the same epitope as an anti-GPC3 antibody comprising a VH domain comprising SEQ ID NO: 65 and a VL domain comprising SEQ ID NO: 75. In some embodiments, an antibody is provided that binds to the same epitope as an anti-GPC3 antibody comprising a VH domain comprising SEQ ID NO: 66 and a VL domain comprising SEQ ID NO: 76. In some embodiments, an antibody is provided that binds to the same epitope as an anti-GPC3 antibody comprising a VH domain comprising SEQ ID NO: 67 and a VL domain comprising SEQ ID NO: 77. In some embodiments, an antibody is provided that binds to the same epitope as an anti-GPC3 antibody comprising a VH domain comprising SEQ ID NO: 68 and a VL domain comprising SEQ ID NO: 78. In some embodiments, an antibody is provided that binds to the same epitope as an anti-GPC3 antibody comprising a VH domain comprising SEQ ID NO: 69 and a VL domain comprising SEQ ID NO: 79. In some embodiments, an antibody is provided that binds to the same epitope as an anti-GPC3 antibody comprising a VH domain comprising SEQ ID NO: 70 and a VL domain comprising SEQ ID NO: 80.In some embodiments, an antibody is provided that binds to the same epitope as an anti-GPC3 antibody comprising a VH domain comprising SEQ ID NO: 117 and a VL domain comprising SEQ ID NO: 123. In some embodiments, an antibody is provided that binds to the same epitope as an anti-GPC3 antibody comprising a VH domain comprising SEQ ID NO: 118 and a VL domain comprising SEQ ID NO: 123. In some embodiments, an antibody is provided that binds to the same epitope as an anti-GPC3 antibody comprising a VH domain comprising SEQ ID NO: 119 and a VL domain comprising SEQ ID NO: 123. In some embodiments, an antibody is provided that binds to the same epitope as an anti-GPC3 antibody comprising a VH domain comprising SEQ ID NO: 120 and a VL domain comprising SEQ ID NO: 123. In some embodiments, an antibody is provided that binds to the same epitope as an anti-GPC3 antibody comprising a VH domain comprising SEQ ID NO: 121 and a VL domain comprising SEQ ID NO: 123. In some embodiments, an antibody is provided that binds to the same epitope as an anti-GPC3 antibody comprising a VH domain comprising SEQ ID NO: 122 and a VL domain comprising SEQ ID NO: 123. In some embodiments, an antibody is provided that binds to the same epitope as an anti-GPC3 antibody comprising a VH domain comprising SEQ ID NO: 117 and a VL domain comprising SEQ ID NO: 124. In some embodiments, an antibody is provided that binds to the same epitope as an anti-GPC3 antibody comprising a VH domain comprising SEQ ID NO: 118 and a VL domain comprising SEQ ID NO: 124. In some embodiments, an antibody is provided that binds to the same epitope as an anti-GPC3 antibody comprising a VH domain comprising SEQ ID NO: 119 and a VL domain comprising SEQ ID NO: 124. In some embodiments, an antibody is provided that binds to the same epitope as an anti-GPC3 antibody comprising a VH domain comprising SEQ ID NO: 120 and a VL domain comprising SEQ ID NO: 124. In some embodiments, an antibody is provided that binds to the same epitope as an anti-GPC3 antibody comprising a VH domain comprising SEQ ID NO: 121 and a VL domain comprising SEQ ID NO: 124. In some embodiments, an antibody is provided that binds to the same epitope as an anti-GPC3 antibody comprising a VH domain comprising SEQ ID NO: 122 and a VL domain comprising SEQ ID NO: 124.In some embodiments, an antibody is provided that binds to the same epitope as an anti-GPC3 antibody comprising a VH domain comprising SEQ ID NO: 117 and a VL domain comprising SEQ ID NO: 125. In some embodiments, an antibody is provided that binds to the same epitope as an anti-GPC3 antibody comprising a VH domain comprising SEQ ID NO: 118 and a VL domain comprising SEQ ID NO: 125. In some embodiments, an antibody is provided that binds to the same epitope as an anti-GPC3 antibody comprising a VH domain comprising SEQ ID NO: 119 and a VL domain comprising SEQ ID NO: 125. In some embodiments, an antibody is provided that binds to the same epitope as an anti-GPC3 antibody comprising a VH domain comprising SEQ ID NO: 120 and a VL domain comprising SEQ ID NO: 125. In some embodiments, an antibody is provided that binds to the same epitope as an anti-GPC3 antibody comprising a VH domain comprising SEQ ID NO: 121 and a VL domain comprising SEQ ID NO: 125. In some embodiments, an antibody is provided that binds to the same epitope as an anti-GPC3 antibody comprising a VH domain comprising SEQ ID NO: 122 and a VL domain comprising SEQ ID NO: 125. In some embodiments, the antibody is an scFv.

[0145] In some embodiments, the present disclosure provides an anti-GPC3 antibody or an antigen-binding fragment thereof that specifically binds to GPC3 competitively with any one of the anti-GPC3 antibodies described herein. In some embodiments, an antibody is provided that specifically binds to GPC3 competitively with an anti-GPC3 antibody comprising a VH domain comprising SEQ ID NO: 61 and a VL domain comprising SEQ ID NO: 71. In some embodiments, an antibody is provided that specifically binds to GPC3 competitively with an anti-GPC3 antibody comprising a VH domain comprising SEQ ID NO: 62 and a VL domain comprising SEQ ID NO: 72. In some embodiments, an antibody is provided that specifically binds to GPC3 competitively with an anti-GPC3 antibody comprising a VH domain comprising SEQ ID NO: 63 and a VL domain comprising SEQ ID NO: 73. In some embodiments, an antibody is provided that specifically binds to GPC3 competitively with an anti-GPC3 antibody comprising a VH domain comprising SEQ ID NO: 64 and a VL domain comprising SEQ ID NO: 74. In some embodiments, an antibody is provided that specifically binds to GPC3 competitively with an anti-GPC3 antibody comprising a VH domain comprising SEQ ID NO: 65 and a VL domain comprising SEQ ID NO: 75. In some embodiments, an antibody is provided that specifically binds to GPC3 competitively with an anti-GPC3 antibody comprising a VH domain comprising SEQ ID NO: 66 and a VL domain comprising SEQ ID NO: 76. In some embodiments, an antibody is provided that specifically binds to GPC3 competitively with an anti-GPC3 antibody comprising a VH domain comprising SEQ ID NO: 67 and a VL domain comprising SEQ ID NO: 77. In some embodiments, an antibody is provided that specifically binds to GPC3 competitively with an anti-GPC3 antibody comprising a VH domain comprising SEQ ID NO: 68 and a VL domain comprising SEQ ID NO: 78. In some embodiments, an antibody is provided that specifically binds to GPC3 competitively with an anti-GPC3 antibody comprising a VH domain comprising SEQ ID NO: 69 and a VL domain comprising SEQ ID NO: 79. In some embodiments, an antibody is provided that specifically binds to GPC3 competitively with an anti-GPC3 antibody comprising a VH domain comprising SEQ ID NO: 70 and a VL domain comprising SEQ ID NO: 80. In some embodiments, an antibody is provided that specifically binds to GPC3 competitively with an anti-GPC3 antibody comprising a VH domain comprising SEQ ID NO: 117 and a VL domain comprising SEQ ID NO: 123.In some embodiments, an antibody is provided that specifically binds to GPC3 competitively with an anti-GPC3 antibody comprising a VH domain comprising SEQ ID NO: 118 and a VL domain comprising SEQ ID NO: 123. In some embodiments, an antibody is provided that specifically binds to GPC3 competitively with an anti-GPC3 antibody comprising a VH domain comprising SEQ ID NO: 119 and a VL domain comprising SEQ ID NO: 123. In some embodiments, an antibody is provided that specifically binds to GPC3 competitively with an anti-GPC3 antibody comprising a VH domain comprising SEQ ID NO: 120 and a VL domain comprising SEQ ID NO: 123. In some embodiments, an antibody is provided that specifically binds to GPC3 competitively with an anti-GPC3 antibody comprising a VH domain comprising SEQ ID NO: 121 and a VL domain comprising SEQ ID NO: 123. In some embodiments, an antibody is provided that specifically binds to GPC3 competitively with an anti-GPC3 antibody comprising a VH domain comprising SEQ ID NO: 122 and a VL domain comprising SEQ ID NO: 123. In some embodiments, an antibody is provided that specifically binds to GPC3 competitively with an anti-GPC3 antibody comprising a VH domain comprising SEQ ID NO: 117 and a VL domain comprising SEQ ID NO: 124. In some embodiments, an antibody is provided that specifically binds to GPC3 competitively with an anti-GPC3 antibody comprising a VH domain comprising SEQ ID NO: 118 and a VL domain comprising SEQ ID NO: 124. In some embodiments, an antibody is provided that specifically binds to GPC3 competitively with an anti-GPC3 antibody comprising a VH domain comprising SEQ ID NO: 119 and a VL domain comprising SEQ ID NO: 124. In some embodiments, an antibody is provided that specifically binds to GPC3 competitively with an anti-GPC3 antibody comprising a VH domain comprising SEQ ID NO: 120 and a VL domain comprising SEQ ID NO: 124. In some embodiments, an antibody is provided that specifically binds to GPC3 competitively with an anti-GPC3 antibody comprising a VH domain comprising SEQ ID NO: 121 and a VL domain comprising SEQ ID NO: 124. In some embodiments, an antibody is provided that specifically binds to GPC3 competitively with an anti-GPC3 antibody comprising a VH domain comprising SEQ ID NO: 122 and a VL domain comprising SEQ ID NO: 124. In some embodiments, an antibody is provided that specifically binds to GPC3 competitively with an anti-GPC3 antibody comprising a VH domain comprising SEQ ID NO: 117 and a VL domain comprising SEQ ID NO: 125.In some embodiments, an antibody is provided that specifically binds to GPC3 competitively with an anti-GPC3 antibody comprising a VH domain comprising SEQ ID NO: 118 and a VL domain comprising SEQ ID NO: 125. In some embodiments, an antibody is provided that specifically binds to GPC3 competitively with an anti-GPC3 antibody comprising a VH domain comprising SEQ ID NO: 119 and a VL domain comprising SEQ ID NO: 125. In some embodiments, an antibody is provided that specifically binds to GPC3 competitively with an anti-GPC3 antibody comprising a VH domain comprising SEQ ID NO: 120 and a VL domain comprising SEQ ID NO: 125. In some embodiments, an antibody is provided that specifically binds to GPC3 competitively with an anti-GPC3 antibody comprising a VH domain comprising SEQ ID NO: 121 and a VL domain comprising SEQ ID NO: 125. In some embodiments, an antibody is provided that specifically binds to GPC3 competitively with an anti-GPC3 antibody comprising a VH domain comprising SEQ ID NO: 122 and a VL domain comprising SEQ ID NO: 125. In some embodiments, the antibody is an scFv.

[0146] In some embodiments, the present specification provides a GPC3-binding protein comprising any one of the anti-GPC3 antibodies described above. In some embodiments, the GPC3-binding protein is a monoclonal antibody, including a mouse, chimeric, humanized, or human antibody. In some embodiments, the anti-GPC3 antibody is an antibody fragment, such as an scFv. In some embodiments, the GPC3-binding protein is a fusion protein comprising the anti-GPC3 antibody provided herein. In other embodiments, the GPC3-binding protein is a multispecific antibody comprising the anti-GPC3 antibody provided herein. Other exemplary GPC3-binding molecules are described in more detail in the following sections.

[0147] In some embodiments, the anti-GPC3 antibodies or antigen binding proteins according to any of the above embodiments may incorporate any of the features, alone or in combination, as described in Sections 5.2.2 to 5.2.8 below.

[0148] 5.2.2. Antibody fragments As used herein, the term "antibody" also includes various antibody fragments thereof. Antibodies provided herein include, but are not limited to, immunoglobulin molecules and immunologically active portions of immunoglobulin molecules. The immunoglobulin molecules provided herein can be of any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) of immunoglobulin molecules. In some embodiments, the antibody is an IgG antibody. In some embodiments, the IgG antibody is an IgG1 antibody. In some embodiments, the IgG antibody is an IgG2, IgG3, or IgG4 antibody.

[0149] Antibody variants and derivatives include antibody functional fragments that retain the ability to bind to antigen. Exemplary functional fragments include Fab fragments (e.g., antibody fragments containing an antigen-binding domain and comprising a portion of a light chain and a heavy chain cross-linked by a disulfide bond); Fab' (e.g., antibody fragments containing a single antigen-binding domain comprising a Fab and an additional portion of a heavy chain up to the hinge region); F(ab')2 (e.g., two Fab' molecules joined by an interchain disulfide bond at the hinge region of the heavy chain; these Fab' molecules may target the same or different epitopes); bispecific Fab (e.g., a fragment containing two antigen-binding domains Fab molecules, each of which may target a different epitope; single chains containing variable regions, also known as scFvs (e.g., variable regions that determine antigen binding from a single light and heavy chain of an antibody linked together by a chain of, for example, 10-25 amino acids); disulfide-linked Fvs, or dsFvs (e.g., variable regions that determine antigen binding from a single light and heavy chain of an antibody linked together by a disulfide bond); camelized VHs (e.g., in which some amino acids at the VH interface are different from the heavy chain of a naturally occurring camel antibody). variable regions that determine antigen binding from a single heavy chain of an antibody, such as those found in a scFv; bispecific scFvs (e.g., scFv or dsFv molecules that have two antigen-binding domains, each of which can target a different epitope); diabodies (e.g., dimerized scFvs formed when the VH domain of a first scFv assembles with the VL domain of a second scFv, and the VL domain of the first scFv assembles with the VH domain of the second scFv; these two antigen-binding regions of a diabody can target the same or different epitopes). triabodies (e.g., trimerized scFvs formed similarly to diabodies, but now three antigen-binding domains are created in a single complex; these three antigen-binding domains may target the same or different epitopes); and tetrabodies (e.g., tetramerized scFvs formed similarly to diabodies, but now four antigen-binding domains are created in a single complex; these four antigen-binding domains may target the same or different epitopes).

[0150] Various techniques have been developed for producing antibody fragments. Traditionally, such fragments were obtained by proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., 1992, J. Biochem. Biophys. Methods 24:107-17; and Brennan et al., 1985, Science 229:81-83). However, such fragments can now be produced directly by recombinant host cells. For example, Fab, Fv, and scFv antibody fragments can all be expressed in and secreted from E. coli or yeast cells, thus allowing for the facile production of large amounts of these fragments. Antibody fragments can also be isolated from the antibody phage libraries discussed above. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (Carter et al., 1992, Bio / Technology 10:163-67). Alternatively, F(ab')2 fragments can be directly isolated from recombinant host cell culture. Fab and F(ab')2 fragments with increased in vivo half-lives containing salvage receptor-binding epitope residues are described, for example, in U.S. Pat. No. 5,869,046. Other techniques for producing antibody fragments will be apparent to those skilled in the art. In certain embodiments, the antibody is a single-chain Fv fragment (scFv) (see, e.g., WO 93 / 16185; U.S. Pat. Nos. 5,571,894 and 5,587,458). Fvs and scFvs have intact combining sites without constant regions; therefore, they may be suitable for reducing nonspecific binding during in vivo use. scFv fusion proteins can be constructed to fuse an effector protein to either the amino or carboxy terminus of an scFv (see, e.g., Borrebaeck, ed., supra). Antibody fragments can also be "linear antibodies," e.g., as described in the references cited above. Such linear antibodies can be monospecific or multispecific, e.g., bispecific.

[0151] In some specific embodiments, the antibodies provided herein are scFvs comprising any one of the VH domains described above, including, for example, SEQ ID NOS: 61-70 and 117-122, and any one of the VL domains described above, including, for example, SEQ ID NOS: 71-80 and 123-125. More specifically, in some embodiments, provided herein are scFvs comprising the amino acid sequence of SEQ ID NO: 81. In some embodiments, provided herein are scFvs comprising the amino acid sequence of SEQ ID NO: 82. In some embodiments, provided herein are scFvs comprising the amino acid sequence of SEQ ID NO: 83. In some embodiments, provided herein are scFvs comprising the amino acid sequence of SEQ ID NO: 84. In some embodiments, provided herein are scFvs comprising the amino acid sequence of SEQ ID NO: 85. In some embodiments, provided herein are scFvs comprising the amino acid sequence of SEQ ID NO: 86. In some embodiments, provided herein are scFvs comprising the amino acid sequence of SEQ ID NO: 87. In some embodiments, provided herein are scFvs comprising the amino acid sequence of SEQ ID NO: 88. In some embodiments, provided herein is an scFv comprising the amino acid sequence of SEQ ID NO: 89. In some embodiments, provided herein is an scFv comprising the amino acid sequence of SEQ ID NO: 90. In some embodiments, provided herein is an scFv comprising the amino acid sequence of SEQ ID NO: 128. In some embodiments, provided herein is an scFv comprising the amino acid sequence of SEQ ID NO: 129.

[0152] In some embodiments, provided herein is an scFv having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 81. In some embodiments, provided herein is an scFv having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 82. In some embodiments, provided herein is an scFv having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 83. In some embodiments, provided herein is an scFv having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 84. In some embodiments, provided herein is an scFv having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 85.In some embodiments, provided herein is an scFv having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 86. In some embodiments, provided herein is an scFv having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 87. In some embodiments, provided herein is an scFv having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 88. In some embodiments, provided herein is an scFv having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 89. In some embodiments, provided herein is an scFv having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 90.In some embodiments, provided herein is an scFv having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 128. In some embodiments, provided herein is an scFv having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 129.

[0153] Humanized Antibodies The antibodies described herein include humanized antibodies. Humanized antibodies, such as the humanized antibodies disclosed herein, can be prepared by, but are not limited to, CDR-grafting (EP 239,400; WO 91 / 09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089), veneering, or resurfacing (EP 592,106 and 519,596; Padlan, Molecular Immunology 28(4 / 5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); and Roguska et al., PNAS 91:969-973 (1994)), chain shuffling (U.S. Pat. No. 5,565,332), and methods such as those described in, for example, U.S. Pat. Nos. 6,407,213, 5,766,886, WO 9317105, Tan et al., J. Immunol. 169:1119 25 (2002), Caldas et al., Protein Eng. 13(5):353-60 (2000), Morea et al., Methods 20(3):267 79 (2000), Baca et al., J. Biol. Chem. 272(16):10678-84 (1997), Roguska et al., Protein Eng. 9(10):895 904 (1996), Couto et al. al., Cancer Res. 55(23 Supp):5973s-5977s (1995), Couto et al., Cancer Res. 55(8):1717-22 (1995), Sandhu JS, Gene 150(2):409-10 (1994), and Pedersen et al., J. Mol. Biol. 235(3):959-73 (1994). See also U.S. Patent Application Publication No. 2005 / 0042664 A1 (February 24, 2005), each of which is incorporated herein by reference in its entirety.

[0154] In some embodiments, the antibodies provided herein may be humanized antibodies that bind to GPC3, including human GPC3. For example, a humanized single-chain antibody of the present disclosure may comprise one or more CDRs set forth in SEQ ID NOS: 61-80 and 117-125. Various methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody may have one or more amino acid residues introduced into it from a non-human source. Such non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization can be performed by substituting hypervariable region sequences for the corresponding sequences of a human antibody, for example, according to the methods of Jones et al., Nature 321:522-25 (1986); Riechmann et al., Nature 332:323-27 (1988); and Verhoeyen et al., Science 239:1534-36 (1988)). In a specific embodiment, humanization of the antibodies provided herein is performed as described in Section 6 below.

[0155] In some cases, humanized antibodies are constructed by CDR grafting, in which the amino acid sequences of the CDRs of a parent non-human antibody are grafted onto a human antibody framework. For example, Padlan et al. revealed that only about one-third of the residues in the CDRs actually contact the antigen, and called them "specificity-determining residues," or SDRs (Padlan et al., FASEB J. 9:133-39 (1995)). In the SDR grafting technique, only the SDR residues are grafted onto a human antibody framework (see, for example, Kashmiri et al., Methods 36:25-34 (2005)).

[0156] To reduce antigenicity, the selection of human variable domains to be used in making humanized antibodies can be important. For example, according to the so-called "best-fit" method, the sequence of the variable domain of a non-human antibody is screened against the entire library of known human variable domain sequences. The human sequence that is closest to the non-human antibody can be selected as the human framework for the humanized antibody (Sims et al., J. Immunol. 151:2296-308 (1993); and Chothia et al., J. Mol. Biol. 196:901-17 (1987)). Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. This same framework can be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA 89:4285-89 (1992); and Presta et al., J. Immunol. 151:2623-32 (1993)). In some cases, the framework is selected from the most abundant human subclass, V L 6 Subgroup I(V L 6I) and V H Subgroup III(V H III) are derived from the consensus sequence. Alternatively, human germline genes are used as the source of the framework regions.

[0157] In an alternative paradigm based on CDR comparison, called superhumanization, FR homology is irrelevant. This method involves comparing non-human sequences with a functional human germline gene repertoire. Next, genes encoding the same or closely related canonical structures as the mouse sequences are selected. Subsequently, within the range of genes that share canonical structures with non-human antibodies, those with the highest homology within the CDRs are selected as FR donors. Finally, non-human CDRs are grafted onto these FRs (see, e.g., Tan et al., J. Immunol. 169:1119-25 (2002)).

[0158] Furthermore, it is generally desirable that antibodies be humanized with retention of their affinity for the antigen and other favorable biological properties. To achieve this goal, one method involves the preparation of humanized antibodies by analyzing the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available that illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. These include, for example, WAM (Whitelegg and Rees, Protein Eng. 13:819-24 (2002)), Modeller (Sali and Blundell, J. Mol. Biol. 234:779-815 (1993)), and Swiss PDB Viewer (Guex and Peitsch, Electrophoresis 18:2714-23 (1997)). Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, for example, the analysis of residues that influence the ability of the candidate immunoglobulin to bind to its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the hypervariable region residues are directly and most substantially involved in influencing antigen binding.

[0159] Another antibody humanization method is based on a metric of antibody humanness called human string content (HSC). This method compares the mouse sequence to a repertoire of human germline genes and scores differences as HSC. The target sequence is then humanized by maximizing its HSC rather than using a global identity measure, creating multiple diverse humanized variants (Lazar et al., Mol. Immunol. 44:1986-98 (2007)).

[0160] In addition to the methods described above, empirical methods can be used to generate and select humanized antibodies. Such methods include methods based on generating large libraries of humanized variants and selecting the best clones using enrichment or high-throughput screening techniques. Antibody variants can be isolated from phage, ribosomal, and yeast display libraries and by bacterial colony screening (see, e.g., Hoogenboom, Nat. Biotechnol. 23:1105-16 (2005); Dufner et al., Trends Biotechnol. 24:523-29 (2006); Feldhaus et al., Nat. Biotechnol. 21:163-70 (2003); and Schlapschy et al., Protein Eng. Des. Sel. 17:847-60 (2004)).

[0161] In the FR library approach, a set of residue variants is introduced at specific positions within the FR, followed by screening of the library to select the FR that best supports the grafted CDR. The substituted residues may include some or all of the "vernier" residues identified as likely to contribute to CDR structure (see, e.g., Foote and Winter, J. Mol. Biol. 224:487-99 (1992)), or from a more limited set of target residues identified by Baca et al., J. Biol. Chem. 272:10678-84 (1997).

[0162] In FR shuffling, instead of creating a combinatorial library of selected residue variants, entire FRs are combined with non-human CDRs (see, e.g., Dall'Acqua et al., Methods 36:43-60 (2005)). One-step FR shuffling methods can be used. Such methods have been shown to be efficient, with the resulting antibodies exhibiting improved biochemical and physicochemical properties, including enhanced expression, increased affinity, and thermal stability (see, e.g., Damschroder et al., Mol. Immunol. 44:3049-60 (2007)).

[0163] The "humaneering" method is based on the experimental identification of essential minimal specificity determinants (MSDs) and the evaluation of sequential substitution and binding of non-human fragments into a human FR library. This methodology typically results in epitope retention and identification of antibodies from multiple subclasses that possess distinctive human V-segment CDRs.

[0164] "Human engineering" methods involve modifying non-human antibodies or antibody fragments by making specific changes to the antibody's amino acid sequence to produce modified antibodies that have reduced immunogenicity in humans, yet retain the desired binding properties of the original non-human antibody. Broadly, this technique involves classifying amino acid residues in non-human antibodies into "low risk," "intermediate risk," or "high risk" residues. This classification is performed using a global risk / reward calculation, in which the expected benefit (e.g., with respect to immunogenicity in humans) of making a particular substitution is determined compared to the risk that the substitution will affect the folding of the resulting antibody. By aligning amino acid sequences from the variable regions of a non-human antibody with the corresponding regions of specific or consensus human antibody sequences, specific human amino acid residues to be substituted at given positions (e.g., low risk or intermediate risk) in the non-human antibody sequence can be selected. Based on the alignment, amino acid residues at low risk or intermediate risk positions in the non-human sequence can be substituted for the corresponding residue in the human antibody sequence. Techniques for producing human modified proteins are described in further detail in Studnicka et al., Protein Engineering 7:805-14 (1994); U.S. Patent Nos. 5,766,886; 5,770,196; 5,821,123; and 5,869,619; and WO 93 / 11794.

[0165] For example, composite human antibodies can be generated using Composite Human Antibody™ technology (Antitope Ltd., Cambridge, UK). To generate composite human antibodies, fragments of multiple human antibody variable region sequences are engineered to avoid T-cell epitopes, thereby minimizing the immunogenicity of the resulting antibody.

[0166] A deimmunized antibody is an antibody from which T cell epitopes have been removed. Methods for producing deimmunized antibodies have been described. See, for example, Jones et al., Methods Mol Biol. 525:405-23 (2009), xiv, and De Groot et al., Cell. Immunol. 244:148-153 (2006). A deimmunized antibody comprises a variable region depleted of T cell epitopes and a human constant region. Briefly, the variable region of an antibody is cloned, and then overlapping peptides derived from the variable region of the antibody are tested in a T cell proliferation assay to identify T cell epitopes. The T cell epitopes are identified by in silico methods that identify peptide binding to human MHC class II. Mutations are introduced into the variable region that abolish binding to human MHC class II. The mutated variable region is then used to generate a deimmunized antibody.

[0167] 5.2.4. Antibody Variants In some embodiments, one or more amino acid sequence modifications of the antibodies that bind to GPC3 described herein are contemplated. For example, it may be desirable to optimize the binding affinity and / or other biological properties of the antibody, including, but not limited to, specificity, thermostability, expression level, effector function, glycosylation, reduced immunogenicity, or solubility. Thus, in addition to the antibodies that bind to GPC3 described herein, it is contemplated that variants of the antibodies that bind to GPC3 described herein may be prepared. For example, antibody variants may be prepared by introducing appropriate nucleotide changes into the encoding DNA and / or by synthesis of the desired antibody or polypeptide. Those skilled in the art will understand that amino acid modifications may alter post-translational processes of the antibody.

[0168] chemical modification In some embodiments, the antibodies provided herein are chemically modified, for example, by covalently attaching any type of molecule to the antibody. Antibody derivatives can include antibodies that have been chemically modified, for example, by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, or conjugation to one or more immunoglobulin domains (e.g., Fc or portions of Fc). Any of a number of chemical modifications can be performed by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formulation, metabolic synthesis of tunicamycin, etc. Additionally, the antibody can contain one or more non-classical amino acids.

[0169] In some embodiments, the antibodies provided herein are altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody can conveniently be accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

[0170] When the antibodies provided herein are fused to an Fc region, the carbohydrate attached thereto can be varied. Natural antibodies produced by mammalian cells typically contain biantennary oligosaccharides, generally N-linked, attached to Asn297 in the CH2 domain of the Fc region. See, e.g., Wright et al., TIBTECH 15:26-32 (1997). The oligosaccharides can contain various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharides in the binding molecules provided herein can be made to generate variants with certain improved properties.

[0171] In other embodiments, when the antibodies provided herein are fused to an Fc region, the antibody variants provided herein can have a carbohydrate structure lacking fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan at Asn297 relative to the sum of all glycostructures (e.g., complex, hybrid, and high-mannose structures) attached to Asn297, as measured by MALDI-TOF mass spectrometry, e.g., as described in WO 2008 / 077546. Asn297 refers to the asparagine residue located at approximately position 297 in the Fc region (EU numbering of Fc region residues); however, due to slight sequence variations in antibodies, Asn297 can also be located ± approximately 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylation variants may exhibit improved ADCC function. See, e.g., U.S. Patent Application Publication Nos. 2003 / 0157108 and 2004 / 0093621.Examples of literature relating to "defucosylated" or "fucose-deficient" antibody variants include U.S. Patent Application Publication No. 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; U.S. Patent Application Publication No. 2003 / 0115614; U.S. Patent Application Publication No. 2002 / 0164328; U.S. Patent Application Publication No. 2004 / 0093621; U.S. Patent Application Publication No. 2004 / 0132140 ...5614; U.S. Patent Application Publication No. 2004 / 0135614; U.S. Patent Application Publication No. 2004 / 0132140; U.S. Patent Application Publication No. 2004 / 0132140; U.S. Patent Application Publication No. 2004 / 01356 No. 2004 / 0110704; U.S. Patent Application Publication No. 2004 / 0110282; U.S. Patent Application Publication No. 2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO 2005 / 053742; WO 2002 / 031140; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004). Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells, which are deficient in protein fucosylation (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application Publication No. 2003 / 0157108; and WO 2004 / 056312), and knockout cell lines such as α-1,6-fucosyltransferase gene FUT8 knockout CHO cells (see, e.g., Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO 2003 / 085107).

[0172] Biantennary oligosaccharides are further provided in the binding molecules, including antibodies, provided herein, where biantennary oligosaccharides attached to the Fc region are biantennary-linked via GlcNAc. Such variants may exhibit reduced fucosylation and / or improved ADCC function. Examples of such variants are described, for example, in WO 2003 / 011878 (Jean-Mairet et al.); U.S. Pat. No. 6,602,684 (Umana et al.); and U.S. Patent Application Publication No. 2005 / 0123546 (Umana et al.). Variants having at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such variants may exhibit improved CDC function. Such variants are described, for example, in WO 1997 / 30087; WO 1998 / 58964; and WO 1999 / 22764.

[0173] In molecules comprising the antibody and an Fc region, one or more amino acid modifications can be introduced into the Fc region, thereby generating an Fc region variant. The Fc region variant can comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) containing an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0174] In some embodiments, the present application contemplates variants that retain some, but not all, effector functions, making them desirable candidates for applications in which the in vivo half-life of the binding molecule is important, but certain effector functions (such as complement and ADCC) are unnecessary or deleterious. Reduced / depleted CDC and / or ADCC activity can be confirmed by performing in vitro and / or in vivo cytotoxicity assays. For example, Fc receptor (FcR) binding assays can be performed to ensure that a binding molecule lacks FcγR binding (and thus likely lacks ADCC activity) but retains FcRn binding ability. Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); U.S. Pat. No. 5,821,337 (see, Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be used (see, e.g., ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc. Mountain View, CA); and CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays may also be performed to confirm that the antibody is unable to bind C1q and therefore lacks CDC activity.See, e.g., C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). Determination of FcRn binding and in vivo clearance / half-life can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0175] Binding molecules with reduced effector function include those with substitutions at one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant in which residues 265 and 297 are substituted with alanine (U.S. Pat. No. 7,332,581).

[0176] Certain variants with improved or reduced binding to FcRs have been described (see, e.g., U.S. Pat. No. 6,737,056; WO 2004 / 056312; and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001)).

[0177] In some embodiments, the variants comprise an Fc region with one or more amino acid substitutions that improve ADCC, e.g., substitutions at Fc region positions 298, 333, and / or 334 (EU numbering of residues). In some embodiments, changes are made to the Fc region that result in altered (i.e., either improved or decreased) C1q binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0178] Binding molecules with increased half-life and improved binding to the neonatal Fc receptor (FcRn), which is involved in the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are described in U.S. Patent Application Publication No. 2005 / 0014934A1 (Hinton et al.). These molecules comprise an Fc region with one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include those having a substitution at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, e.g., a substitution at Fc region residue 434 (U.S. Pat. No. 7,371,826). See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; and WO 94 / 29351 for other examples of Fc region variants.

[0179] In some embodiments, it may be desirable to create a cysteine-engineered antibody, in which one or more residues of the antibody are replaced with cysteine ​​residues. In some embodiments, the replaced residues are located at accessible sites of the antibody. By replacing these residues with cysteine, reactive thiol groups are thus located at accessible sites of the antibody, which can be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create immunoconjugates, as further described herein.

[0180] Substitutions, deletions, or insertions Mutations can be substitutions, deletions, or insertions of one or more codons encoding the antibody or polypeptide that result in a change in the amino acid sequence compared to the original antibody or polypeptide. Target sites for substitutional mutagenesis include the CDRs and FRs.

[0181] Amino acid substitutions may result in the replacement of one amino acid with another amino acid having similar structural and / or chemical properties, such as the replacement of leucine with serine, and may be, for example, conservative amino acid substitutions. Standard techniques known to those of skill in the art can be used to introduce mutations into the nucleotide sequences encoding the molecules provided herein, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis, which result in amino acid substitutions. Insertions or deletions may optionally range from about 1 to 5 amino acids. In certain embodiments, substitutions, deletions, or insertions comprise fewer than 25 amino acid substitutions, fewer than 20 amino acid substitutions, fewer than 15 amino acid substitutions, fewer than 10 amino acid substitutions, fewer than 5 amino acid substitutions, fewer than 4 amino acid substitutions, fewer than 3 amino acid substitutions, or fewer than 2 amino acid substitutions compared to the original molecule. In specific embodiments, substitutions are conservative amino acid substitutions made at one or more predicted non-essential amino acid residues. Tolerable variations may be determined by systematically making amino acid insertions, deletions, or substitutions in a sequence and testing the resulting variants for activity exhibited by the parent antibody.

[0182] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing multiple residues, as well as intrasequence insertions of single or multiple amino acid residues. An exemplary terminal insertion is an antibody with an N-terminal methionyl residue.

[0183] The present disclosure includes antibodies generated by conservative amino acid substitutions. In conservative amino acid substitutions, an amino acid residue is replaced with an amino acid residue having a side chain with a similar charge. As described above, families of amino acid residues with similarly charged side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resulting mutants can be screened for biological activity to identify mutants that retain activity. After mutagenesis, the encoded protein can be expressed and the activity of the protein can be determined. Conservative substitutions (e.g., within amino acid groups with similar properties and / or side chains) can be made so that properties are maintained or not significantly altered. Exemplary substitutions are shown in Table 2 below. [Table 2]

[0184] Amino acids can be grouped by similarities in the properties of their side chains (see, e.g., Lehninger, Biochemistry 73-75 (2d ed. 1975)): (1) nonpolar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu (E); and (4) basic: Lys (K), Arg (R), His (H). Alternatively, naturally occurring residues can be divided into groups based on shared side chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that affect chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe. For example, any cysteine ​​residue not involved in maintaining the proper conformation of the antibody can also be substituted with another amino acid, such as alanine or serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Non-conservative substitutions would involve exchanging a member of one of these classes for another.

[0185] Certain substitutional variants involve substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further study will have modified (e.g., improved) certain biological properties relative to the parent antibody (e.g., increased affinity, decreased immunogenicity) and / or will substantially retain certain biological properties of the parent antibody. Exemplary substitutional variants are affinity matured antibodies, which may be conveniently generated using, for example, phage display-based affinity maturation techniques such as those described herein. Briefly, one or more CDR residues are mutated and the variant antibodies are displayed on phage and screened for detailed biological activity (e.g., binding affinity).

[0186] For example, to improve antibody affinity, changes (e.g., substitutions) can be made in CDRs. Such changes can be made in CDR "hot spots," i.e., residues encoded by codons that undergo frequent mutation during somatic maturation (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or in SDRs (a-CDRs), where the resulting mutant antibodies or fragments thereof are tested for binding affinity. Affinity maturation by construction of and reselection from secondary libraries is described, for example, in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. This library is then screened to identify any antibody variants with the desired affinity. Another method for introducing diversity involves CDR-targeted approaches, in which several CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding can be specifically identified using, for example, alanine scanning mutagenesis or modeling. A more detailed description of affinity maturation is provided in the following section.

[0187] In some embodiments, substitutions, insertions, or deletions may occur within one or more CDRs, so long as such changes do not substantially reduce the ability of the antibody to bind to the antigen. For example, conservative changes (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made to a CDR. In some embodiments of the variant antibody sequences provided herein, each CDR is either unaltered or does not contain more than one, two, or three amino acid substitutions.

[0188] A useful method for identifying residues or regions in an antibody that can be targeted for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells, Science, 244:1081-1085 (1989). In this method, a residue or group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) is identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the antibody's interaction with the antigen is affected. Further substitutions can be introduced at amino acid positions that demonstrate functional sensitivity to the initial substitutions. Alternatively, or in addition, a crystal structure of the antigen-antibody complex is determined to identify contact points between the antibody and antigen. Such contact residues and neighboring residues can be targeted or excluded as candidates for substitution. Mutants can be screened to determine whether they contain desired properties.

[0189] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g., for ADEPT) or a polypeptide which increases the serum half-life of the antibody.

[0190] Mutations can be made using methods known in the art, such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. Cloned DNA can be subjected to site-directed mutagenesis (see, e.g., Carter, Biochem J. 237:1-7 (1986); and Zoller et al., Nucl. Acids Res. 10:6487-500 (1982)), cassette mutagenesis (see, e.g., Wells et al., Gene 34:315-23 (1985)), or other known techniques to generate antibody variant DNA.

[0191] 5.2.5. In Vitro Affinity Maturation In some embodiments, antibody variants exhibiting improved properties, such as affinity, stability, or expression level, compared to the parent antibody can be prepared by in vitro affinity maturation. Similar to natural prototypes, in vitro affinity maturation is based on the principle of mutation and selection. Antibody libraries are displayed on the surface of organisms (e.g., phage, bacteria, yeast, or mammalian cells) or in association (e.g., covalently or noncovalently) with their encoding mRNA or DNA. Affinity selection of the displayed antibodies makes it possible to isolate organisms or complexes carrying the genetic information encoding the antibody. Typically, two or three rounds of mutation and selection using display methods such as phage display yield antibody fragments with affinities in the low nanomolar range. Affinity-matured antibodies can have nanomolar or even picomolar affinities for their target antigens.

[0192] Phage display is a widely adopted method for antibody display and selection. Antibodies are displayed on the surface of Fd or M13 bacteriophage as fusions with bacteriophage coat proteins. Selection involves binding of the phage-displayed antibodies to their targets by exposure to antigen, a process called "panning." Antigen-bound phage are recovered and used to infect bacteria to generate phage for further rounds of selection. For reviews, see, e.g., Hoogenboom, Methods. Mol. Biol. 178:1-37 (2002); and Bradbury and Marks, J. Immunol. Methods 290:29-49 (2004).

[0193] In the yeast display system (see, e.g., Boder et al., Nat. Biotech. 15:553-57 (1997); and Chao et al., Nat. Protocols 1:755-68 (2006)), antibodies are fused to the adhesive subunit of the yeast agglutinin protein Aga2p, which attaches to the yeast cell wall via a disulfide bond with Aga1p. When proteins are displayed via Aga2p, they are projected away from the cell surface, minimizing the possibility of interactions with other molecules on the yeast cell wall. Antibodies with improved affinity or stability are selected by screening the library using magnetic separation and flow cytometry. Binding to the soluble antigen of interest is determined by labeling yeast with biotinylated antigen and a secondary reagent, such as streptavidin conjugated to a fluorophore. Changes in antibody surface expression can be measured by immunofluorescence labeling of either hemagglutinin or c-Myc epitope tags flanking single-chain antibodies (e.g., scFvs). Expression has been shown to correlate with the stability of the displayed protein, thus allowing antibodies to be selected for improved stability as well as affinity (see, e.g., Shusta et al., J. Mol. Biol. 292:949-56 (1999)). An additional advantage of yeast display is that displayed proteins are folded in the endoplasmic reticulum of eukaryotic yeast cells, utilizing endoplasmic reticulum chaperones and quality control machinery. Once maturation is complete, antibody affinity can be conveniently "titrated" while still displayed on the yeast surface, eliminating the need to express and purify each clone. A theoretical limitation of yeast surface display is the potentially small functional library size compared to other display methods; however, recent approaches have demonstrated the use of a yeast cell mating system to generate functional libraries of up to 10 14 Combinatorial diversity of estimated size has been generated (see, e.g., US Patent Application Publication No. 2003 / 0186374; and Blaise et al., Gene 342:211-18 (2004)).

[0194] In ribosome display, antibody-ribosome-mRNA (ARM) complexes are generated for selection in a cell-free system. This DNA library encoding a specific library of antibodies is genetically fused to a spacer sequence lacking a stop codon. This spacer sequence, once translated, remains associated with peptidyl-tRNA and occupies the ribosomal tunnel, allowing the protein of interest to protrude from the ribosome and fold. The resulting mRNA-ribosome-protein complex can bind to a surface-bound ligand, allowing the antibody and its encoding mRNA to be simultaneously isolated by ligand affinity capture. The ribosome-bound mRNA is then reverse-transcribed back to cDNA, which can then be subjected to mutagenesis and used in subsequent rounds of selection (see, e.g., Fukuda et al., Nucleic Acids Res. 34:e127 (2006)). In mRNA display, a covalent bond is established between an antibody and mRNA using puromycin as an adapter molecule (Wilson et al., Proc. Natl. Acad. Sci. USA 98:3750-55 (2001)).

[0195] Because these methods are performed entirely in vitro, they offer two main advantages over other selection techniques. First, the diversity of the library is not limited by the transformation efficiency of bacterial cells, but only by the number of ribosomes and different mRNA molecules present in the test tube. Second, because the library does not need to be transformed after any diversification step, random mutations can be easily induced after each selection round, for example, by non-proofreading polymerases.

[0196] In some embodiments, a mammalian display system may be used.

[0197] Diversity can be introduced into the CDRs of an antibody library in a targeted manner or by random introduction. The former approach involves sequentially targeting all CDRs of an antibody by high-level or low-level mutagenesis, or by targeting isolated hotspots of somatic hypermutation (see, e.g., Ho et al., J. Biol. Chem. 280:607-17 (2005)) or residues suspected of affecting affinity based on experimentation or structural reasons. Diversity can also be introduced by replacing naturally diverse regions in DNA shuffling or similar techniques (see, e.g., Lu et al., J. Biol. Chem. 278:43496-507 (2003); U.S. Patent Nos. 5,565,332 and 6,989,250). Alternative techniques target hypervariable loops that extend into framework region residues (see, e.g., Bond et al., J. Mol. Biol. 348:699-709 (2005)), employ loop deletions and insertions in the CDRs, or use hybridization-based diversification (see, e.g., U.S. Patent Application Publication No. 2004 / 0005709). Additional methods for generating diversity in CDRs are described, for example, in U.S. Patent No. 7,985,840. Additional methods that may be used for generating antibody libraries and / or antibody affinity maturation are disclosed, for example, in U.S. Pat. Nos. 8,685,897 and 8,603,930, and U.S. Patent Application Publication Nos. 2014 / 0170705, 2014 / 0094392, 2012 / 0028301, 2011 / 0183855, and 2009 / 0075378, each of which is incorporated herein by reference.

[0198] Screening of libraries can be accomplished by a variety of techniques known in the art, for example, antibodies can be immobilized on solid supports, columns, pins, or cellulose / poly(vinylidene fluoride) membranes / other filters, expressed on host cells attached to adsorption plates or used for cell sorting, or conjugated to biotin and captured on streptavidin-coated beads, or used in any other method of panning display libraries.

[0199] For reviews of in vitro affinity maturation methods, see, e.g., Hoogenboom, Nature Biotechnology 23:1105-16 (2005); Quiroz and Sinclair, Revista Ingeneria Biomedia 4:39-51 (2010); and references therein.

[0200] 5.2.6. Antibody Modification Covalent modifications of antibodies are included within the scope of the present disclosure. Covalent modifications involve reacting targeted amino acid residues of the antibody with organic derivatizing agents capable of reacting with selected side chains or the N- or C-terminal residues of the antibody. Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of the hydroxyl group of seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine, and histidine side chains (see, e.g., Creighton, Proteins: Structure and Molecular Properties 79-86 (1983)), acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.

[0201] Other types of covalent modifications of antibodies within the scope of this disclosure include altering the native glycosylation pattern of the antibody or polypeptide, as described above (see, e.g., Beck et al., Curr. Pharm. Biotechnol. 9:482-501 (2008); and Walsh, Drug Discov. Today 15:773-80 (2010)), and linking the antibody to one of a variety of nonproteinaceous polymers, such as polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, e.g., by the methods described in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192; or 4,179,337. Antibodies that bind GPC3 of the present disclosure can also be genetically fused or conjugated to one or more immunoglobulin constant regions or portions thereof (e.g., Fc) to extend half-life and / or confer known Fc-mediated effector functions.

[0202] The single-chain antibodies that bind GPC3 of the present disclosure can also be modified to form chimeric molecules comprising a single-chain antibody that binds GPC3 fused to another heterologous polypeptide or amino acid sequence, such as an epitope tag (see, e.g., Terpe, Appl. Microbiol. Biotechnol. 60:523-33 (2003)) or the Fc region of an IgG molecule (see, e.g., Aruffo, Antibody Fusion Proteins 221-42 (Chamow and Ashkenazi eds., 1999)). Single-chain antibodies that bind GPC3 can also be used to generate GPC3-binding chimeric antigen receptors (CARs), as described in more detail below.

[0203] Also provided herein is a fusion protein comprising a single-chain antibody that binds to GPC3 of the present disclosure and a heterologous polypeptide. In some embodiments, the heterologous polypeptide that is genetically fused or chemically conjugated to the antibody is useful for targeting the antibody to cells that have GPC3 expressed on their cell surface.

[0204] Also provided herein is a panel of antibodies that bind to the GPC3 antigen. In specific embodiments, the antibody panel has different association rates, different dissociation rates, different affinities for the GPC3 antigen, and / or different specificities for the GPC3 antigen. In some embodiments, the panel comprises or consists of about 10 to about 1,000 antibodies or more. The antibody panel can be used in assays such as ELISA, for example, in 96-well or 384-well plates.

[0205] 5.2.7. Antibody Preparation Methods for preparing antibodies have been described. See, for example, Els Pardon et al., Nature Protocol, 9(3):674 (2014). Antibodies (such as scFv fragments) may be obtained using methods known in the art, such as by immunizing a camelid species (such as a camel or llama) and obtaining hybridomas therefrom, or by cloning a library of antibodies using molecular biology techniques known in the art, followed by selection by ELISA on individual clones from the unselected library, or by using phage display.

[0206] Antibodies provided herein can be produced by culturing cells transformed or transfected with a vector containing nucleic acid encoding the antibody. Polynucleotide sequences encoding the polypeptide components of the antibodies of the present disclosure can be obtained using standard recombinant techniques. Desired polynucleotide sequences can be isolated and sequenced from antibody-producing cells, such as hybridoma cells or B cells. Alternatively, polynucleotides can be synthesized using a nucleotide synthesizer or PCR techniques. Once obtained, the polypeptide-encoding sequence is inserted into a recombinant vector capable of replicating in a host cell and expressing heterologous polynucleotides. For purposes of the present disclosure, many vectors available and known in the art can be used. The selection of an appropriate vector will depend primarily on the size of the nucleic acid to be inserted into the vector and the particular host cell to be transformed with the vector. Suitable host cells for expressing the antibodies of the present disclosure include prokaryotes such as archaebacteria and eubacteria, including gram-negative or gram-positive organisms, eukaryotic microorganisms such as filamentous fungi or yeast, invertebrate cells such as insects or plant cells, and vertebrate cells such as mammalian host cell lines. Host cells are transformed with the above-described expression vectors and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. Antibodies produced by the host cells are purified using standard protein purification methods as known in the art.

[0207] Antibody production methods, including vector construction, expression, and purification, are further described in Plueckthun et al., Antibody Engineering: Producing antibodies in Escherichia coli: From PCR to fermentation 203-52 (McCafferty et al. eds., 1996); Kwong and Rader, E. coli Expression and Purification of Fab Antibody Fragments, in Current Protocols in Protein Science (2009); Tachibana and Takekoshi, Production of Antibody Fab Fragments in Escherichia coli, in Antibody Expression and Production (Al-Rubeai ed., 2011); and Therapeutic Monoclonal Antibodies: From Bench to Clinic (An ed., 2009).

[0208] Of course, it is contemplated that alternative methods well known in the art can be used to prepare anti-GPC3 antibodies. For example, the appropriate amino acid sequence, or a portion thereof, can be prepared by direct peptide synthesis using solid-phase techniques (see, for example, Stewart et al., Solid-Phase Peptide Synthesis (1969); and Merrifield, J. Am. Chem. Soc. 85:2149-54 (1963)). Manual techniques or automated in vitro protein synthesis can be performed. The desired anti-GPC3 antibody can be produced by chemically synthesizing various portions of the anti-GPC3 antibody separately and combining them using chemical or enzymatic methods. Alternatively, the antibody can be purified from cells or body fluids, such as milk, of transgenic animals modified to express the antibody, as disclosed, for example, in U.S. Patent Nos. 5,545,807 and 5,827,690.

[0209] Polyclonal antibodies Polyclonal antibodies are generally raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. A protein immunogenic in the immunizing species, such as keyhole limpet hemocyanin (KLH), serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor, is conjugated with a bifunctional or derivatizing agent, such as maleimidobenzoyl sulfosuccinimide ester (conjugation via cysteine ​​residues), N-hydroxysuccinimide (via lysine residues), glutaraldehyde, succinic anhydride, SOCl, or R. 1 NC-NR (wherein R and R 1 are independently lower alkyl groups) to conjugate the relevant antigen. Examples of adjuvants that may be utilized include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl lipid A, synthetic trehalose dicorynomycolate). The immunization protocol may be selected by one skilled in the art without undue experimentation.

[0210] For example, animals are immunized against an antigen, immunogenic conjugate, or derivative by combining, for example, 100 μg or 5 μg of protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later, the animals are boosted with one-fifth to one-tenth the amount of the original peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. Seven to 14 days later, the animals are bled and the serum is assayed for antibody titer. Animals are boosted until the titer plateaus. Conjugates can also be made as protein fusions in recombinant cell culture. Aggregating agents such as alum are also suitable for enhancing the immune response.

[0211] Monoclonal antibodies Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies representing the population are identical except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation), which may be present in minor amounts. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of individual antibodies.

[0212] For example, the monoclonal antibodies may be made using the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or may be made by recombinant DNA methods (U.S. Pat. No. 4,816,567).

[0213] In the hybridoma method, a suitable host animal is immunized to elicit lymphocytes that produce, or are capable of producing, antibodies that will specifically bind to the immunizing protein. Alternatively, lymphocytes can be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

[0214] The immunizing agent will typically include an antigen protein or a fusion variant thereof. Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986), pp. 59-103. Immortalized cell lines are usually transformed mammalian cells. The hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused parental myeloma cells. Preferred immortalized myeloma cells are those that fuse efficiently, support stable high-level antibody production by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium.

[0215] The culture medium in which the hybridoma cells are growing is assayed for the production of monoclonal antibodies against the antigen. The culture medium in which the hybridoma cells are cultured can be assayed for the presence of monoclonal antibodies against the desired antigen. Such techniques and assays are known in the art. For example, binding affinity can be determined by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).

[0216] After hybridoma cells that produce antibodies of the desired specificity, affinity, and / or activity are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods (Goding, supra). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, hybridoma cells can be grown in vivo as tumors in mammals.

[0217] The monoclonal antibodies secreted by the subclones are preferably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0218] Monoclonal antibodies can also be made by recombinant DNA methods such as those described in U.S. Pat. No. 4,816,567 and as described above. DNA encoding monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of mouse antibodies). Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA can be placed into an expression vector, which is then transfected into host cells such as Escherichia coli (E. coli) cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not naturally produce immunoglobulin proteins, thereby synthesizing monoclonal antibodies in such recombinant host cells. Review articles on recombinant expression in bacteria of DNA encoding the antibody include Skerra et al., Curr. Opinion in Immunol., 5:256-262 (1993) and Pliickthun, Immunol. Rev. 130:151-188 (1992).

[0219] In a further embodiment, antibodies can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al., Nature, 348:552-554 (1990), Clackson et al., Nature, 352:624-628 (1991), and Marks et al., J. Mol. Biol., 222:581-597 (1991). Subsequently, the literature has described chain shuffling (Marks et al., Bio / Technology, 10:779-783 (1992)) and the production of high affinity (nM range) human antibodies by combinatorial infection and in vivo recombination as strategies for constructing very large phage libraries (Waterhouse et al., Nucl. Acids Res., 21:2265-2266 (1993)). Therefore, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolating monoclonal antibodies.

[0220] The DNA can also be modified, for example, by substituting the coding sequence (U.S. Pat. No. 4,816,567; Morrison, et al., Proc. Natl. Acad. Sci. USA, 81:6851 (1984)) or by covalently ligating all or part of the coding sequence of a non-immunoglobulin polypeptide to the coding sequence. Such a non-immunoglobulin polypeptide can be substituted to create a chimeric bivalent antibody containing one antigen-binding site specific for one antigen and another antigen-binding site specific for a different antigen.

[0221] Chimeric or hybrid antibodies can also be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolane and methyl-4-mercaptobutyrimidate.

[0222] Recombinant production in prokaryotic cells Polynucleic acid sequences encoding antibodies of the present disclosure can be obtained using standard recombinant techniques. The desired polynucleic acid sequence can be isolated from antibody-producing cells, such as hybridoma cells, and sequenced. Alternatively, polynucleotides can be synthesized using a nucleotide synthesizer or PCR techniques. Once obtained, the polypeptide-encoding sequence is inserted into a recombinant vector capable of replicating and expressing heterologous polynucleotides in a prokaryotic host. For purposes of the present disclosure, many vectors available and known in the art can be used. The selection of an appropriate vector will depend primarily on the size of the nucleic acid to be inserted into the vector and the specific host cell to be transformed with the vector. Each vector contains various components depending on its function (amplification or expression of the heterologous polynucleotide, or both) and its compatibility with the particular host cell in which it resides. Vector components generally include, but are not limited to, an origin of replication, a selectable marker gene, a promoter, a ribosome binding site (RBS), a signal sequence, the heterologous nucleic acid insert, and a transcription termination sequence.

[0223] Generally, in connection with these hosts, plasmid vectors containing replicon and control sequences derived from species compatible with the host cell are used. The vector usually carries a replication site as well as marking sequences capable of providing phenotypic selection in transformed cells. For example, E. coli is typically transformed with pBR322, a plasmid derived from an E. coli species. Examples of pBR322 derivatives used to express specific antibodies are described in detail in Carter et al., U.S. Pat. No. 5,648,237.

[0224] Additionally, in connection with these hosts, transformation vectors may be used, such as phage vectors containing replicon and control sequences compatible with the host microorganism. For example, bacteriophages such as GEM™-11 may be utilized to generate recombinant vectors that can be used to transform susceptible host cells, such as E. coli LE392.

[0225] The expression vector of the present application may contain two or more promoter-cistron pairs, each encoding a polypeptide component. A promoter is a non-translated regulatory sequence located upstream (5') of a cistron that controls its expression. Prokaryotic promoters are typically divided into two classes: inducible and constitutive. An inducible promoter is a promoter that initiates an increase in the transcription level of the cistron under its control in response to a change in culture conditions, such as the presence or absence of a nutrient or a change in temperature.

[0226] Numerous promoters recognized by a variety of potential host cells are well known. The promoter of choice can be operably linked to the cistron DNA encoding the antibody by removing the promoter from the source DNA by restriction enzyme digestion and inserting the isolated promoter sequence into the vector of the present application. Both the native promoter sequence and many heterologous promoters can be used to direct amplification and / or expression of the target gene. Generally, heterologous promoters allow for greater transcription and higher yields of the expressed target gene compared to the native target polypeptide promoter, and therefore, in some embodiments, heterologous promoters are utilized.

[0227] Suitable promoters for use in prokaryotic hosts include the PhoA promoter, β-lactamase and lactose promoter systems, tryptophan (trp) promoter systems, and hybrid promoters such as the tac or trc promoter. However, other promoters that function in bacteria (such as other known bacterial or phage promoters) are similarly suitable. Their nucleic acid sequences have been published, allowing those skilled in the art to operably ligate them to a cistron encoding a target peptide, providing any necessary restriction sites by using linkers or adapters (Siebenlist et al., Cell 20:269 (1980)).

[0228] In one embodiment, each cistron present in a recombinant vector contains a secretory signal sequence component that directs translocation of an expressed polypeptide across a membrane. Generally, the signal sequence may be a component of the vector, or it may be a part of the target polypeptide DNA inserted into the vector. For purposes of the present invention, the signal sequence selected should be one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. For prokaryotic host cells that do not recognize and process the native signal sequence for a heterologous polypeptide, the signal sequence can be substituted by a prokaryotic signal sequence selected from the group consisting of, for example, alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II (STII) leaders, LamB, PhoE, PelB, OmpA, and MBP.

[0229] In some embodiments, production of the antibodies of the present disclosure can occur in the cytoplasm of the host cell, and therefore the presence of a secretory signal sequence within each cistron is not required. - The strains provide cytoplasmic conditions that favor disulfide bond formation, thus allowing proper folding and assembly of the expressed protein subunits.

[0230] Prokaryotic host cells suitable for expressing the antibodies of the present disclosure include archaebacteria and eubacteria, such as gram-negative or gram-positive organisms. Examples of useful bacteria include Escherichia (e.g., E. coli), Bacilli (e.g., B. subtilis), Enterobacteriaceae, Pseudomonas species (e.g., P. aeruginosa), Salmonella typhimurium, Serratia marcescens, Klebsiella, Proteus, Shigella, Rhizobium, Vitreoscilla, or Paracoccus. In some embodiments, gram-negative cells are used. In one embodiment, E. coli cells are used as the host. Exemplary E. coli strains include strain W3110 (Bachmann, Cellular and Molecular Biology, vol. 2 (Washington, DC: American Society for Microbiology, 1987), pp. 1190-1219; ATCC Accession No. 27,325), and the genotype W3110 AfhuA (AtonA) ptr3 lac Iq lacL8 AompT A (nmpc-fepE) degP41 kan RExamples of suitable strains include E. coli 294 (ATCC 31,446), E. coli B, E. coli 1776 (ATCC 31,537), and E. coli RV308 (ATCC 31,608), as well as their derivatives. These examples are illustrative rather than limiting. Methods for constructing derivatives of any of the above-mentioned bacteria with defined genotypes are known in the art and are described, for example, in Bass et al., Proteins, 8:309-314 (1990). Generally, the appropriate bacterium should be selected taking into account the replicability of the replicon in the bacterial cells. For example, when well-known plasmids such as pBR322, pBR325, pACYC177, or pKN410 are used to supply the replicon, E. coli, Serratia, or Salmonella species can be suitably used as hosts.

[0231] Typically, the host cell should secrete minimal amounts of proteolytic enzymes, and additional protease inhibitors may be included in the cell culture if desired.

[0232] Host cells are transformed with the above-described expression vectors and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. Transformation refers to the introduction of DNA into a prokaryotic host so that the DNA is replicable, either as an extrachromosomal element or by chromosomal integrant. Depending on the host cell used, transformation is carried out using standard techniques appropriate for such cells. Generally, a calcium treatment utilizing calcium chloride is used for bacterial cells that contain significant cell wall barriers. Another transformation method utilizes polyethylene glycol / DMSO. Yet another technique that may be used is electroporation.

[0233] Prokaryotic cells used to produce the antibodies of the present application are grown in a medium known in the art that is suitable for culturing the selected host cells. An example of a suitable medium is Luria Broth (LB) plus necessary nutrient supplements. In some embodiments, the medium also contains a selection agent, which is chosen based on the construction of the expression vector to selectively allow growth of prokaryotic cells containing the expression vector. For example, ampicillin is added to the medium to grow cells that express an ampicillin resistance gene.

[0234] Any necessary supplements other than carbon, nitrogen, and inorganic phosphate sources may also be included at appropriate concentrations, introduced alone or in mixture with another supplement or medium, such as a complex nitrogen source. Optionally, the culture medium may contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, thioglycolic acid, dithioerythritol, and dithiothreitol. Prokaryotic host cells are cultured at a suitable temperature and pH.

[0235] When an inducible promoter is used in the expression vector of the present application, protein expression is induced under conditions suitable for promoter activation. In one embodiment of the present application, the PhoA promoter is used to control the transcription of the polypeptide. Accordingly, the transformed host cell is cultured in a phosphate-limited medium for induction. Preferably, the phosphate-limited medium is CRAP medium (see, for example, Simmons et al., J. Immunol. Methods 263:133-147 (2002)). As known in the art, various other inducers can be used depending on the vector construct used.

[0236] The expressed antibodies of the present disclosure are secreted into the periplasm of the host cells and recovered therefrom. Protein recovery typically involves disruption of the microorganisms, generally by means such as osmotic shock, sonication, or lysis. After cell disruption, cell debris or whole cells can be removed by centrifugation or filtration. The protein can be further purified, for example, by affinity resin chromatography. Alternatively, the protein can be carried into the culture medium and isolated therein. For further purification of the produced protein, the cells can be removed from the culture medium, and the culture supernatant can be filtered and concentrated. The expressed polypeptide can be further isolated and identified using well-known methods such as polyacrylamide gel electrophoresis (PAGE) and Western blot assays.

[0237] Alternatively, proteins are produced in large quantities by fermentation processes. A variety of large-scale fed-batch fermentation procedures are available for the production of recombinant proteins. Various fermentation conditions can be modified to improve the production yield and quality of the antibodies of the present disclosure. For example, chaperone proteins have been demonstrated to promote proper folding and solubility of heterologous proteins produced in bacterial host cells. Chen et al., J Bio Chem 274:19601-19605 (1999); U.S. Patent No. 6,083,715; U.S. Patent No. 6,027,888; Bothmann and Pluckthun, J. Biol. Chem. 275:17100-17105 (2000); Ramm and Pluckthun, J. Biol. Chem. 275:17106-17113 (2000); Arie et al., Mol. Microbiol. 39:199-210 (2001).

[0238] To minimize proteolysis of expressed heterologous proteins (especially those sensitive to proteolysis), certain host strains deficient in proteases can be used in the present invention, as described, for example, in U.S. Pat. Nos. 5,264,365; 5,508,192; and Hara et al., Microbial Drug Resistance, 2:63-72 (1996). Escherichia coli (E. coli) strains deficient in proteases and transformed with plasmids overexpressing one or more chaperone proteins can be used as host cells in the antibody-encoding expression systems of the present application.

[0239] The antibodies produced herein can be further purified to obtain substantially homogeneous preparations for further assays and uses. Standard protein purification methods known in the art can be used. The following procedures are exemplary of suitable purification procedures: fractionation on an immunoaffinity or ion exchange column, ethanol precipitation, reverse-phase HPLC, chromatography on silica or on a cation exchange resin such as DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and gel filtration using, for example, Sephadex G-75. For immunoaffinity purification of binding molecules of the present disclosure, some embodiments can use, for example, Protein A immobilized on a solid phase. The solid phase to which Protein A is immobilized is preferably a column comprising a glass or silica surface, more preferably a controlled-pore glass column or a silicic acid column. In some embodiments, the column is coated with a reagent such as glycerol to prevent nonspecific adhesion of contaminants. The solid phase is then washed to remove contaminants nonspecifically bound to the solid phase. Finally, the antibody of interest is recovered from the solid phase by elution.

[0240] Recombinant production in eukaryotic cells For eukaryotic expression, vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes and an enhancer element, a promoter, and a transcription termination sequence.

[0241] Vectors used in eukaryotic hosts may also contain an insert encoding a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide. Preferably, the heterologous signal sequence selected is one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. In mammalian cell expression, mammalian signal sequences as well as viral secretory leaders, such as the herpes simplex gD signal, are available. DNA for such precursor regions can be ligated in-reading frame to DNA encoding the antibody of the present application.

[0242] Generally, the origin of replication component is not needed for mammalian expression vectors (although the SV40 origin may typically be used only because it contains the early promoter).

[0243] Expression and cloning vectors may contain a selection gene, also called a selectable marker. Selection genes can encode proteins that confer resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate, or tetracycline; complement auxotrophic deficiencies; or supply critical nutrients not available from complex media.

[0244] One example of a selection scheme utilizes a drug to arrest growth of the host cell. Cells successfully transformed with a heterologous gene produce a protein that confers drug resistance and thus survive the selection regimen. Examples of such dominant selection use the drugs neomycin, mycophenolic acid, and hygromycin.

[0245] Another example of a suitable selectable marker for mammalian cells is one that enables the identification of cells competent to incorporate nucleic acid encoding the antibody of the present application. For example, cells transformed with a DHFR selection gene are initially identified by culturing all transformants in a culture medium containing methotrexate (Mtx), a competitive antagonist of DHFR. An exemplary suitable host cell when wild-type DHFR is used is a Chinese hamster ovary (CHO) cell line deficient in DHFR activity. Alternatively, host cells transformed or co-transformed with a DNA sequence encoding a polypeptide, a wild-type DHFR protein, and another selectable marker, such as aminoglycoside 3'-phosphotransferase (APH), (particularly wild-type hosts containing endogenous DHFR) can be selected by growing the cells in a medium containing a selection agent for the selectable marker, such as an aminoglycoside antibiotic.

[0246] Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to a nucleic acid encoding the desired polypeptide sequence. Eukaryotic genes have an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated. Many genes may contain additional sequences found 70 to 80 bases upstream from the start of transcription. The 3' end of most eukaryotic genes may be a signal for addition of a poly(A) tail to the 3' end of the coding sequence. All of these sequences can be inserted into eukaryotic expression vectors.

[0247] Transcription of polypeptides from vectors in mammalian host cells can be controlled by promoters obtained, for example, from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as adenovirus type 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retroviruses, hepatitis B virus, and simian virus 40 (SV40), from heterologous mammalian promoters, such as the actin promoter or immunoglobulin promoters, and from heat shock promoters, provided that such promoters are compatible with the host cell system.

[0248] Transcription of a DNA encoding an antibody of the present disclosure by higher eukaryotes is often increased by inserting an enhancer sequence into the vector. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin). Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. See also Yaniv, Nature 297:17-18 (1982) for a discussion of enhancing elements for activation of eukaryotic promoters. The enhancer may be spliced ​​into the vector at a position 5' or 3' to the polypeptide-coding sequence, but is preferably located at a site 5' from the promoter.

[0249] Expression vectors used in eukaryotic host cells (yeast cells, fungal cells, insect cells, plant cells, animal cells, human cells, or nucleated cells from other multicellular organisms) also contain sequences necessary for the termination of transcription and stabilization of mRNA. Such sequences are commonly available from the 5' and, occasionally, 3' untranslated regions of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding the polypeptide. One useful transcription termination component is the bovine growth hormone polyadenylation region.

[0250] Suitable host cells for cloning or expressing the DNA in the vectors herein include the higher eukaryotic cells described herein, including vertebrate host cells. Propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are the SV40-transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned to grow in suspension culture, Graham et al., J. Gen. Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TR1 cells (Mather et al., Annals NY Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatocellular carcinoma line (Hep G2).

[0251] Host cells can be transformed with the above-described expression or cloning vectors for antibody production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

[0252] The host cells used to produce the antibodies of the present application may be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), minimal essential medium (MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's modified Eagle's medium (DMEM), Sigma) are suitable for culturing the host cells. In addition, the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al. Any of the media described in U.S. Pat. Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655, or 5,122,469, WO 90 / 03430, WO 87 / 00195, or U.S. Pat. No. Re. 30,985 may be used as a culture medium for the host cells. Any of these media may optionally contain hormones and / or other growth factors (insulin, transferrin, or The culture medium may be supplemented with other nutrients such as epidermal growth factor, salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as the drug GENTAMYCIN™), trace elements (usually defined as inorganic compounds present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to one of skill in the art. Culture conditions, such as temperature, pH, and the like, will be those conventionally used with the host cell selected for expression and will be apparent to one of skill in the art.

[0253] Using recombinant techniques, antibodies can be produced intracellularly, in the periplasmic space, or directly secreted into the culture medium. If the antibody is produced intracellularly, as a first step, particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. If the antibody is secreted into the culture medium, supernatants from such expression systems are generally first concentrated using commercially available protein concentration filters, such as Amicon or Millipore Pellicon ultrafiltration units. Protease inhibitors, such as PMSF, can be included in any of the foregoing steps to inhibit proteolysis, and antibiotics can be included to prevent the growth of adventitious contaminants.

[0254] Protein compositions prepared from cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique. The matrix to which the affinity ligand is attached is most often agarose, although other matrices are also available. Mechanically stable matrices, such as controlled-pore glass or poly(styrene-divinyl)benzene, allow for faster flow rates and shorter processing times than can be achieved with agarose. Other protein purification techniques, such as fractionation on ion-exchange columns, ethanol precipitation, reverse-phase HPLC, chromatography on silica, heparin SEPHAROSE™, chromatography on anion- or cation-exchange resins (such as polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation, are also available, depending on the antibody to be recovered. Following one or more optional preliminary purification steps, the mixture containing the antibody of interest and contaminants can be subjected to low-pH hydrophobic interaction chromatography.

[0255] 5.2.8. Other Binding Molecules, Including Antibodies In another aspect, the present disclosure provides a binding molecule comprising the anti-GPC3 antibody provided herein.In addition to the chimeric antigen receptor (CAR) provided herein as described in Section 5.3 below, in some embodiments, the antibody against GPC3 provided herein is part of another binding molecule.Exemplary binding molecules of the present disclosure are described below.

[0256] fusion proteins In various embodiments, the antibodies provided herein can be genetically fused or chemically conjugated to another agent, e.g., a protein-based entity. The antibody can be chemically conjugated to the agent or otherwise non-covalently conjugated to the agent. The agent can be a peptide or an antibody (or fragment thereof).

[0257] Thus, in some embodiments, provided herein are antibodies that are recombinantly fused or chemically conjugated (covalently or non-covalently conjugated) with a heterologous protein or polypeptide (or fragments thereof, e.g., polypeptides of about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, or about 500 amino acids) to create fusion proteins, and uses thereof. Specifically, provided herein are fusion proteins comprising an antigen-binding fragment (e.g., CDR1, CDR2, and / or CDR3) of an antibody provided herein and a heterologous protein, polypeptide, or peptide.

[0258] Additionally, the antibodies provided herein can be fused to marker or "tag" sequences, such as peptides, to facilitate purification. In specific embodiments, the marker or tag amino acid sequences are a hexahistidine peptide, a hemagglutinin ("HA") tag, and a "FLAG" tag.

[0259] Methods for fusing or conjugating moieties (including polypeptides) to antibodies are known (e.g., Arnon et al., "Monoclonal Antibodies for Immunotargeting of Drugs in Cancer Therapy," in Monoclonal Antibodies and Cancer Therapy 243-56 (Reisfeld et al. eds., 1985); Hellstrom et al., "Antibodies for Drug Delivery," in Controlled Drug Delivery 623-53 (Robinson et al. eds., 2d ed. 1987); Thorpe, "Antibody Carriers of Cytotoxic Agents in Cancer Therapy: A Review," in Monoclonal Antibodies: Biological and Clinical Applications 475-506 (Pinchera et al. eds., 1985); "Analysis, Results, and Future Prospect of the Therapeutic Use of Radiolabeled Antibodies in Cancer Therapy," in Monoclonal Antibodies for Cancer Detection and Therapy 303-16(Baldwin et al.eds.,1985);Thorpe et al.,Immunol.Rev.62:119-58 (1982); U.S. Patent Nos. 5,336,603; 5,622,929; 5,359,046; 5,349,053; 5,447,851; 5,723,125; 5,783,181; 5,908,626; 5,844,095; and 5,112,9 46; EP 307,434; EP 367,166; EP 394,827; WO 91 / 06570, WO 96 / 04388, WO 96 / 22024, WO 97 / 34631, and WO 99 / 04813; Ashkenazi (See, e.g., Traunecker et al., Proc. Natl. Acad. Sci. USA, 88:10535-39 (1991); Traunecker et al., Nature, 331:84-86 (1988); Zheng et al., J. Immunol. 154:5590-600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA 89:11337-41 (1992)).

[0260] Fusion proteins can be created, for example, by the techniques of gene shuffling, motif shuffling, exon shuffling, and / or codon shuffling (collectively referred to as "DNA shuffling"). DNA shuffling can be used to alter the activities of antibodies as provided herein, including, for example, antibodies with higher affinity and lower dissociation rates (see, e.g., U.S. Pat. Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458; Patten et al., Curr. Opinion Biotechnol. 8:724-33 (1997); Harayama, Trends Biotechnol. 16(2):76-82 (1998); Hansson et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzo and Blasco, Biotechniques 24(2):308-13 (1998)). Antibodies, or the encoded antibodies, may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion, or other methods prior to recombination. Polynucleotides encoding the antibodies provided herein may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc., of one or more heterologous molecules.

[0261] In some embodiments, an antibody provided herein is conjugated to a second antibody to form an antibody heteroconjugate.

[0262] In various embodiments, the antibody is genetically fused to the agent. Genetic fusion can be achieved by placing a linker (e.g., a polypeptide) between the antibody and the agent. The linker can be a flexible linker.

[0263] In various embodiments, the antibody is genetically conjugated to the therapeutic molecule, where a hinge region links the antibody to the therapeutic molecule.

[0264] Also provided herein are methods for producing the various fusion proteins provided herein. The various methods described above in Section 5.2.7 may also be used to produce the fusion proteins provided herein.

[0265] In specific embodiments, the fusion proteins provided herein are recombinantly expressed. Recombinant expression of the fusion proteins provided herein may require the construction of an expression vector containing a polynucleotide encoding the protein or a fragment thereof. Once a polynucleotide encoding a protein or a fragment thereof provided herein is obtained, a vector for the production of the molecule can be generated by recombinant DNA technology using techniques well known in the art. Thus, described herein are methods for preparing proteins by expressing a polynucleotide containing a coding nucleotide sequence. Methods well known to those skilled in the art can be used to construct expression vectors containing the coding sequence and appropriate transcriptional and translational control signals. Such methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Also provided are replicable vectors containing a nucleotide sequence encoding a fusion protein provided herein, or a fragment thereof, or a CDR thereof, operably linked to a promoter.

[0266] The expression vector can be transferred into a host cell by conventional techniques and the transfected cells then cultured by conventional techniques to produce the fusion protein provided herein. Thus, also provided herein is a host cell containing a polynucleotide encoding a fusion protein provided herein, or a fragment thereof, operably linked to a heterologous promoter.

[0267] A variety of host-expression vector systems can be utilized to express the fusion proteins provided herein. Such host-expression systems represent vehicles by which a coding sequence of interest can be produced and subsequently purified, but also represent cells that, when transformed or transfected with the appropriate nucleotide coding sequence, can express the fusion proteins provided herein in situ. These include, but are not limited to, microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing the coding sequence; yeast (e.g., Saccharomyces) transformed with recombinant yeast expression vectors containing the coding sequence; and yeast cells (e.g., Saccharomyces cerevisiae) transformed with recombinant yeast expression vectors containing the coding sequence. Examples of suitable expression systems include Pichia; insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing the coding sequence; plant cell systems infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus, CaMV, tobacco mosaic virus, TMV) containing the coding sequence or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid); or mammalian cell systems (e.g., COS, CHO, BHK, 293, NSO, and 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genomes of mammalian cells (e.g., metallothionein promoters) or mammalian viral promoters (e.g., adenovirus late promoters; vaccinia virus 7.5K promoters). For expression of recombinant fusion proteins, bacterial cells such as Escherichia coli or eukaryotic cells can be used, particularly for expression of whole recombinant antibody molecules. For example, mammalian cells such as Chinese hamster ovary cells (CHO) are effective expression systems for antibodies or their variants when coupled with vectors such as the major immediate-early gene promoter element from human cytomegalovirus. In specific embodiments, expression of a nucleotide sequence encoding a fusion protein provided herein is regulated by a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

[0268] In bacterial systems, several expression vectors can be advantageously selected depending on the intended use for expressing the fusion protein. For example, when producing large quantities of such fusion proteins to make pharmaceutical compositions of the fusion protein, a vector that directs high expression of a fusion protein product that is easily purified may be desirable. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther et al., EMBO 12:1791 (1983)) (coding sequences can be individually ligated in-frame with the lacZ coding region into the vector to produce a fusion protein); pIN vectors (Inouye & Inouye, Nucleic Acids Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem. 24:5503-5509 (1989)); pGEX vectors can also be used to express foreign polypeptides as fusion proteins with glutathione 5-transferase (GST). Generally, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to matrix glutathione agarose beads, followed by elution in the presence of free glutathione. pGEX vectors are designed to contain thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

[0269] Several viral-based expression systems are available for mammalian host cells. When adenovirus is used as an expression vector, the coding sequence of interest can be ligated to an adenovirus transcription / translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into a non-essential region of the viral genome (e.g., region E1 or E3) will result in recombinant virus that is viable in infected hosts and capable of expressing the fusion protein (see, e.g., Logan & Shenk, Proc. Natl. Acad. Sci. USA 8 1:355-359 (1984)). Specific initiation signals may also be required for efficient translation of the inserted coding sequence. Such signals include the ATG initiation codon and adjacent sequences. Furthermore, to ensure translation of the entire insert, the initiation codon must be in phase with the reading frame of the desired coding sequence. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see, e.g., Bittner et al., Methods in Enzymol. 153:51-544 (1987)).

[0270] In addition, a host cell strain may be chosen that modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the expressed foreign protein. To this end, eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include, but are not limited to, CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT2O, and T47D, NS0 (a murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O, and HsS78Bst cells.

[0271] Stable expression can be used for long-term, high-yield production of recombinant proteins. For example, cell lines that stably express a fusion protein can be engineered. Rather than using expression vectors containing viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer sequence, transcription terminator, polyadenylation site, etc.) and a selectable marker. After introduction of the exogenous DNA, the engineered cells can be grown in an enriched medium for 1-2 days and then switched to a selective medium. The selectable marker in the recombinant plasmid confers resistance to selection, allowing the cells to stably integrate the plasmid into their chromosomes and grow to form foci that can then be cloned and expanded into cell lines. Advantageously, cell lines that express the fusion protein can be engineered using this method. Such engineered cell lines can be particularly useful for screening and evaluating compositions that interact directly or indirectly with the binding molecule.

[0272] A number of selection systems may be used, including, but not limited to, herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223 (1977)), hypoxanthine guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adenine phosphoribosyltransferase (Lowy et al., Cell 22:8-17 (1980)) genes, which can be used in tk-, hgprt-, or aprt- cells, respectively. Additionally, antimetabolite resistance can be used as a selection criterion for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl. Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072 (1981)); neo, which confers resistance to the aminoglycoside G-418 (Wu and Wu, Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); May, TIB TECH 11(5):155-215 (1993); and hygro, which confers resistance to hygromycin (Santerre et al., Gene 30:147 (1984)).Desired recombinant clones can be selected by routine application of methods generally known in the field of recombinant DNA technology, as described, for example, in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and Chapters 12 and 13, Dracopoli et al. (eds.), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), which are incorporated herein by reference in their entireties.

[0273] The expression level of a fusion protein can be increased by vector amplification (for a review, see Bebbington and Hentschel, "The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning," Vol. 3 (Academic Press, New York, 1987)). If the marker in the vector system expressing the fusion protein is amplifiable, increasing the level of inhibitor present in the host cell culture will increase the copy number of the marker gene. Because the amplified region is linked to the fusion protein gene, production of the fusion protein will also increase (Crouse et al., Mol. Cell. Biol. 3:257 (1983)).

[0274] A host cell can be co-transfected with multiple expression vectors provided herein. The vectors can contain identical selectable markers, thereby enabling equal expression of each encoded polypeptide. Alternatively, a single vector can be used that encodes and is capable of expressing multiple polypeptides. The coding sequence can comprise cDNA or genomic DNA.

[0275] After being produced by recombinant expression, the fusion proteins provided herein can be purified by any method known in the art for the purification of polypeptides (e.g., immunoglobulin molecules), such as, for example, chromatography (e.g., ion exchange, affinity, particularly for specific antigens after Protein A, size-exclusion column chromatography, and kappa-selection affinity chromatography), centrifugation, differential solubility, or any other standard technique for purifying proteins. Additionally, the fusion protein molecules provided herein can be fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification.

[0276] Immunoconjugates In some embodiments, the present disclosure also provides immunoconjugates comprising any of the anti-GPC3 antibodies described herein conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitors, toxins (e.g., protein toxins of bacterial, fungal, plant, or animal origin, enzymatically active toxins or fragments thereof), or radioactive isotopes.

[0277] In some embodiments, the immunoconjugate is an antibody-drug conjugate (ADC), wherein the antibody is coupled to a drug such as, but not limited to, a maytansinoid (see U.S. Pat. Nos. 5,208,020, 5,416,064, and EP 0 425 235 B1 ); auristatins, such as the monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483, 5,780,588, and 7,498,298); dolastatins; calicheamicin or its derivatives (see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode et al., Cancer Res. Res. 58:2925-2928 (1998); anthracyclines such as daunomycin or doxorubicin (Kratz et al., Current Med. Chem. 13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters 16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005); Nagy et al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); King et al. al., J. Med. Chem. 45:4336-4343 (2002); and U.S. Patent No. 6,630,579); methotrexate; vindesine; taxanes such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; trichothecenes; and CC1065.

[0278] In some embodiments, the immunoconjugate comprises an antibody as described herein conjugated to an enzymatically active toxin or fragment thereof, including, but not limited to, diphtheria A chain, a nonbinding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolacca americana proteins (PAPI, PAPII, and PAP-S), bitter gourd inhibitor, curcin, crotin, saponaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the trichothecenes.

[0279] In some embodiments, immunoconjugates include antibodies as described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioisotopes are available for producing radioconjugates. Examples include At 211 , I 131 , I 125 , Y 90 ,Re 186 ,Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 and radioactive isotopes of Lu. When a radioconjugate is used for detection, it may contain a radioactive atom, such as tc99m or I123, for scintigraphic studies, or again a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

[0280] Conjugates of antibodies and cytotoxic agents can be made using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bisazide compounds (such as bis(p-azidobenzoyl)hexanediamine), bisdiazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as 2,6-toluene diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to antibodies. See WO 94 / 11026.

[0281] The linker may be a "cleavable linker" that facilitates release of the conjugated drug within the cell, although non-cleavable linkers are also contemplated herein. Linkers for use in the conjugates of the present disclosure include, without limitation, acid-labile linkers (e.g., hydrazone linkers), disulfide-containing linkers, peptidase-sensitive linkers (e.g., peptide linkers containing amino acids such as valine and / or citrulline, such as citrulline-valine or phenylalanine-lysine), photolabile linkers, dimethyl linkers, thioether linkers, or hydrophilic linkers designed to circumvent multidrug transporter-mediated resistance.

[0282] The immunoconjugates or ADCs herein contemplate such conjugates prepared with crosslinker reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate), which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, Ill., USA).

[0283] In other embodiments, the antibodies provided herein are conjugated or recombinantly fused to, for example, diagnostic molecules. Such diagnosis and detection can be achieved by coupling the antibody to a detectable substance, including, but not limited to, various enzymes such as horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; prosthetic groups such as, but not limited to, streptavidin / biotin or avidin / biotin; fluorescent substances such as, but not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; luminescent substances such as, but not limited to, luminol; bioluminescent substances such as, but not limited to, luciferase, luciferin, or aequorin; chemiluminescent substances such as γ-, Auger-, β-, α-, or positron-emitting radioisotopes of 225Ac.

[0284] 5.3. Chimeric Antigen Receptors In another aspect, provided herein is a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain comprising an antibody (e.g., scFv) provided herein that binds to GPC3. Exemplary CARs comprising this scFv fragment are exemplified in Section 6 below.

[0285] In some embodiments, the chimeric antigen receptor (CAR) provided herein comprises a polypeptide comprising: (a) an extracellular antigen-binding domain comprising one or more antibodies that specifically bind to GPC3 as provided herein, and optionally one or more additional binding domains; (b) a transmembrane domain; and (c) an intracellular signaling domain. Each component and additional region is described in further detail below.

[0286] 5.3.1. Extracellular Antigen-Binding Domains The extracellular antigen-binding domain of the CAR described herein comprises one or more (such as any one of 1, 2, 3, 4, 5, 6, or more) antibodies, which can be fused to each other directly by peptide bonds or via peptide linkers.

[0287] antibody The CAR of the present disclosure comprises an extracellular antigen-binding domain comprising one or more of the antibodies provided herein. These antibodies may be of the same or different origin and may be of the same or different size. Exemplary antibodies include, but are not limited to, the scFv fragments described in Section 5.2 above.

[0288] In some embodiments, a CAR is provided herein comprising a polypeptide comprising: (a) an extracellular antigen-binding domain comprising an anti-GPC3 antibody (e.g., an scFv); (b) a transmembrane domain; and (c) an intracellular signaling domain, wherein the anti-GPC3 antibody is, for example, an anti-GPC3 antibody as described in Section 5.2 above, including those comprising one or more CDRs of Tables 3 and 4.

[0289] More specifically, in some embodiments, a CAR is provided herein comprising a polypeptide comprising: (a) an extracellular antigen-binding domain comprising an anti-GPC3 antibody (e.g., an scFv fragment); (b) a transmembrane domain; and (c) an intracellular signaling domain, wherein the anti-GPC3 antibody (e.g., scFv) comprises: (i) an HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 11, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 21, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 31, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 41, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 42. 2 and LCDR3 comprising the amino acid sequence of SEQ ID NO: 51, or (ii) an HCDR1 comprising the amino acid sequence of SEQ ID NO: 2, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 12, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 22, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 32, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 42, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 52, or (iii) an HCDR1 comprising the amino acid sequence of SEQ ID NO: 3, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 13, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 23, and an amino acid sequence of SEQ ID NO: 33. an LCDR1 comprising the amino acid sequence of SEQ ID NO: 43, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 53; or (iv) an HCDR1 comprising the amino acid sequence of SEQ ID NO: 4, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 14, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 24, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 34, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 44, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 54; or (v) an HCDR1 comprising the amino acid sequence of SEQ ID NO: 5, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 15, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 25 an HCDR3 comprising the amino acid sequence of SEQ ID NO: 35, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 35, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 45, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 55, or (vi) an HCDR1 comprising the amino acid sequence of SEQ ID NO: 6, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 16, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 26, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 36, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 46, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 56, or (vii) an HCDR1 comprising the amino acid sequence of SEQ ID NO: 7,(viii) an HCDR1 comprising the amino acid sequence of SEQ ID NO: 8, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 18, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 28, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 38, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 48, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 58, or (ix) an HCDR1 comprising the amino acid sequence of SEQ ID NO: 9, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 19, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 20, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 21, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 22, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 23, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 24, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 25, or (x) an HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 29, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 39, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 49, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 59, or (x) an HCDR1 comprising the amino acid sequence of SEQ ID NO: 10, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 20, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 30, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 40, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 50, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 60. In some embodiments, the antibody (e.g., scFv) comprises a VH domain selected from the group of SEQ ID NOs: 61-70 and 117-122, and a VL domain selected from the group of SEQ ID NOs: 71-80 and 123-125. In a more specific embodiment, the antibody is an scFv comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 81 to 90, 128, and 129.

[0290] In other embodiments, the extracellular antigen-binding domain further comprises one or more additional antigen-binding domains that bind to one or more additional antigens, for example, one, two, three, four or more additional antibody binding regions (antibodies) that target one or more additional antigens.

[0291] In some embodiments, the one or more additional antigens targeted by the CAR of the present disclosure are cell surface molecules. Antibodies can be selected to recognize antigens that serve as cell surface markers on target cells associated with a particular disease state. In some embodiments, the antigen is a tumor antigen. In some embodiments, the tumor antigen is a tumor-specific antigen (TSA) or tumor-associated antigen (TAA). TSAs are unique to tumor cells and are not present on other cells in the body. TAA-associated antigens are not unique to tumor cells but are instead expressed on normal cells under conditions that do not allow for immune tolerance to the antigen. Expression of an antigen on a tumor can occur under conditions that allow the immune system to respond to the antigen. A TAA can be an antigen expressed on normal cells during fetal development, when the immune system is immature and unable to respond, or a TAA can be an antigen that is normally present only at very low levels on normal cells but is expressed at much higher levels on tumor cells.

[0292] In addition to the antigen-binding domain of the extracellular domain, the CARs provided herein can further comprise one or more of a linker (e.g., a peptide linker), a transmembrane domain, a hinge region, a signal peptide, an intracellular signaling domain, a costimulatory signaling domain, each of which is described in more detail below.

[0293] For example, in some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell (such as a T cell). In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. In some embodiments, the intracellular signaling domain comprises a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain is derived from a costimulatory molecule selected from the group consisting of CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand for CD83, and combinations thereof. In some embodiments, the costimulatory signaling domain is derived from CD137. In some embodiments, the GPC3 CAR further comprises a hinge domain (such as a CD8α hinge domain) located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain. In some embodiments, the GPC3 CAR further comprises a signal peptide (such as a CD8α signal peptide) located at the N-terminus of the polypeptide. In some embodiments, the polypeptide comprises, from N-terminus to C-terminus, a CD8α signal peptide, an extracellular antigen-binding domain, a CD8α hinge domain, a CD8α transmembrane domain, a costimulatory signaling domain derived from CD137, and a primary intracellular signaling domain derived from CD3ζ. In some embodiments, the GPC3 CAR is monospecific. In some embodiments, the GPC3 CAR is monovalent. In some embodiments, the GPC3 CAR is multispecific. In some embodiments, the GPC3 CAR is multivalent.

[0294] Peptide Linker When multiple antibodies are present in the CAR, the various antibodies can be fused to each other with a peptide linker.In some embodiments, the antibodies are fused directly to each other without any peptide linker.The peptide linkers connecting different antibodies can be the same or different.Different domains of the CAR can also be fused to each other with a peptide linker.

[0295] Each peptide linker of a CAR can have the same or different length and / or sequence depending on the structural and / or functional characteristics of the antibody and / or various domains. Each peptide linker can be independently selected and optimized. The length, degree of flexibility, and / or other properties of one or more peptide linkers used in a CAR can have some effect on properties, including, but not limited to, affinity, specificity, or avidity for one or more particular antigens or epitopes. For example, a longer peptide linker can be selected to ensure that two adjacent domains do not sterically interfere with each other. In some embodiments, a short peptide linker can be placed between the transmembrane domain and the intracellular signaling domain of a CAR. In some embodiments, the peptide linker contains flexible residues (such as glycine and serine) to allow adjacent domains to move freely relative to each other. For example, a glycine-serine duo may be a suitable peptide linker.

[0296] The peptide linker can be of any suitable length. In some embodiments, the peptide linker is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100 amino acids in length or more. In some embodiments, the peptide linker is no more than about 100, 75, 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 amino acids in length or less. In some embodiments, the length of the peptide linker is from about 1 amino acid to about 10 amino acids, from about 1 amino acid to about 20 amino acids, from about 1 amino acid to about 30 amino acids, from about 5 amino acids to about 15 amino acids, from about 10 amino acids to about 25 amino acids, from about 5 amino acids to about 30 amino acids, from about 10 amino acids to about 30 amino acids, from about 30 amino acids to about 50 amino acids, from about 50 amino acids to about 100 amino acids, or from about 1 amino acid to about 100 amino acids.

[0297] The peptide linker may have a naturally occurring or non-naturally occurring sequence. For example, a sequence derived from the hinge region of a heavy chain-only antibody may be used as a linker. See, for example, WO 1996 / 34103. In some embodiments, the peptide linker is a flexible linker. Exemplary flexible linkers include, but are not limited to, glycine polymers (G) n , glycine-serine polymers, glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Other linkers known in the art, for example, as described in International Publication No. WO 2016014789, International Publication No. WO 2015158671, International Publication No. WO 2016102965, US Patent Application Publication No. WO 20150299317, International Publication No. WO 2018067992, US Patent No. 7,741,465, Colcher et al., J. Nat. Cancer Inst. 82:1191-1197 (1990), and Bird et al., Science 242:423-426 (1988), the disclosures of each of which are incorporated herein by reference, can also be included in the CARs provided herein.

[0298] 5.3.2. Transmembrane Domains The CAR of the present disclosure comprises a transmembrane domain that can be directly or indirectly fused to an extracellular antigen-binding domain. The transmembrane domain can be derived from either a natural source or a synthetic source. As used herein, "transmembrane domain" refers to any protein structure that is thermodynamically stable in a cell membrane, preferably a eukaryotic cell membrane. A transmembrane domain suitable for use in the CAR described herein can be obtained from a naturally occurring protein. Alternatively, it can be a synthetic, non-naturally occurring protein segment, such as a hydrophobic protein segment that is thermodynamically stable in a cell membrane.

[0299] Transmembrane domains are classified based on their three-dimensional structure. For example, transmembrane domains can form an α-helix, a complex of two or more α-helices, a β-barrel, or any other stable structure capable of spanning a cellular phospholipid bilayer. Additionally, or alternatively, transmembrane domains can be classified based on their topology, including the number of passes the transmembrane domain makes across the membrane and the orientation of the protein. For example, a single-pass transmembrane protein crosses the cellular membrane once, while a multi-pass transmembrane protein crosses the cellular membrane at least twice (e.g., 2, 3, 4, 5, 6, 7, or more times). Membrane proteins can be defined as type I, type II, or type III depending on their termini relative to the inside and outside of the cell and the topology of one or more transmembrane segments. Type I membrane proteins have only one membrane-spanning segment and are oriented such that the N-terminus of the protein is on the extracellular side of the cellular lipid bilayer and the C-terminus of the protein is on the cytoplasmic side. Type II membrane proteins also have only one membrane-spanning segment, but are oriented so that the C-terminus of the protein is on the extracellular side of the cell's lipid bilayer and the N-terminus of the protein is on the cytoplasmic side. Type III membrane proteins have multiple membrane-spanning segments and can be further subdivided based on the number of transmembrane segments and the location of the N- and C-termini.

[0300] In some embodiments, the transmembrane domain of a CAR described herein is derived from a type I single-pass membrane protein. In some embodiments, the transmembrane domain of a multi-pass membrane protein may also be compatible for use in the CAR described herein. A multi-pass membrane protein may comprise a complex (at least 2, 3, 4, 5, 6, 7, or more) alpha helix or beta sheet structure. In some embodiments, the N- and C-termini of a multi-pass membrane protein are present on opposite sides of a lipid bilayer, e.g., the N-terminus of the protein is present on the cytoplasmic side of the lipid bilayer and the C-terminus of the protein is present on the extracellular side.

[0301] In some embodiments, the transmembrane domain of the CAR is selected from the group consisting of the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (C D278), 4-1BB(CD137), GITR, CD40, BAFFR, HVEM(LIGHTR), SLAMF7, NKp80(KLRF1), CD160, GPC3, IL-2Rβ , IL-2Rγ, IL-7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, C D103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR 2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY 55), PSGL1, CDIOO (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C. In some embodiments, the transmembrane domain is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1.

[0302] In some specific embodiments, the transmembrane domain is derived from CD8α, hi some embodiments, the transmembrane domain is the transmembrane domain of CD8α comprising the amino acid sequence of SEQ ID NO:103.

[0303] The transmembrane domain used in the CARs described herein can also comprise at least a portion of a synthetic, non-naturally occurring protein segment. In some embodiments, the transmembrane domain is a synthetic, non-naturally occurring alpha helix or beta sheet. In some embodiments, the protein segment is at least about 20 amino acids, e.g., at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 amino acids, or more. Examples of synthetic transmembrane domains are known in the art, for example, in U.S. Pat. No. 7,052,906 and WO 2000 / 032776 (the relevant disclosures of which are incorporated herein by reference).

[0304] The transmembrane domains provided herein may comprise a transmembrane region and a cytoplasmic region located C-terminal to the transmembrane domain. The cytoplasmic region of the transmembrane domain may comprise three or more amino acids, which in some embodiments facilitates orientation of the transmembrane domain in a lipid bilayer. In some embodiments, one or more cysteine ​​residues are present in the transmembrane region of the transmembrane domain. In some embodiments, one or more cysteine ​​residues are present in the cytoplasmic region of the transmembrane domain. In some embodiments, the cytoplasmic region of the transmembrane domain comprises a positively charged amino acid. In some embodiments, the cytoplasmic region of the transmembrane domain comprises the amino acids arginine, serine, and lysine.

[0305] In some embodiments, the transmembrane region of the transmembrane domain comprises hydrophobic amino acid residues. In some embodiments, the transmembrane domain of a CAR provided herein comprises an artificial hydrophobic sequence. For example, a triplet of phenylalanine, tryptophan, and valine may be present at the C-terminus of the transmembrane domain. In some embodiments, the transmembrane region comprises mostly hydrophobic amino acid residues, such as alanine, leucine, isoleucine, methionine, phenylalanine, tryptophan, or valine. In some embodiments, the transmembrane region is hydrophobic. In some embodiments, the transmembrane region comprises a poly-leucine-alanine sequence. The hydropathy, i.e., hydrophobic or hydrophilic properties, of a protein or protein segment can be determined by any method known in the art, for example, Kyte and Doolittle hydropathy analysis.

[0306] 5.3.3. Intracellular Signaling Domains The CAR of the present disclosure comprises an intracellular signaling domain (ISD). The intracellular signaling domain is involved in activating at least one of the normal effector functions of immune effector cells expressing the CAR. The term "effector function" refers to a specialized function of a cell. For example, the effector function of a T cell can be cytolytic activity or helper activity, including cytokine secretion. Thus, the term "cytoplasmic signaling domain" refers to a portion of a protein that transmits an effector function signal and directs a cell to perform a specialized function. Typically, the entire cytoplasmic signaling domain can be used, but in many cases, it is not necessary to use the entire chain. To the extent that a truncated portion of the cytoplasmic signaling domain is used, such a truncated portion can be used in place of the intact chain, so long as it transmits the effector function signal. Thus, the term cytoplasmic signaling domain is intended to include any truncated portion of the cytoplasmic signaling domain that is sufficient to transmit the effector function signal.

[0307] In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell. In some embodiments, the CAR comprises an intracellular signaling domain consisting essentially of a primary intracellular signaling domain of an immune effector cell. A "primary intracellular signaling domain" refers to a cytoplasmic signaling sequence that induces immune effector functions, playing a stimulatory role. In some embodiments, the primary intracellular signaling domain contains a signaling motif known as an immunoreceptor tyrosine-based activation motif, or ITAM. As used herein, "ITAM" refers to a conserved protein motif typically present in the tails of signaling molecules expressed in many immune cells. This motif may contain two repeats of the amino acid sequence YxxL / I, separated by 6-8 amino acids (where each x is independently any amino acid), resulting in the conserved motif YxxL / Ix(6-8)YxxL / I. ITAMs within signaling molecules are important for intracellular signal transduction, which is mediated, at least in part, by phosphorylation of tyrosine residues in the ITAM upon activation of the signaling molecule. ITAMs may also serve as docking sites for other proteins involved in signaling pathways. Exemplary ITAM-containing primary cytoplasmic signaling sequences include those derived from CD3ζ, FcRγ (FCER1G), FcRβ (Fcε Rib), CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d.

[0308] In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. In some embodiments, the intracellular signaling domain consists of the cytoplasmic signaling domain of CD3ζ. In some embodiments, the primary intracellular signaling domain is the cytoplasmic signaling domain of wild-type CD3ζ. In some embodiments, the primary intracellular signaling domain of CD3ζ comprises the amino acid sequence of SEQ ID NO: 105. In some embodiments, the primary intracellular signaling domain of wild-type CD3ζ. In some embodiments, the primary intracellular signaling domain is a functional mutant of the cytoplasmic signaling domain of CD3ζ comprising one or more mutations, such as Q65K.

[0309] 5.3.4. Costimulatory Signaling Domains Many immune effector cells require costimulation in addition to antigen-specific signal stimulation to promote cell proliferation, differentiation, and survival, and to activate cellular effector functions. In some embodiments, a CAR comprises at least one costimulatory signaling domain. The term "costimulatory signaling domain," as used herein, refers to at least a portion of a protein that mediates signal transduction within a cell to induce an immune response, such as an effector function. The costimulatory signaling domain of the chimeric receptor described herein can be a cytoplasmic signaling domain from a costimulatory protein, which transmits signals and regulates responses mediated by immune cells, such as T cells, NK cells, macrophages, neutrophils, or eosinophils. The "costimulatory signaling domain" can be the cytoplasmic portion of a costimulatory molecule. The term "costimulatory molecule" refers to a cognate binding partner on an immune cell (such as a T cell) that specifically binds to a costimulatory ligand and thereby mediates a costimulatory response by the immune cell, such as, but not limited to, proliferation and survival.

[0310] In some embodiments, the intracellular signaling domain comprises a single costimulatory signaling domain. In some embodiments, the intracellular signaling domain comprises two or more (e.g., about any of 2, 3, 4, or more) costimulatory signaling domains. In some embodiments, the intracellular signaling domain comprises two or more of the same costimulatory signaling domain. In some embodiments, the intracellular signaling domain comprises two or more costimulatory signaling domains from different costimulatory proteins, such as any two or more costimulatory proteins described herein. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain (e.g., the cytoplasmic signaling domain of CD3ζ) and one or more costimulatory signaling domains. In some embodiments, the one or more costimulatory signaling domains and the primary intracellular signaling domain (e.g., the cytoplasmic signaling domain of CD3ζ) are fused to each other via an optional peptide linker. The primary intracellular signaling domain and the one or more costimulatory signaling domains can be arranged in any suitable order. In some embodiments, the one or more costimulatory signaling domains are located between the transmembrane domain and the primary intracellular signaling domain (e.g., the cytoplasmic signaling domain of CD3ζ). Multiple costimulatory signaling domains can provide additive or synergistic stimulatory effects.

[0311] Activation of a costimulatory signaling domain in a host cell (e.g., an immune cell) can induce the cell to increase or decrease cytokine production and secretion, phagocytic properties, proliferation, differentiation, survival, and / or cytotoxicity. The costimulatory signaling domain of any costimulatory molecule may be compatible for use in the CARs described herein. The type or types of costimulatory signaling domains are selected based on factors such as the type of immune effector cell on which the effector molecule will be expressed (e.g., T cells, NK cells, macrophages, neutrophils, or eosinophils) and the desired immune effector function (e.g., ADCC effect). Examples of costimulatory signaling domains for use in CARs include, without limitation, members of the B7 / CD28 family (e.g., B7-1 / CD80, B7-2 / CD86, B7-H1 / PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BTLA / CD272, CD28, CTLA-4, Gi24 / VISTA / B7-H5, ICOS / CD278, PD-1, PD-L2 / B7-DC, and PDCD6); members of the TNF superfamily (e.g., 4-1BB / TNFSF9 / CD137, 4-1BB ligand / TNFSF9, BAFF / BLyS / TNFSF13B, BAFFR / TNFRSF13C, and CTLA-4); , CD27 / TNFRSF7, CD27 ligand / TNFSF7, CD30 / TNFRSF8, CD30 ligand / TNFSF8, CD40 / TNFRSF5, CD40 / TNFRSF5, CD40 ligand / TNFSF5, DR3 / TNFRSF25, GITR / TNFRSF18, GITR ligand / TNFSF18, HVEM / TNFRSF14, LIGHT / TNFSF14, lymphotoxin-α / TNF-β, OX40 / TNFRSF4, OX40 ligand / TNFSF4, RELT / TNFRSF19L, TACI / TNFRSF13B, TL1A / TNFSF15, TNF-α, and TNFRII / TNFRSF1B);SLAM family members (e.g., 2B4 / CD244 / SLAMF4, BLAME / SLAMF8, CD2, CD2F-10 / SLAMF9, CD48 / SLAMF2, CD58 / LFA-3, CD84 / SLAMF5, CD229 / SLAMF3, CRACC / SLAMF7, NTB-A / SLAMF6, and SLAM / CD150); and any other costimulatory molecules, e.g., CD2, CD7, CD53, CD82 / Kai-1, CD90 / Thy1, CD96, CD160, CD200, CD300a / LMIR1, HLA class I, HLA-DR, Ikaros, integrin α4 / CD49d, integrin α4β1, integrin α4β7 / LPAM-1, LAG-3, TCL1A, TCL1B, CRTAM, DAP12, Dectin-1 / CLEC7A, DPPIV / CD26, EphB6, TIM-1 / KIM-1 / HAVCR, TIM-4, TSLP, TSLPR, lymphocyte function-associated antigen-1 (LFA-1), and NKG2C;

[0312] In some embodiments, the one or more costimulatory signaling domains are selected from the group consisting of CD27, CD28, CD137, OX40, CD30, CD40, CD3, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds to CD83 (such as CD83 and MD2).

[0313] In some embodiments, the intracellular signaling domain in a CAR of the present disclosure comprises a costimulatory signaling domain derived from CD137 (i.e., 4-1BB). In some embodiments, the intracellular signaling domain comprises the cytoplasmic signaling domain of CD3ζ and the costimulatory signaling domain of CD137. In some embodiments, the intracellular signaling domain comprises the costimulatory signaling domain of CD137 comprising the amino acid sequence of SEQ ID NO: 104.

[0314] Mutants of any of the costimulatory signaling domains described herein, such that the costimulatory signaling domain has the ability to modulate the immune response of an immune cell, are also within the scope of the present disclosure. In some embodiments, the costimulatory signaling domain comprises up to 10 amino acid residue (e.g., 1, 2, 3, 4, 5, or 8) mutations when compared to its wild-type counterpart. Such costimulatory signaling domains comprising one or more amino acid mutations may be referred to as mutants. Mutating amino acid residues in a costimulatory signaling domain may result in increased signaling and enhanced stimulation of an immune response compared to a costimulatory signaling domain that does not contain the mutation. Mutating amino acid residues in a costimulatory signaling domain may result in decreased signaling and reduced stimulation of an immune response compared to a costimulatory signaling domain that does not contain the mutation.

[0315] Hinge Area The CAR of the present disclosure may comprise a hinge domain located between the extracellular antigen-binding domain and the transmembrane domain. A hinge domain is generally an amino acid segment found between two domains of a protein, and may provide flexibility to the protein, allowing one or both domains to move relative to each other. Any amino acid sequence that provides such flexibility and movement of the extracellular antigen-binding domain relative to the transmembrane domain of the effector molecule may be used.

[0316] The hinge domain can contain about 10 to 100 amino acids, e.g., about 15 to 75 amino acids, 20 to 50 amino acids, or 30 to 60 amino acids. In some embodiments, the hinge domain can be at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 amino acids in length.

[0317] In some embodiments, the hinge domain is the hinge domain of a naturally occurring protein. The hinge domain of any protein known in the art to contain a hinge domain is compatible for use in the chimeric receptors described herein. In some embodiments, the hinge domain is at least a portion of the hinge domain of a naturally occurring protein and confers flexibility to the chimeric receptor. In some embodiments, the hinge domain is derived from CD8α. In some embodiments, the hinge domain is a portion of the hinge domain of CD8α, e.g., a fragment comprising at least 15 (e.g., 20, 25, 30, 35, or 40) consecutive amino acids of the hinge domain of CD8α. In some embodiments, the hinge domain of CD8α comprises the amino acid sequence of SEQ ID NO: 102.

[0318] Hinge domains of antibodies, such as IgG, IgA, IgM, IgE, or IgD antibodies, are also compatible for use in the pH-dependent chimeric receptor systems described herein. In some embodiments, the hinge domain is a hinge domain that connects the constant domains CH1 and CH2 of an antibody. In some embodiments, the hinge domain is of an antibody and comprises the hinge domain of an antibody and one or more constant regions of the antibody. In some embodiments, the hinge domain comprises the hinge domain of an antibody and the CH3 constant region of the antibody. In some embodiments, the hinge domain comprises the hinge domain of an antibody and the CH2 and CH3 constant regions of the antibody. In some embodiments, the antibody is an IgG, IgA, IgM, IgE, or IgD antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the hinge region comprises the hinge region and the CH2 and CH3 constant regions of an IgG1 antibody. In some embodiments, the hinge region comprises the hinge region and CH3 constant region of an IgG1 antibody.

[0319] Non-naturally occurring peptides may also be used as hinge domains for the chimeric receptors described herein. In some embodiments, the hinge domain between the C-terminus of the extracellular ligand-binding domain and the N-terminus of the transmembrane domain of an Fc receptor is a peptide linker such as a (GxS)n linker, where x and n can independently be an integer between 3 and 12, including 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or greater.

[0320] 5.3.6. Signal Peptide A CAR of the present disclosure may include a signal peptide (also known as a signal sequence) at the N-terminus of the polypeptide. Generally, a signal peptide is a peptide sequence that targets a polypeptide to a desired site within a cell. In some embodiments, the signal peptide will target the effector molecule to the secretory pathway of a cell, allowing for incorporation and anchoring of the effector molecule into the lipid bilayer. Signal peptides, including signal sequences of naturally occurring proteins or synthetic, non-naturally occurring signal sequences, that are compatible for use in the CARs described herein will be apparent to those of skill in the art. In some embodiments, the signal peptide is derived from a molecule selected from the group consisting of CD8α, GM-CSF receptor α, and IgG1 heavy chain. In some embodiments, the signal peptide is derived from CD8α. In some embodiments, the signal peptide of CD8α comprises the amino acid sequence of SEQ ID NO: 101.

[0321] 5.3.7. Polypeptides Comprising CAR and One or More Additional Domains As shown in Section 6 below, engineered immune effector cells expressing a CAR and exogenously introduced p40 and CCL-19 unexpectedly provided superior efficacy. For example, p40 and CCL-19 armored CAR-T cells exhibited higher efficacy at lower doses compared to corresponding naked CAR-T cells.

[0322] Thus, in another aspect, provided herein is a polypeptide comprising a CAR provided herein and at least one of the p40 subunit of IL-12 and CCL-19. In some embodiments, provided herein is a polypeptide comprising a CAR provided herein and p40. In some embodiments, provided herein is a polypeptide comprising a CAR provided herein and CCL-19. In other embodiments, provided herein is a polypeptide comprising a CAR provided herein and both p40 and CCL-19. In some embodiments, p40 is human p40 comprising the amino acid sequence of SEQ ID NO: 135, and / or CCL-19 is human CCL-19 comprising the amino acid sequence of SEQ ID NO: 136.

[0323] In the present polypeptides, CAR, p40, and / or CCL-19 can be arranged in any order. For example, when CAR, p40, and CCL-19 are all present in a polypeptide, in some embodiments, the polypeptides provided herein comprise, from N-terminus to C-terminus, CAR, p40, and CCL-19; in some embodiments, the polypeptides provided herein comprise, from N-terminus to C-terminus, CAR, CCL-19, and p40; in some embodiments, the polypeptides provided herein comprise, from N-terminus to C-terminus, CCL-19, p40, and CAR; in some embodiments, the polypeptides provided herein comprise, from N-terminus to C-terminus, p40, CCL-19, and CAR; in some embodiments, the polypeptides provided herein comprise, from N-terminus to C-terminus, p40, CAR, and CCL-19; and in some embodiments, the polypeptides provided herein comprise, from N-terminus to C-terminus, CCL-19, CAR, and p40.

[0324] In some embodiments of the various polypeptides provided herein, CAR, p40, and / or CCL-19 are linked to each other via a peptide linker. In some embodiments, the peptide linker is a self-cleaving peptide, such as the 2A self-cleaving peptide, such that CAR, p40, and / or CCL-19 are cleaved into separate polypeptides in the cell. Members of the 2A peptide family are named after the viruses in which they were first described. For example, F2A, the first described 2A peptide, is derived from foot-and-mouth disease virus. This self-cleaving 18-22 amino acid long 2A peptide mediates "ribosomal skipping" between proline and glycine residues, inhibiting peptide bond formation without affecting downstream translation. These peptides allow multiple proteins to be encoded as a polyprotein, which dissociates into its component proteins upon translation. Self-cleaving peptides are found in members of the Picornaviridae family of viruses, including aphthoviruses, such as foot-and-mouth disease virus (FMDV), equine rhinitis A virus (ERAV), thosea asigna virus (TaV), and porcine teschovirus-1 (PTV-1) (see Donnelly et al., J. Gen. Virol., 82:1027-101 (2001); Ryan et al., J. Gen. Virol., 72:2727-2732 (2001)), and cardioviruses, such as Theiloviruses (e.g., Theiler's murine encephalomyelitis) and encephalomyocarditis virus. The 2A peptides derived from FMDV, ERAV, PTV-1, and TaV, sometimes referred to as "F2A," "E2A," "P2A," and "T2A," respectively, are encompassed by the present disclosure, as described, for example, in Donnelly et al., J. Gen. Virol., 78:13-21 (1997); Ryan and Drew, EMBO J., 13:928-933 (1994); Szymczak et al., Nature Biotech., 5:589-594 (2004); Hasegawa et al., Stem Cells, 25(7):1707-12 (2007).In yet other embodiments, the intein-mediated protein splicing system is used herein, e.g., as described in Shah and Muir, Chem Sci., 5(1):446-461 (2014) and Topilina and Mills, Mobile DNA, 5(5) (2014). Other methods known in the art can also be used with the present constructs.

[0325] In some embodiments, the 2A self-cleaving peptide is selected from the group consisting of F2A, E2A, P2A, T2A, or variants thereof. In some embodiments, the self-cleaving peptide is a 2A self-cleaving peptide P2A fragment comprising the amino acid sequence of SEQ ID NO: 138. In a specific embodiment, the self-cleaving peptide is a T2A fragment comprising the amino acid sequence of SEQ ID NO: 139.

[0326] In some specific embodiments, p40 and CCL-19 are linked by a first self-cleaving peptide. In some embodiments, the first self-cleaving peptide is a T2A fragment of the IL-12 p40 subunit comprising the amino acid sequence of SEQ ID NO: 139. In some embodiments, the domain comprising the p40 subunit of IL-12 and CCL-19 provided herein comprises the amino acid sequence of SEQ ID NO: 134. In some embodi...

Claims

1. An antibody or antigen-binding fragment thereof that binds to glypican-3 (GPC3), (i) an HCDR1 comprising the amino acid sequence of SEQ ID NO: 9, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 19, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 29, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 39, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 49, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 59; or (ii) an HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 11, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 21, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 31, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 41, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 51; or (iii) an HCDR1 comprising the amino acid sequence of SEQ ID NO: 2, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 12, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 22, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 32, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 42, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 52; or (iv) an HCDR1 comprising the amino acid sequence of SEQ ID NO: 3, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 13, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 23, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 33, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 43, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 53; or (v) an HCDR1 comprising the amino acid sequence of SEQ ID NO: 4, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 14, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 24, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 34, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 44, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 54; or (vi) an HCDR1 comprising the amino acid sequence of SEQ ID NO: 5, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 15, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 25, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 35, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 45, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 55; or (vii) an HCDR1 comprising the amino acid sequence of SEQ ID NO: 6, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 16, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 26, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 36, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 46, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 56; or (viii) an HCDR1 comprising the amino acid sequence of SEQ ID NO: 7, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 17, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 27, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 37, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 47, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 57; or (ix) an HCDR1 comprising the amino acid sequence of SEQ ID NO: 8, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 18, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 28, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 38, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 48, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 58; or (x) an HCDR1 comprising the amino acid sequence of SEQ ID NO: 10, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 20, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 30, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 40, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 50, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 60; The antibody or antigen-binding fragment thereof, comprising:

2. (i) a VH domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 119, and a VL domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 124; or (ii) a VH domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 62 and a VL domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 72; or (iii) a VH domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 63 and a VL domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 73; or (iv) a VH domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 64 and a VL domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 74; or (v) a VH domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 65 and a VL domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 75; or (vi) a VH domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 66 and a VL domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 76; or (vii) a VH domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 67 and a VL domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 77; or (viii) a VH domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 68 and a VL domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 78; or (ix) a VH domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 69 and a VL domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 79; or (x) a VH domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 70 and a VL domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 80; or (xi) a VH domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 117 and a VL domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 123; or (xii) a VH domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 118 and a VL domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 123; or (xiii) a VH domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 119 and a VL domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 123; or (xiv) a VH domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 120 and a VL domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 123; or (xv) a VH domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 121 and a VL domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 123; or (xvi) a VH domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 122 and a VL domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 123; or (xvii) a VH domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 117 and a VL domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 124; or (xviii) a VH domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 118 and a VL domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 124; or (xix) a VH domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 61 and a VL domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 71; or (xx) a VH domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 120 and a VL domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 124; or (xxi) a VH domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 121 and a VL domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 124; or (xxii) a VH domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 122 and a VL domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 124; or (xxiii) a VH domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 117 and a VL domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 125; or (xxiv) a VH domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 118 and a VL domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 125; or (xxv) a VH domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 119 and a VL domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 125; or (xxvi) a VH domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 120 and a VL domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 125; or (xxvii) a VH domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 121 and a VL domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 125; or (xxviii) a VH domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 122 and a VL domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:

125.

2. The antibody or antigen-binding fragment thereof of claim 1, optionally comprising: (i) a VH domain comprising the amino acid sequence of SEQ ID NO: 119 and a VL domain comprising the amino acid sequence of SEQ ID NO: 124; or (ii) a VH domain comprising the amino acid sequence of SEQ ID NO: 62 and a VL domain comprising the amino acid sequence of SEQ ID NO: 72; or (iii) a VH domain comprising the amino acid sequence of SEQ ID NO: 63 and a VL domain comprising the amino acid sequence of SEQ ID NO: 73; or (iv) a VH domain comprising the amino acid sequence of SEQ ID NO: 64 and a VL domain comprising the amino acid sequence of SEQ ID NO: 74; or (v) a VH domain comprising the amino acid sequence of SEQ ID NO: 65 and a VL domain comprising the amino acid sequence of SEQ ID NO: 75; or (vi) a VH domain comprising the amino acid sequence of SEQ ID NO: 66 and a VL domain comprising the amino acid sequence of SEQ ID NO: 76; or (vii) a VH domain comprising the amino acid sequence of SEQ ID NO: 67 and a VL domain comprising the amino acid sequence of SEQ ID NO: 77; or (viii) a VH domain comprising the amino acid sequence of SEQ ID NO: 68 and a VL domain comprising the amino acid sequence of SEQ ID NO: 78; or (ix) a VH domain comprising the amino acid sequence of SEQ ID NO: 69 and a VL domain comprising the amino acid sequence of SEQ ID NO: 79; or (x) a VH domain comprising the amino acid sequence of SEQ ID NO: 70 and a VL domain comprising the amino acid sequence of SEQ ID NO: 80; or (xi) a VH domain comprising the amino acid sequence of SEQ ID NO: 117 and a VL domain comprising the amino acid sequence of SEQ ID NO: 123; or (xii) a VH domain comprising the amino acid sequence of SEQ ID NO: 118 and a VL domain comprising the amino acid sequence of SEQ ID NO: 123; or (xiii) a VH domain comprising the amino acid sequence of SEQ ID NO: 119 and a VL domain comprising the amino acid sequence of SEQ ID NO: 123; or (xiv) a VH domain comprising the amino acid sequence of SEQ ID NO: 120 and a VL domain comprising the amino acid sequence of SEQ ID NO: 123; or (xv) a VH domain comprising the amino acid sequence of SEQ ID NO: 121 and a VL domain comprising the amino acid sequence of SEQ ID NO: 123; or (xvi) a VH domain comprising the amino acid sequence of SEQ ID NO: 122 and a VL domain comprising the amino acid sequence of SEQ ID NO: 123; or (xvii) a VH domain comprising the amino acid sequence of SEQ ID NO: 117 and a VL domain comprising the amino acid sequence of SEQ ID NO: 124; or (xviii) a VH domain comprising the amino acid sequence of SEQ ID NO: 118 and a VL domain comprising the amino acid sequence of SEQ ID NO: 124; or (xix) a VH domain comprising the amino acid sequence of SEQ ID NO: 61 and a VL domain comprising the amino acid sequence of SEQ ID NO: 71; or (xx) a VH domain comprising the amino acid sequence of SEQ ID NO: 120 and a VL domain comprising the amino acid sequence of SEQ ID NO: 124; or (xxi) a VH domain comprising the amino acid sequence of SEQ ID NO: 121 and a VL domain comprising the amino acid sequence of SEQ ID NO: 124; or (xxii) a VH domain comprising the amino acid sequence of SEQ ID NO: 122 and a VL domain comprising the amino acid sequence of SEQ ID NO: 124; or (xxiii) a VH domain comprising the amino acid sequence of SEQ ID NO: 117 and a VL domain comprising the amino acid sequence of SEQ ID NO: 125; or (xxiv) a VH domain comprising the amino acid sequence of SEQ ID NO: 118 and a VL domain comprising the amino acid sequence of SEQ ID NO: 125; or (xxv) a VH domain comprising the amino acid sequence of SEQ ID NO: 119 and a VL domain comprising the amino acid sequence of SEQ ID NO: 125; or (xxvi) a VH domain comprising the amino acid sequence of SEQ ID NO: 120 and a VL domain comprising the amino acid sequence of SEQ ID NO: 125; or (xxvii) a VH domain comprising the amino acid sequence of SEQ ID NO: 121 and a VL domain comprising the amino acid sequence of SEQ ID NO: 125; or (xxviii) a VH domain comprising the amino acid sequence of SEQ ID NO: 122 and a VL domain comprising the amino acid sequence of SEQ ID NO: 125 The antibody or antigen-binding fragment thereof, comprising:

3. 3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, which is a Fab fragment, a Fab' fragment, a F(ab)'2 fragment, a single-chain variable fragment (scFv), or a disulfide-stabilized variable fragment (dsFv), and / or which comprises an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 129, 81-90, and 128, and / or which is an IgG and / or which is a humanized antibody.

4. A method for detecting GPC3 in a tissue sample, comprising contacting the tissue sample with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 and detecting binding of the antibody to the tissue sample, wherein an increase in binding of the antibody to the tissue sample compared to binding of the antibody to a control sample indicates detection of GPC3 in the tissue sample; and / or wherein the antibody is directly labeled with a detectable marker; and / or further comprising contacting the tissue sample with a secondary antibody that specifically binds to the antibody and detecting binding of the secondary antibody. wherein increased binding of the secondary antibody to the tissue sample compared to binding of the secondary antibody to a control sample detects GPC3 in the tissue sample; and / or wherein the tissue sample comprises cells from hepatocellular carcinoma (HCC), melanoma, ovarian clear cell carcinoma (OCCC), yolk sac tumor (YST), neuroblastoma, hepatoblastoma, nephroblastoma (Wilms' tumor), lung squamous cell carcinoma, lung adenocarcinoma, large cell lung carcinoma, small cell lung carcinoma, testicular nonseminomatous germ cell tumor, liposarcoma, cervical intraepithelial neoplasia, adrenal adenoma, schwannoma, embryonal tumor, gastric cancer, colorectal cancer, thyroid cancer, and / or esophageal cancer.

5. (a) an extracellular antigen-binding domain comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3; (b) a transmembrane domain; and (c) intracellular signaling domain A chimeric antigen receptor (CAR) comprising a polypeptide comprising:

6. 6. The CAR according to claim 5, wherein the antibody or antigen-binding fragment thereof is an scFv, and / or further comprises a signal peptide located at the N-terminus of the polypeptide, optionally wherein the signal peptide is derived from a molecule selected from the group consisting of CD8α, GM-CSF receptor α, and an IgG1 heavy chain; and / or further comprises a hinge domain located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain, optionally wherein the hinge domain is derived from CD8α; and / or the transmembrane domain is selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1. the CAR and / or the intracellular signaling domain derived from a molecule comprises a costimulatory signaling domain, optionally wherein the costimulatory signaling domain is derived from a costimulatory molecule selected from the group consisting of CD27, CD28, CD137, OX40, CD30, CD40, CD3, HVEM, ICOS, Myd88, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand for CD83, and a combination thereof; and / or the CAR wherein the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell, optionally wherein the primary intracellular signaling domain is derived from CD3ζ.

7. (i) the amino acid sequence of SEQ ID NO: 91, or (ii) the amino acid sequence of SEQ ID NO: 92, or (iii) the amino acid sequence of SEQ ID NO: 93, or (iv) the amino acid sequence of SEQ ID NO: 94, or (v) the amino acid sequence of SEQ ID NO: 95, or (vi) the amino acid sequence of SEQ ID NO: 96, or (vii) the amino acid sequence of SEQ ID NO: 97, or (viii) the amino acid sequence of SEQ ID NO: 98; or (ix) the amino acid sequence of SEQ ID NO: 99; or (x) the amino acid sequence of SEQ ID NO: 100, or (xi) the amino acid sequence of SEQ ID NO: 107, or (xii) the amino acid sequence of SEQ ID NO: 108, or (xiii) the amino acid sequence of SEQ ID NO: 109, or (xiv) the amino acid sequence of SEQ ID NO: 110, or (xv) the amino acid sequence of SEQ ID NO: 111, or (xvi) the amino acid sequence of SEQ ID NO: 112, or (xvii) the amino acid sequence of SEQ ID NO: 113, or (xviii) the amino acid sequence of SEQ ID NO: 114, or (xix) the amino acid sequence of SEQ ID NO: 115, or (xx) the amino acid sequence of SEQ ID NO: 116, or (xxi) the amino acid sequence of SEQ ID NO: 130; or (xxii) the amino acid sequence of SEQ ID NO: 131 The CAR according to claim 5 or 6, comprising a polypeptide comprising:

8. A polypeptide comprising the CAR according to any one of claims 5 to 7, further comprising a p40 subunit of IL-12 and / or CCL-19, wherein optionally, p40 is human p40 comprising the amino acid sequence of SEQ ID NO: 135, and optionally, CCL-19 is human CCL-19 comprising the amino acid sequence of SEQ ID NO: 136; and / or the polypeptide, and / or p40 and CCL-19 are linked by a first self-cleaving peptide, and optionally, the first self-cleaving peptide has the amino acid sequence of SEQ ID NO:

139. and / or the polypeptide wherein the p40 subunit of IL-12 and CCL-19 are present in a domain comprising the amino acid sequence of SEQ ID NO: 134, and optionally the CAR is linked to the domain by a second self-cleaving peptide, and optionally the second self-cleaving peptide is a 2A self-cleaving peptide P2A fragment comprising the amino acid sequence of SEQ ID NO: 138; and / or the polypeptide comprising the amino acid sequence of SEQ ID NO:

133.

9. A nucleic acid molecule comprising a sequence encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, the CAR according to any one of claims 5 to 7, or the polypeptide according to claim 8.

10. A vector comprising the nucleic acid molecule of claim 9, which is optionally a viral or non-viral vector.

11. 11. A host cell transformed with the vector of claim 10, wherein the host cell is optionally an immune effector cell selected from the group consisting of a T cell, a NK cell, a peripheral blood mononuclear cell (PBMC), a hematopoietic stem cell, a pluripotent stem cell, an embryonic stem cell, and combinations thereof.

12. An immune effector cell expressing (i) the CAR according to any one of claims 5 to 7, (ii) exogenously introduced p40, and (iii) exogenously introduced CCL-19.

13. A composition comprising a therapeutically effective amount of the antibody or antigen-binding fragment thereof of any one of claims 1 to 3, the nucleic acid molecule of claim 9, the vector of claim 10, the host cell of claim 11, or the immune effector cell of claim 12, and a pharmaceutically acceptable carrier.

14. 14. The composition of claim 13 for use in a method of treating a patient having a cancer that expresses GPC3, the method comprising administering the composition to the patient, wherein the cancer is optionally selected from the group consisting of hepatocellular carcinoma (HCC), melanoma, ovarian clear cell carcinoma (OCCC), yolk sac tumor (YST), neuroblastoma, hepatoblastoma, nephroblastoma (Wilm's tumor), lung squamous cell carcinoma, lung adenocarcinoma, large cell lung carcinoma, small cell lung carcinoma, testicular nonseminomatous germ cell tumor, liposarcoma, cervical intraepithelial neoplasia, adrenal adenoma, schwannoma, embryonal tumor, gastric cancer, colorectal cancer, thyroid cancer, and esophageal cancer, and optionally the host cells or immune effector cells are obtained from the patient, and optionally the host cells or immune effector cells are obtained from a healthy donor.

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