Methods and compositions comprising oncolytic viruses expressing GPC3 and GPC3-targeted therapies

Abstract

Described herein, inter alia, are oncolytic viruses expressing a glypican-3 (GPC3) and methods for treating a patient suffering from a solid tumor by administering an oncolytic virus expressing a GPC3, optionally also administering a GPC3-targeting therapy (e.g., T cell engager targeted to GPC3, GPC3 CAR immune cells, GPC3 antibody or antibody fragment).

Description

[0001] Attorney Docket No. 40056-0091 WO1 Methods and Compositions Comprising Oncolytic Viruses Expressing GPC3 and GPC3-Targeted Therapies

[0002] CLAIM OF PRIORITY

[0003] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 756,366, filed on February 10, 2025, The entire contents of the foregoing are incorporated herein by reference.

[0004] TECHNICAL FIELD

[0005] This disclosure relates to methods of treating a subject having a solid tumor comprising administering an oncolytic virus (OV) expressing a glypican-3 (GPC3) and optionally a GPC3-targeted therapy that binds to GPC3 (e g., a bispecific T cell engager (TCE); a population of immune cells expressing a chimeric antigen receptor that binds to GPC3; or an anti-GPC3 antibody, antibody fragment, or antibody drug conjugate),

[0006] BACKGROUND

[0007] A universal tumor-agnostic cancer therapy remains elusive in the field of immunotherapy. There have been impressive patient specific immunotherapies with recent FDA approvals including chimeric antigen receptor (CAR) T cell therapies. Limitations include the autologous nature of these cells and the length of time required for manufacturing. The benefits of bispecific T cell engagers (called TCEs interchangeably herein) are their off-the-shelf availability and the ability to acutely tune targeting with dosing regimens [1, 2], TCEs have emerged as a promising immunotherapy strategy for the treatment of B-cell hematological malignancies. Blinatumomab, an FDA approved TCE composed of a CD19 scFv joined to a CD3 scFv that drives endogenous T-cell mediated immune responses against malignant cells, has shown durable clinical responses for the treatment of B-cell acute lymphoblastic leukemia (B-ALL) and non-Hodgkin's lymphoma (1, 2).

[0008] Cancer remains a major global health challenge and is currently the second leading cause of death worldwide (Bray et. al. (2024) CA Cancer J Clin 74, 229-263). Solid tumors account for nearly 90% of all malignancies, underscoring their overwhelming contribution to cancer burden. Checkpoint blockade, chimeric antigen receptor (C / XR)-T cells, bispecific T cell engagers (BiTEs), monoclonal antibodies, and antibody -drug conjugates (ADCs) have all demonstrated remarkable activity by¬ harnessing immune effectors with unprecedented precision (30-34). While hematologic malignancies have been particularly amenable to these strategies, the translation of success into solid tumors has beenAttorney Docket No. 40056-0091 WO1 more limited. Challenges facing solid tumor therapies using CAR T cells and TCE therapies are driven by a relative lack amenable and targetable tumor antigens (3, 4). The shared expression of solid tumor antigens on normal tissue and their heterogeneous, and nonuniform, expression patterns on tumors limits the potential for effective and durable anti-tumor responses (5, 6). In addition, many solid tumors are immunologically “cold” and limit T cell trafficking and anti-tumor functionality, a phenomenon uncommonly observed in hematological malignancies (7-9), Further, most solid tumors have a complex microenvironment that represents a challenge for cancer therapies

[0024] , Thus, improved and more accessible immunotherapies remain to be explored.

[0009] SUMMARY

[0010] The present disclosure is based, at least in part, on the discovery that an oncolytic virus (OV) expressing a glypican-3 (GPC3) antigen can redirect GPC3-targeted therapies, such as GPC3-targeted bispecific T cell engagers (GPC3 TCEs) and chimeric antigen receptor (CAR)-engmeered immune cell therapies targeting GPC3 (e g., GPC3 CAR T cells), to drive anti-tumor responses of endogenous T cells against multiple solid tumor types. GPC3 is a cell surface heparan sulfate proteoglycan that may play a role in the control of cell division and growth regulation. Human GPC3 (e.g,, Genbank Accession Nos: NP 004475.1, NP_ OI 158089.1, NP_001158090.1, NP_001158091.1, XP_054182809.1, and

[0011] XP 016884902.1; UmProt ID Nos: P51654-1, P51654-2, and P51654-3; etc.) is a cell-surface glycoprotein in which heparan sulfate glycosaminoglycan chains are covalently linked to a protein core that is anchored to the membrane via a glycosyl phosphatidylinositol linkage. There are four known variants / isoforms of human GPC3. The sequences are known, including GenBank Accession numbers: NM_001164617 and NP_001158089 (variant I, VI); NM_004484 and NP__004475 (variant 2, V2); NM 001164618 and NP 001158090 (variant 3, V3); and NM 001164619 and NP 001158091 (variant 4, V4).

[0012] Amongst other things, the data in this application demonstrate robust cell surface GPC3 expression on multiple tumor types infected with an OV carrying a GPC3-encoding gene (referred to as OV-GPC3 and CF33-GPC3 throughout). This GPC3 expression is shown to promote activation and tumor killing by T cells when treated with a GPC3-TCE or a population of GPC3 CAR T cells.

[0013] Described herein, inter alia, are oncolytic viruses (OV) expressing a glypican-3 (GPC3), wherein the OV expressing a GPC3 (OV-GPC3) comprises a nucleotide sequence comprising:Attorney Docket No. 40056-0091 WO1 (i) nucleotides 6,302 - 80,395 of SEQ ID NO: Al, A3, or A4 (or 8,000 - 80,300 of SEQ ID NO: Al, A3, or A4) having no more than 300 single nucleotide modifications (e.g,, modifications that do not change the amino acid sequence of the encoded proteins);

[0014] (ii) a nucleotide sequence encoding a human GPC3; and

[0015] (lii) nucleotides 82,356 - 190,102 of SEQ ID NO: Al, A3, or A4 (or 82,400 - 189,000 of SEQ ID NO: Al, A3, or A4) having no more than 300 single nucleotide modifications (e.g., modifications that do not change the amino acid sequence of the encoded proteins).

[0016] In some embodiments, the OV-GPC3 nucleotide sequence comprises:

[0017] (i) nucleotides 6,302 - 80,395 of SEQ ID NO: Al, A3, or A4 (or 8,000 - 80,300 of SEQ ID NO: Al, A3, or A4) having no more than 250, 200, 150, 100, 75, 50, or 30 single nucleotide modifications (e.g., nucleotide substitutions), wherein the modifications do not change the amino acid sequence of the encoded proteins; and

[0018] (ii ) nucleotides 82,356 - 190,102 of SEQ ID NO: Al, A3, or A4 (or 82,400 - 189,000 of SEQ ID NO: Al, A3, or A4) having no more than 250, 200, 150, 100, 75, 50, or 30 single nucleotide modifications (e.g., nucleotide substitutions), wherein the modifications do not change the ammo acid sequence of the encoded proteins.

[0019] In various embodiments: the GPC3 comprises an amino acid sequence selected from SEQ ID NOs: 1-6 and 1B-6B; the GPC3 comprises at least one or at least two amino acid substitutions selected from F41E, L66A, W260R, Y264K, L268E, M269S, Y277A, Y408D, L421E, W423A, L428R, Y432A using SEQ ID NO: 1 as a reference sequence (e.g., F41E corresponds to F17E in SEQ ID NO:1B, F41E m SEQ ID NO:4, and F17E in SEQ ID NO:5B); the GPC3 comprises at least one amino acid substitution selected from F41E, L66A, W260R, Y264K, L268E, M269S, Y277A, Y408D, L421E, W423A, L428R, Y432A using SEQ ID NO: 1 as a reference sequence (e.g., F41E corresponds to F17E in SEQ ID NO:1B, F41E in SEQ ID NO:4, and F17E in SEQ ID NO:5B); the GPC3 comprises two amino acid substitutions selected from F41E, L66A, W260R, Y264K, L268E, M269S, Y277A, Y408D, L421E, W423A, L428R, Y432A using SEQ ID NO: 1 as a reference sequence (e.g., F41E corresponds to F17E in SEQ ID NO: IB, F41E in SEQ ID NO:4, and F17E in SEQ ID NO:5B); the two amino acid substitutions are F41E and W260R; the GPC3 comprises the amino acid sequence of SEQ ID NO: 3 or 3B.

[0020] In some embodiments, the OV-GPC3 further comprises a promoter that is operably linked to the sequence encoding the human GPC3.

[0021] In some embodiments, the nucleotide sequence comprises:Attorney Docket No. 40056-0091 WO1 (i) nucleotides 6,302 - 80,395 of SEQ ID NO: Al, A3, or A4 and nucleotides 82,356 - 190,102 of SEQ ID NO: Al, A3, or A4; or

[0022] (li) nucleotides 8,000 - 80,300 of SEQ ID NO: Al, A3, or A4 and nucleotides 82,400 - 189,000 of SEQ ID NO: Al, A3, or A4; or

[0023] (lii) nucleotides 9,000 - 80,000 of SEQ ID NO: Al, A3, or A4 and nucleotides 83,000 - 187,000 of SEQ ID NO: Al, A3, or A4.

[0024] Described herein, inter alia, are methods of administering an oncolytic virus (OV) expressing a GPC3 (OV-GPC3) disclosed herein. In some embodiments, the methods further comprise administering a therapeutic agent targeted to the GPC3, for example: a bispecific T cell engager, a chimeric antigen receptor, an antibody or an antibody-drug conjugate. The GPC3 includes variants 1-4 (VI -V4, respectively), truncations thereof, and mutations thereof (e.g., one or more point mutations in the amino acid sequence of any of GPC3-V1, GPC3-V2, GPC3-V3, and GPC-V4, for example, that reduces binding to Wnt (e.g., any one or more of F41E, W260R, Y264K, and M269S in GPC3-V2)). In some embodiments, the GPC3 can include one or more mutations that severely reduce or essentially eliminate signaling activity (e.g., at amino acid F4I, W260, Y264, M269, and combinations thereof in GPC3-V2 SEQ ID NO: 1, corresponding to amino acids FT 7, W236, Y240, M245 and combinations thereof in mature GPC3-V2 (SEQ ID NO: IB).

[0025] The oncolytic viruses as described herein include a transgene encoding a human GPC3 (e.g., GPC3-V2) having the sequence (including a signal sequence):

[0026] MAGTVRTACLVVAMLLSLDFPGQAQPPPPPPDATCHQVRSFFQRLQPGLKWVPETPVPGSDLQVCLPKG PTCCSRKMEEKYQLTARLNMEQLLQSASMELKFLIIQNAAVFQEAFEIVVRHAKNYTNAMFKNNYPSLT PQAFEFVGEFFTDVSLYILGSDINVDDMVNELFDSLFPVIYTQLMNPGLPDSALDINECLRGARRDLKVFGNFPKLIMTQVSKSLQVTRIFLQALNLGIEVINTTDHLKFSKDCGRMLTRMWYCSYCQGLMMVKPCGGY CNVVMQGCMAGVVEIDKYWREYILSLEELVNGMYRIYDMENVLLGLFSTIHDSIQYVQKNAGKLTTTIG KLCAHSQQRQYRSAYYPEDLFIDKKVLKVAHVEHEETLSSRRRELIQKLKSFISFYSALPGYICSHSPVAE NDTLCWNGQELVERYSQKAARNGMKNQFNLHELKMKGPEPVVSQIIDKLKHINQLLRTMSMPKGRVL DKNLDEEGFESGDCGDDEDECIGGSGDGMIKVKNQLRFLAELAYDLDVDDAPGNSQQATPKDNEISTFH NLGNVHSPLKLLTSMAISVVCFFFLVH (SEQ ID NO: 1). The transgene can encode the mature form of human GPC3-V2 having the sequence:

[0027] QPPPPPPDATCHQVRSFFQRLQPGLKWVPEI'PVPGSDLQVCLPKGPTCCSRKMEEKYQLTARLNMEQLL QSASMELKFLIIQNAAVFQEAFEIVVRHAKNYTNAMFKNNYPSLTPQAFEFVGEFFTDVSLYILGSDINVD DMVNELFDSLFPVIYTQLMNPGLPDSALDINECLRGARRDLKVFGNFPKLIMTQVSKSLQVTRIFLQALN LGIEVINTTDHLKFSKDCGRMLTRMWYCSYCQGLMMVKPCGGYCNVVMQGCMAGVVEIDKYWREYIAttorney Docket No. 40056-0091 WO1 LSLEELVNGMYRIYDMENVLLGLFSTIHDSIQYVQKNAGKLTTTIGKLCAHSQQRQYRSAYYPEDLFIDK KVLKVAHVEHEETLSSRRRELIQKLKSF1SFYSALPGYICSHSPVAENDTLCWNGQELVERYSQKAARNG MKNQFNLHELKMKGPEPVVSQIIDKLKHINQLLRTMSMPKGRVLDKNLDEEGFESGDCGDDEDECIGGSGDGMIKVKNQLRFLAELAYDLDVDDAPGNSQQATPKDNEISTFHNLGNVHSPLKLLTSMAISVVCFFFLVH (SEQ ID NO: 1B), which can be preceded by a signal sequence that permits cell surface expression of GPC3-V2.

[0028] Described herein, inter alia, are methods of killing solid tumor cancer cells comprising: administering to the subject an effective amount of an oncolytic virus expressing a GPC3 (OV-GPC3); and optionally, administering to the subject a therapy, such as a bispecific T cell engager (TCE), antibody, or CAR T cell, that binds to GPC3 (e.g., the form of GPC3 encoded by the OV-GPC3). Also described herein are methods of treating a subject having a solid tumor comprising: administering to the subject an effective amount of an oncolytic virus expressing GPC3 (0V-GPC3); and optionally, administering to the subject a therapy (e.g., an immune cell, antibody, antibody fragment, antibodyconjugate (e.g., antibody-drug conjugate), or a bispecific T cell engager (TCE)) that binds to GPC3 (e.g., the form of GPC3 encoded by the 0V-GPC3). In some embodiments, the therapy that binds GPC3 is any one or more of: a population of immune cells expressing a GPC3-targeted scFv (e.g., GPC3 CAR T cells), a GPC3-targeted TCE, a GPC3 antibody or antibody fragment, a GPC3 antibody-drug conjugate; e.g., ERY-974, ABP-110, Codrituzumab (GC33), hYP7, HN3, Anti-Glypican 3 antibody [SP86](ab95363), ECT-204, Anti-CD3 / MUCl -armed-cytokine induced killer cells, GPC-3298306, MDX-1414, B010-A, HLX-63, LQ-102, GSK2857916, BMS-986182, and BMS-986183).

[0029] Also described herein, inter alia, are methods of treating a subject having a solid tumor and / or methods of killing solid tumor cancer cells in a subject comprising:

[0030] administering to the subject an effective amount of an oncolytic virus expressing a human GPC3 (0V-GPC3), wherein the nucleotide sequence of 0V-GPC3 comprises:

[0031] (a) a nucleotide sequence comprising at least 25, 50, 60, 70, 80, 90, 100, 110, 120, or 121 of S

[0032]

[0033] EQ ID NOs: XI, X17, X26, X27, X28, X32, X34, X36, X41, X52, X53, X65, X66, X76, X77, X79, X81, X82, X87, X90, X91, X92, X93, X104, X108, X110, X111, X116, X118, X120, X123, X128, X129, X130, X138, X140, X142, X143, X145, X146, X147, X149, X150, X151, X152, X153, X154, X155, X156, X157, X158, X160, X161, X165, X168, X169, X170, X172, X173, X174, X175, X176, X177, X178, X181, X182, X186, X192, X193, X194, X195, X197, X198, X199, X203, X206, X211, X212, X216, X219, X220, X222, X223, X224, X225, X226, X227, X228, X229, X231, X232, X233, X234, X236, X238, X240, X241,

[0034]

[0035] Attorney Docket No. 40056-0091 WO1 X243, X244, X252, X253, X254, X257, X258, X259, X260, X261, X263, X264, X265, X266, X267, X268, X269, X270, X271, X272, X274, and X275, or a variant of one or more thereof having 1, 2, 3, 4, 5, 20, 30, 40, 50, or 100 single nucleotide substitutions, wherein the nucleotide substitutions do not alter the ammo acid sequence of the encoded protein and / or do not alter the function of the encoded protein; or

[0036] a nucleotide sequence encoding at least 25, 50, 60, 70, 80, 90, 100, 110, 120, or 121 proteins having the amino acid sequences of SEQ ID NOs: Z1, Z17, Z26, Z27, Z28, Z32, Z34, Z36, Z41, Z52, Z53, Z55, Z63, Z65, Z66, Z76, Z77, Z79, Z81, Z82, Z87, Z90, Z91, Z92, Z93, Z104, Z108, Z110, Z111, Z116, Z118, Z120, Z123, Z128, Z129, Z130, Z138, Z140, Z142, Z143, Z145, Z146, Z147, Z149, Z150, Z151, Z152, Z153, Z154, Z155, Z156, Z157, Z158, Z160, Z161, Z165, Z168, Z169, Z170, Z172, Z173, Z174, Z175, Z176, Z177, Z178, Z181, Z182, Z186, Z192, Z193, Z194, Z195, Z197, Z198, Z199, Z203, Z206, Z211, Z212, Z216, Z219, Z220, Z222, Z223, Z224, Z225, Z226, Z227, Z228, Z229, Z231, Z232, Z233, Z234, Z236, Z238, Z240, Z241, Z243, Z244, Z252, Z253, Z254, Z257, Z258, Z259, Z260, Z261, Z263, Z264, Z265, Z266, Z267, Z268, Z269, Z270, Z271, Z272, Z274, and Z275, or a variant of one or more thereof having 1, 2, 3, 4, or 5 single amino acid substitutions, wherein the amino acid substitutions are conservative and / or do not alter the function of the protein; and

[0037] (b) a nucleotide sequence encoding a human GPC3 (e.g., any of SEQ ID NOs: 1-6 and 1B-6B). In some embodiments, the method further comprises administering to the subject an effective amount of therapy that binds to GPC3 (e.g., a GPC3 TCE, a population of GPC3-targeted T cells, a population of GPC3-targeted NK cells, a GPC3-targeted antibody or antibody fragment).

[0038] For example, an OV-GPC3 can comprise a nucleotide sequence encoding 50 of SEQ ID NOs: Z1, Z17, Z26, Z27, Z28, Z32, Z34, Z36, Z41, Z52, Z53, Z55, Z63, Z65, Z66, Z76, Z77, Z79, Z81, Z82, Z87, Z90, Z91, Z92, Z93, Z104, Z108, Z110, Z111, Z116, Z118, Z120, Z123, Z128, Z129, Z130, Z138, Z140, Z142, Z143, Z145, Z146, Z147, Z149, Z150, Z151, Z152, Z153, Z154, Z155, Z156, Z157, Z158, Z160, Z161, Z165, Z168, Z169, Z170, Z172, Z173, Z174, Z175, Z176, Z177, Z178, Z181, Z182, Z186, Zl 92, Z193, Z194, Z195, Z197, Z198, Z199, Z203, Z206, Z211, Z212, Z216, Z219, Z220, Z222, Z223, Z224, Z225, Z226, Z227, Z228, Z229, Z231, Z232, Z233, Z234, Z236, Z238, Z240, Z241, Z243, Z244, Z252, Z253, Z254, Z257, Z258, Z259, Z260, Z261, Z263, Z264, Z265, Z266, Z267, Z268, Z269, Z270, Z271, Z272, Z274, and Z275 and of those 50, one or more and up to all fifty can comprises 1, 2, 3, 4, or 5 single amino acid substitution that are conservative and / or do not alter the function of the protein.Attorney Docket No. 40056-0091 WO1 Another example of an 0V-GPC3 is an 0V-GPC3 comprising a nucleotide sequence encoding 50 of SEQ ID NOs: Z1, Z17, Z26, Z27, Z28, Z32, Z34, Z36, Z41, Z52, Z53, Z55, Z63, Z65, Z66, Z76, Z77, Z79, Z81, Z82, Z87, Z90, Z91, Z92, Z93, Z104, Z108, Z110, Z111, Z116, Z118, Z120, Z123, Z128, Z129, Z130, Z138, Z140, Z142, Z143, Z145, Z146, Z147, Z149, Z150, Z151, Z152, Z153, Z154, Z155, Z156, Z157, Z158, Z160, Z161, Z165, Z168, Z169, Z170, Z172, Z173, Z174, Z175, Z176, Z177, Z178, Z181, Z182, Z186, Z192, Z193, Z194, Z195, Z197, Z198, Z199, Z203, Z206, Z211, Z212, Z216, Z219, Z220, Z222, Z223, Z224, Z225, Z226, Z227, Z228, Z229, Z231, Z232, Z233, Z234, Z236, Z238, Z240, Z241, Z243, Z244, Z252, Z253, Z254, Z257, Z258, Z259, Z260, Z261, Z263, Z264, Z265, Z266, Z267, Z268, Z269, Z270, Z271, Z272, Z274, and Z275.

[0039] In another example, the OV-GPC3 comprises a nucleotide sequence comprising all 121 of SEQ ID NOs: XI, XI 7, X26, X27, X28, X32, X34, X36, X41, X52, X53, X55, X63, X65, X66, X76, X77, X79, X81, X82, X87, X90, X91, X92, X93, X104, X108, XI 10, Xlll, X116, X118, X120, X123, X128, X129, X130, X138, X140, X142, X143, X145, X146, X147, X149, XI 50, X151, X152, X153, X154, X155, X156, X157, X158, X160, X161, X165, X168, X169, X170, X172, X173, X174, X175, X176, X177, X178, X181, X182, X186, X192, X193, X194, X195, X197, X198, X199, X203, X206, X211, X212, X216, X219, X220, X222, X223, X224, X225, X226, X227, X228, X229, X231, X232, X233, X234, X236, X238, X240, X241, X243, X244, X252, X253, X254, X257, X258, X259, X260, X261, X263, X264, X265, X266, X267, X268, X269, X270, X271, X272, X274, and X275, and of those 121, one or more (and up to all 121 sequences) have 1, 2, 3, 4, 5, 20, 30, 40, 50, or 100 single nucleotide substitutions, wherein the nucleotide substitutions do not alter the amino acid sequence of the encoded protein and / or do not alter the function of the encoded protein.

[0040] Described herein, inter alia, are methods of treating a subject having a solid tumor comprising: administering to the subject an effective amount of an oncolytic virus expressing a GPC3 (OV-GPC3), the nucleotide sequence of OV-GPC3 comprising: (a) an oncolytic virus nucleotide sequence; and (b) a nucleotide sequence encoding a human GPC3. In some embodiments, the method further comprises administering to the subject an effective amount of a therapy, such as a bispecific T cell engager (TCE) or population of immune cells expressing a CAR, that binds to GPC3.

[0041] In various embodiments of any of the methods or compositions described herein: OV-GPC3 does not encode functional thymidine kinase; the OV-GPC3 comprises a nucleotide sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO: Al over the entire length of SEQ ID NO: Al; the OV-GPC3 comprises a nucleotide sequence that is at least 90%, 95%,Attorney Docket No. 40056-0091 WO1 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO: Al, but lacks the 5’ ITR sequence and the 3’ ITR sequence of SEQ ID NO: Al (i.e,, lacks nucleotides 1 - 6,301 and 190,103 -196,397 of SEQ ID NO: Al; i.e., nucleotides 6,302-190,102 of SEQ ID NO: Al); the OV-GPC3 comprises a nucleotide sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO: A3 over the entire length of SEQ ID NO: A3; the OV-GPC3 comprises a nucleotide sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99,9%, or 100% identical to SEQ ID NO: A3, but lacks the 5’ ITR sequence and the 3’ ITR sequence of SEQ ID NO: A3 (i.e., lacks nucleotides 1 - 6,301 and 190,103 - 196,397 of SEQ ID NO: A3; i.e., nucleotides 6,302-190,102 of SEQ ID NO: A3); the OV-GPC3 comprises a nucleotide sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO: A4 over the entire length of SEQ ID NO: A4; the OV-GPC3 comprises a nucleotide sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO: A4, but lacks the 5’ ITR sequence and the 3’ ITR sequence of SEQ ID NO: A4 (i.e., lacks nucleotides 1 - 6,301 and 190,103 - 196,397 of SEQ ID NO: A4; i.e., nucleotides 6,302-190,102 of SEQ ID NO: A4); the OV-GPC3 comprises a nucleotide sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO: A5 over the entire length of SEQ ID NO: A5; the OV-GPC3 comprises a nucleotide sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO: A5, but lacks the 5’ ITR sequence and the 3’ ITR sequence of SEQ ID NO: A5 (i.e., lacks nucleotides 1 - 6,301 and 191,595 - 198,422 of SEQ ID NO: A5; i.e., nucleotides 6,302-191,421 of SEQ ID NO: A5); the oncolytic virus nucleotide sequence comprises a nucleotide sequence that is at 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100% identical to: a) SEQ ID NO: A2 over the entire length of SEQ ID NO: A2 without the JR2 sequence of SEQ ID NO: A2 (i.e., as identified in FIG. 35; (i.e., not including nucleotides 77682-78084 of SIQ ID NO: A2); or b) SEQ ID NO: A2 over the entire length of SEQ ID NO: A2 but not including the JR2 gene sequence, the 5’ ITR sequence and the 3’ ITR sequence of SEQ ID NO: A2 (i.e., not including nucleotides 77682-78084, nucleotides 1-4054, and nucleotides 185351-189404 of SEQ ID NO: A2); the oncolytic virus nucleotide sequence comprises at least 25, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140 or 150 of the ORF in Table 4 and encodes at least 25, 50, 60, 70, 80, 90, 100, 110, 120, or 121 of SEQ ID NOs: Z1, Z17, Z26, Z27, Z28, Z32, Z34, Z36, Z41, Z52, Z53, Z55, Z63, Z65, Z66, Z76, Z77, Z79, Z81, Z82, Z87, Z90, Z91, Z92, Z93, Z104, Z108, Z110, Z111, Z116, Z118, Z120, Z123, Z128, Z129, Z130, Z138, Z140, Z142, Z143, Z145, Z146, Z147, Z149, Z150, Z151, Z152, Z153, Z154, Z155, Z156, Z157, Z158, Z160, Z161, Z165, Z168, Z169, Z170, Z172, Z173, Z174, Z175, Z176, Z177, Z178, Z181, Z182, Z186, Z192, Z193, Z194, Z195, Z197, Z198, Z199, Z203, Z206,Attorney Docket No. 40056-0091 WO1 Z211, Z212, Z216, Z219, Z220, Z222, Z223, Z224, Z225, Z226, Z227, Z228, Z229, Z231, Z232, Z233, Z234, Z236, Z238, Z240, Z241, Z243, Z244, Z252, Z253, Z254, Z257, Z258, Z259, Z260, Z261, Z263, Z264, Z265, Z266, Z267, Z268, Z269, Z270, Z271, Z272, Z274, and Z275; the oncolytic virus nucleotide sequence has no modifications in the regions encoding at least 25, 50, 60, 70, 80, 90, 100, 110, 120, or 121 of SEQ ID NOs: Z1, Z17, Z26, Z27, Z28, Z32, Z34, Z36, Z41, Z52, Z53, Z55, Z63, Z65, Z66, Z76, Z77, Z79, Z81, Z82, Z87, Z90, Z91, Z92, Z93, Z104, Z108, Z110, Z111, Z116, Z118, Z120, Z123, Z128, Z129, Z130, Z138, Z140, Z142, Z143, Z145, Z146, Z147, Z149, Z150, Z151, Z152, Z153, Z154, Z155, Z156, Z157, Z158, Z160, Z161, Z165, Z168, Z169, Z170, Z172, Z173, Z174, Z175, Z176, Z177, Z178, Z181, Z182, Z186, Z192, Z193, Z194, Z195, Z197, Z198, Z199, Z203, Z206, Z211, Z212, Z216, Z219, Z220, Z222, Z223, Z224, Z225, Z226, Z227, Z228, Z229, Z231, Z232, Z233, Z234, Z236, Z238, Z240, Z241, Z243, Z244, Z252, Z253, Z254, Z257, Z258, Z259, Z260, Z261, Z263, Z264, Z265, Z266, Z267, Z268, Z269, Z270, Z271, Z272, Z274, and Z275, or all of the open reading frame (ORF) m Table 4; the oncolytic virus nucleotide sequence comprises modifications within the regions encoding at least 25, 50, 60, 70, 80, 90, 100, 110, 120, or 121 of SEQ ID NO: Z1, Z17, Z26, Z27, Z28, Z32, Z34, Z36, Z41, Z52, Z53, Z55, Z63, Z65, Z66, Z76, Z77, Z79, Z81, Z82, Z87, Z90, Z91, Z92, Z93, Z104, Z108, Z110, Z111, Z116, Z118, Z120, Z123, Z128, Z129, Z130, Z138, Z140, Z142, Z143, Z145, Z146, Z147, Z149, Z150, Z151, Z152, Z153, Z154, Z155, Z156, Z157, Z158, Z160, Z161, Z165, Z168, Z169, Z170, Z172, Z173, Z174, Z175, Z176, Z177, Zl 78. Z181, Z182, Z186, Z192, Z193, Z194, Z195, Z197, Z198, Z199, Z203, Z206, Z211, Z212, Z216, Z219, Z220, Z222, Z223, Z224, Z225, Z226, Z227, Z228, Z229, Z231, Z232, Z233, Z234, Z236, Z238, Z240, Z241, Z243, Z244, Z252, Z253, Z254, Z257, Z258, Z259, Z260, Z261, Z263, Z264, Z265, Z266, Z267, Z268, Z269, Z270, Z271, Z272, Z274, and Z275, or any ORF identified in Table 4, that do not change the amino acid sequence of the encoded protein; the oncolytic virus nucleotide sequence comprises the regions encoding SEQ ID NOs: Z1, Z17, Z26, Z27, Z28, Z32, Z34, Z36, Z41, Z52, Z53, Z55, Z63, Z65, Z66, Z76, Z77, Z79, Z81, Z82, Z87, Z90, Z91, Z92, Z93, Z104, Z108, Z110, Z111, Z116, Z118, Z120, Z123, Z128, Z129, Z130, Z138, Z140, Z142, Z143, Z145, Z146, Z147, Z149, Z150, Z151, Z152, Z153, Z154, Z155, Z156, Z157, Z158, Z160, Z161, Z165, Z168, Z169, Z170, Z172, Z173, Z174, Z175, Z176, Z177, Z178, Z181, Z182, Z186, Z192, Z193, Z194, Z195, Z197, Z198, Z199, Z203, Z206, Z211, Z212, Z216, Z219, Z220, Z222, Z223, Z224, Z225, Z226, Z227, Z228, Z229, Z231, Z232, Z233, Z234, Z236, Z238, Z240, Z241, Z243, Z244, Z252, Z253, Z254, Z257, Z258, Z259, Z260, Z261, Z263, Z264, Z265, Z266, Z267, Z268, Z269, Z270, Z271, Z272, Z274, and Z275 and at least 130, 140, 150, 160, 170, 180, 190, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, or 275 of the ORF in Table 4; the oncolytic virus nucleotideAttorney Docket No. 40056-0091 WO1 sequence comprises at least 25, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140 or 150 of the ORF in Table 4 or all of the ORF in Table 4; the oncolytic virus nucleotide sequence comprises a nucleotide sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99,99%, or 100% identical to: SEQ ID NO: A2 over the entire length of SEQ ID NO: A2, excluding all or a portion of the JR2 gene sequence (nt 77,603-78-137 of SEQ ID NO: A2) and excluding the ITR sequences, and comprises nucleotide sequences that encode at least 25, 50, 60, 70, 80, 90, 100, 110, 120, or 121 of SEQ ID NOs: Z1, Z17, Z26, Z27, Z28, Z32, Z34, Z36, Z41, Z52, Z53, Z55, Z63, Z65, Z66, Z76, Z77, Z79, Z81, Z82, Z87, Z90, Z91, Z92, Z93, Z104, Z108, Z110, Z111, Z116, Z118, Z120, Z123, Z128, Z129, Z130, Z138, Z140, Z142, Z143, Z145, Z146, Z147, Z149, Z150, Z151, Z152, Z153, Z154, Z155, Z156, Z157, Z158, Z160, Z161, Z165, Z168, Z169, Z170, Z172, Z173, Z174, Z175, Z176, Z177, Z178, Z181, Z182, Z186, Z192, Z193, Z194, Z195, Z197, Z198, Z199, Z203, Z206, Z211, Z212, Z216, Z219, Z220, Z222, Z223, Z224, Z225, Z226, Z227, Z228, Z229, Z231, Z232, Z233, Z234, Z236, Z238, Z240, Z241, Z243, Z244, Z252, Z253, Z254, Z257, Z258, Z259, Z260, Z261, Z263, Z264, Z265, Z266, Z267, Z268, Z269, Z270, Z271, Z272, Z274, and Z275, or a variant of one or more thereof having 1, 2, 3, 4, or 5 single amino acid substitutions. In some embodiments, the single amino acid substitutions are conservative and / or do not alter the function of the protein.

[0042] In various embodiments of any of the methods or compositions described herein: the OV-GPC3 comprises a nucleotide sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99% or 100% identical to: SEQ ID NO: Al over the entire length of SEQ ID NO: Al, and encodes at least 25, 50, 60, 70, 80, 90, 100, 110, 120, or 121 proteins having the amino acid sequences of SEQ ID NO: Z1, Z17, Z26, Z27, Z28, Z32, Z34, Z36, Z41, Z52, Z53, Z55, Z63, Z65, Z66, Z76, Z77, Z79, Z81, Z82, Z87, Z90, Z91, Z92, Z93, Z104, Z108, Z110, Z111, Z116, Z118, Z120, Z123, Z128, Z129, Z130, Z138, Z140, Z142, Z143, Z145, Z146, Z147, Z149, Z150, Z151, Z152, Z153, Z154, Z155, Z156, Z157, Z158, Z160, Z161, Z165, Z168, Z169, Z170, Z172, Z173, Z174, Z175, Z176, Z177, Z178, Z181, Z182, Z186, Z192, Z193, Z194, Z195, Z197, Z198, Z199, Z203, Z206, Z211, Z212, Z216, Z219, Z220, Z222, Z223, Z224, Z225, Z226, Z227, Z228, Z229, Z231, Z232, Z233, Z234, Z236, Z238, Z240, Z241, Z243, Z244, Z252, Z253, Z254, Z257, Z258, Z259, Z260, Z261, Z263, Z264, Z265, Z266, Z267, Z268, Z269, Z270, Z271, Z272, Z274, and Z275, or a variant of one or more thereof having 1, 2, 3, 4, or 5 single amino acid substitutions; the single amino acid substitutions are conservative and / or do not alter the function of the protein; the OV-GPC3 comprises a nucleotide sequence: a) that is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100% identical to: SEQ ID NO: Al over the entire length of SEQ ID NO: Al, but lacks the 5’ ITR sequence and the 3’ ITR sequence of SEQ ID NO: Al; and b)Attorney Docket No. 40056-0091 WO1 encodes at least 25, 50, 60, 70, 80, 90, 100, 110, 120, or 121 proteins having the amino acid sequences of SEQ ID NO: Z1, Z17, Z26, Z27, Z28, Z32, Z34, Z36, Z41, Z52, Z53, Z55, Z63, Z65, Z66, Z76, Z77, Z79, Z81, Z82, Z87, Z90, Z91, Z92, Z93, Z104, Z108, Z110, Z111, Z116, Z118, Z120, Z123, Z128, ZA 29, Z130, Z138, Z140, Z142, Z143, Z145, Z146, Z147, Z149, Z150, Z151, Z152, ZA 53, Z154, Z155, Z156, Z157, Z158, Z160, Z161, Z165, Z168, Z169, Z170, Z172, Z173, Z174, Z175, Z176, Z177, Z178, Z181, Z182, Z186, Z192, Z193, Z194, Z195, Z197, Z198, Z199, Z203, Z206, Z211, Z212, Z216, Z219, Z220, Z222, Z223, Z224, Z225, Z226, Z227, Z228, Z229, Z231, Z232, Z233, Z234, Z236, Z238, Z240, Z241, Z243, Z244, Z252, Z253, Z254, Z257, Z258, Z259, Z260, Z261, Z263, Z264, Z265, Z266, Z267, Z268, Z269, Z270, Z271, Z272, Z274, and Z275, or a variant of one or more thereof having 1, 2, 3, 4, or 5 single amino acid substitutions; the amino acid substitutions are conservative and / or do not alter the function of the protein; the oncolytic virus sequence is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to nucleotides 6,302 - 190,102 of SEQ ID NO: A1 over the entire length of nucleotides 6,302 - 190,102 of SEQ ID NO: A1; the oncolytic virus sequence is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to nucleotides 7,000-188,000 of SEQ ID NO: Al over the entire length of nucleotides 7,000-188,000 SEQ ID NO: Al; the oncolytic virus sequence is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to nucleotides 8,000-187,000 of SEQ ID NO: Al over the entire length of nucleotides 8,000-187,000 SEQ ID NO: Al; the oncolytic virus sequence is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to nucleotides 10,000-185,000 of SEQ ID NO: A1 over the entire length of nucleotides 10,000-185,000 SEQ ID NO: Al; the oncolytic virus sequence is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to nucleotides 10,001-184,901 of SEQ ID NO: Al over the entire length of the oncolytic virus sequence is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to nucleotides 10,001-184,901 of SEQ ID NO: Al; the oncolytic virus sequence is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to nucleotides 6,302 - 190,102 of SEQ ID NO: A3 over the entire length of nucleotides 6,302 - 190,102 of SEQ ID NO: A3; the oncolytic virus sequence is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to nucleotides 7,000-188,000 of SEQ ID N0: A3 over the entire length of nucleotides 7,000-188,000 SEQ ID NO: A3; the oncolytic virus sequence is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99,6%, 99.7%, 99.8%, 99,9%, 99,99%, or 100% identical to nucleotides 8,000-187,000 of SEQ ID N0: A3 over the entire length of nucleotides 8,000-187,000 SEQ ID NO: A3; the oncolytic virusAttorney Docket No. 40056-0091 WO1 sequence is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to nucleotides 10,000-185,000 of SEQ ID NO: A3 over the entire length of nucleotides 10,000-185,000 SEQ ID NO: A3; the oncolytic virus sequence is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99,6%, 99.7%, 99.8%, 99,9%, 99,99%, or 100% identical to nucleotides 10,001-184,901 of SEQ ID NO: A3 over the entire length of the oncolytic virus sequence is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99,6%, 99.7%, 99.8%, 99.9%, 99,99%, or 100% identical to nucleotides 10,001-184,901 of SEQ ID NO: A3; the oncolytic virus sequence is at. least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to nucleotides 6,302 - 190,102 of SEQ ID NO: A4 over the entire length of nucleotides 6,302 - 190,102 of SEQ ID NO: A4; the oncolytic virus sequence is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to nucleotides 7,000-188,000 of SEQ ID NO: A4 over the entire length of nucleotides 7,000-188,000 SEQ ID NO: A4; the oncolytic virus sequence is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to nucleotides 8,000-187,000 of SEQ ID NO: A4 over the entire length of nucleotides 8,000-187,000 SEQ ID NO: A4; the oncolytic virus sequence is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to nucleotides 10,000-185,000 of SEQ ID NO: A4 over the entire length of nucleotides 10,000-185,000 SEQ ID NO: A4; the oncolytic virus sequence is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to nucleotides 10,001-184,901 of SEQ ID NO: A4 over the entire length of the oncolytic virus sequence is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to nucleotides 10,001-184,901 of SEQ ID NO: A4; the oncolytic virus sequence is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to nucleotides 6,302 - 191,594 of SEQ ID NO: A5 over the entire length of nucleotides 6,302 - 191,594 of SEQ ID NO: A5; the oncolytic virus sequence is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to nucleotides 7,000-190,000 of SEQ ID NO: A5 over the entire length of nucleotides 7,000-190,000 SEQ ID NO: A5; the oncolytic virus sequence is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to nucleotides 8,000-187,000 of SEQ ID NO: A5 over the entire length of nucleotides 8,000-187,000 SEQ ID NO: A5; the oncolytic virus sequence is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to nucleotides 10,000-185,000 of SEQ ID NO: A5 over the entire length of nucleotides 10,000-185,000 SEQ ID NO: A5; the oncolytic virus sequence is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to nucleotides 10,001-184,901 ofAttorney Docket No. 40056-0091 WO1 SEQ ID NO: A5 over the entire length of the oncolytic virus sequence is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99,6%, 99.7%, 99.8%, 99.9%, 99,99%, or 100% identical to nucleotides 10,001-184,901 of SEQ ID NO: A5; the OV-GPC3 is at. least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100% identical to: SEQ ID NO: A2 over the entire length of SEQ ID NO: A2 except that the nucleotide sequence encoding GPC3 and a promoter sequence for expressing GPC3 replaces at least 10 contiguous nucleotides of the JR2 gene sequence; and at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 250, 275, 400, 425, 450, 475, or 500 nucleotides (nts) of the J2R gene sequence have been deleted.

[0043] In various embodiments of any of the methods or compositions described herein: the nucleotide sequence encoding GPC3 is inserted into a noncoding region of SEQ ID NO: Al, A2, A3, A4, or A5; the nucleotide sequence encoding GPC3 encodes any of GPC3-V1, GPC3-V2, GPC3-V3, GPC3-V4 (e.g., nucleotide sequence of any one of NM__001164617; NM_004484; NM__001164618; NM __001164619, SEQ ID NOs:l-6, and SEQ ID NOs: 1B-6B); the nucleotide sequence encoding the GPC3 comprises one or two point mutations that decrease or eliminate GPC3 binding to Wnt (e.g., selected from: F41E, L66A, W260R, Y264K, L268E, M269S, Y277A, Y408D, L421E, W423A, L428R, Y432Ain GPC3-V2); the nucleotide sequence encoding GPC3 does not encode the entirety of the GPC3 (e.g., is a truncated GPC3, GPC3t); the GPC3 comprises or consists of an amino acid sequence identical to SEQ ID NO: 1 (or a variant thereof having 1, 2, 3, 4, or 5 single amino acid modifications (e.g., conservative amino acid substitutions, one or more substitutions selected from: F41E, L66A, W260R, Y264K, L268E, M269S, Y277A, Y408D, L421E, W423A, L428R, Y432Am GPC3-V2)); the GPC3 comprises or consists of an amino acid sequence identical to SEQ ID NO: 2, or a variant thereof having 1, 2, 3, 4, or 5 single amino acid modifications (e.g., conservative amino acid substitutions, one or more substitutions selected from: F41E, L66A, W260R, Y264K, L268E, M269S, Y277A, Y408D, L421E, W423A, L428R, Y432A in GPC3-V2); the GPC3 comprises or consists of an amino acid sequence identical to SEQ ID NO: 3, or a variant thereof having 1, 2, 3, 4, or 5 single amino acid modifications (e.g., conservative amino acid substitutions, one or more substitutions selected from: F41E, L66A, W260R, Y264K, L268E, M269S, Y277A, Y408D, L421E, W423A, L428R, Y432A in GPC3-V2); the GPC3 comprises or consists of an amino acid sequence identical to SEQ ID NO: 4, or a variant thereof having 1, 2, 3, 4, or 5 single amino acid modifications (e.g., conservative ammo acid substitutions, one or more substitutions selected from: F41E, L66A, W260R, Y264K, L268E, M269S, Y277A, Y408D, L421E, W423 A, L428R, Y432A in GPC3-V2); the GPC3 comprises or consists of an amino acid sequence identical to SEQ ID NO: 1B (or a variant thereof having 1, 2, 3, 4, or 5 single amino acidAttorney Docket No. 40056-0091 WO1 modifications (e.g., conservative amino acid substitutions, one or more substitutions selected from: F41E, L66A, W260R, Y264K, L268E, M269S, Y277A, Y408D, L421E, W423A, L428R, Y432A in GPC3-V2); the GPC3 comprises or consists of an amino acid sequence identical to SEQ ID NO: 2B, or a variant thereof having 1, 2, 3, 4, or 5 single amino acid modifications (e.g., conservative amino acid substitutions, one or more substitutions selected from: F41E, L66A, W260R, Y264K, L268E, M269S, Y277A, Y408D, L421E, W423A, L428R, Y432A in GPC3-V2); the GPC3 comprises or consists of an amino acid sequence identical to SEQ ID NO: 3B, or a variant thereof having 1, 2, 3, 4, or 5 single amino acid modifications (e.g., conservative amino acid substitutions, one or more substitutions selected from: F41E, L66A, W260R, Y264K, L268E, M269S, Y277A, Y408D, L421E, W423A, L428R, Y432A in GPC3-V2); the GPC3 comprises or consists of an amino acid sequence identical to SEQ ID NO: 4B, or a variant thereof having 1, 2, 3, 4, or 5 single amino acid modifications (e.g., conservative amino acid substitutions, one or more substitutions selected from: F41E, L66A, W260R, Y264K, L268E, M269S, Y277A, Y408D, L421E, W423 A, L428R, Y432A in GPC3-V2); the nucleotide sequence encoding GPC3 is operably linked to a promoter, e.g., a synthetic early promoter.

[0044] In various embodiments of any of the methods described herein: the therapy binds the GPC3 encoded by the OV-GPC3; the therapy comprises SEQ ID NO: 11; the therapy comprises a domain that binds GPC3 (e.g., an scFv targeted to GPC3); the therapy comprises a TCE (also called BiTE) comprising a domain that binds GPC3 and a domain that binds CD3; the domain that binds GPC3 is a GPC3 -targeted scFv; the domain that binds CD3 is a CD3 -targeted scFv; the TCE comprises SEQ ID NO: 11; the therapy comprises a population of GPC3-targeted immune cells (e.g., GPC3 CAR immune cells); the GPC3 CAR immune cells are GPC3 CAR T cells; the GPC3-targeted immune cells (e.g., GPC3 CAR T cells) comprise a domain that binds GPC3 (e.g., a GPC3-binding domain selected from Table Al or A2, or otherwise described herein); the domain that binds GPC3 is a GPC3-targeted scFv; the therapy comprises any one or more of a population of immune cells expressing a GPC3 -targeted scFv (e.g., GPC3 CAR T cells), a GPC3-targeted TCE, a GPC3 antibody or antibody fragment, and a GPC3 antibody-drug conjugate; the therapy comprises any one or more of ERY-974, ABP-110, SEQ ID NO: 11, Codrituzumab (GC33), CAR T cells expressing a CAR that binds to GPC3 (e.g., SEQ ID NOs: DI and D2), hYP7, HN3, Anti-Glypican 3 antibody [SP86](ab95363), ECT-204, Anti-CD3 / MUCl- armed -cytokine induced killer cells, GPC-3298306, MDX-1414, B010-A, HLX-63, LQ-102, GSK2857916, BMS-986182, and BMS-986183.

[0045] Useful therapies that bind to the GPC3 include a population of immune cells expressing a receptor comprising a domain that binds GPC3, an antibody or antibody fragment targeted to GPC3, anAttorney Docket No. 40056-0091 WO1 antibody-drug conjugate comprising a domain that binds GPC3, a polypeptide comprising a domain that binds GPC, and a TCE (BiTE) comprising a domain that binds GPC3 and a domain that binds CD3.

[0046] In some embodiments, the domain that binds GPC3 comprises:

[0047] a VH comprising an amino acid sequence selected from SEQ ID NOs: B8, Bl 6, B26, B33, B41, B49, B55, B63, and B71, or a variant thereof having no more than 5 single amino acid substitutions, wherein the substitutions are not in the CDRs, and

[0048] a VL comprising an amino acid sequence selected from SEQ ID NOs: B4, B12, B20, B27, B37, B45, B53, B59, B67, and B75, or a variant thereof having no more than 5 single amino acid substitutions, wherein the substitutions are not in the CDRs.

[0049] In some embodiments, the domain that binds GPC3 (e.g., a GPC3-targeted scFv) comprises any one of the following:

[0050] a variable light chain region having CDR1, CDR2, and CDR3 regions comprising SEQ ID NOs: BI-B3 and a variable heavy chain region having CDR1, CDR2, and CDR3 regions comprising SEQ ID NOs: B5-B7;

[0051] a variable light chain region having CDR1, CDR2, and CDR3 regions comprising SEQ ID NOs: B9-B11 and a variable heavy chain region having CDR1, CDR2, and CDR3 regions comprising SEQ ID NOs: B13-B15;

[0052] a single domain having CDR1, CDR2, and CDR3 comprising SEQ ID NOs: B17-B19;

[0053] a single domain having CDR1, CDR2, and CDR3 comprising SEQ ID NOs: B21-B23;

[0054] a variable light chain region having CDR1, CDR2, and CDR3 regions comprising SEQ ID NOs: B32-B34 and a variable heavy chain region having CDR1, CDR2, and CDR3 regions comprising SEQ ID NOs: B29-B31;

[0055] a variable light chain region having CDR1, CDR2, and CDR3 regions comprising SEQ ID NOs: B38-B40 and a variable heavy chain region having CDR1, CDR2, and CDR3 regions comprising SEQ ID NOs: B34-B36;

[0056] a variable light chain region having CDR1, CDR2, and CDR3 regions comprising SEQ ID NOs: B46-B48 and a variable heavy chain region having CDR1, CDR2, and CDR3 regions comprising SEQ ID NOs: B42-B44;

[0057] a variable light chain region having CDR1, CDR2, and CDR3 regions comprising SEQ ID NOs: B46, B54, and B48 and a variable heavy chain region having CDR1, CDR2, and CDR3 regions comprising SEQ ID NOs: B50-B52;Attorney Docket No. 40056-0091 WO1 a variable light chain region having CDR1, CDR2, and CDR3 regions comprising SEQ ID NOs: B60-B62 and a variable heavy chain region having CDR1, CDR2, and CDR3 regions comprising SEQ ID NOs: B56-B58;

[0058] a variable light chain region having CDR1, CDR2, and CDR3 regions comprising SEQ ID NOs: B68-B70 and a variable heavy chain region having CDR1, CDR2, and CDR3 regions comprising SEQ ID NOs: B64-B66; and

[0059] a variable light chain region having CDR1, CDR2, and CDR3 regions comprising SEQ ID NOs: B76-B78 and a variable heavy chain region having CDR1, CDR2, and CDR3 regions comprising SEQ ID NOs: B72-B74.

[0060] In various embodiments, the GPC3 targeting domain comprises any one of the following:

[0061] a variable light chain region comprising SEQ ID NO: B4 and a variable heavy region comprising SEQ ID NO: B8;

[0062] a variable light chain region comprising SEQ ID NO: B12 and a variable heavy region comprising SEQ ID NO: Bl 6;

[0063] a single domain comprising SEQ ID NO: 20;

[0064] an scFv comprising SEQ ID NO: B24;

[0065] a variable light chain region comprising SEQ ID NO: B27 and a variable heavy region comprising SEQ ID NO: B26;

[0066] an scFv comprising SEQ ID NO:28;

[0067] a variable light chain region comprising SEQ ID NO: B37 and a variable heavy region comprising SEQ ID NO: B33;

[0068] a variable light chain region comprising SEQ ID NO: B45 and a variable heavy region comprising SEQ ID NO: B41;

[0069] a variable light chain region comprising SEQ ID NO: B53 and a variable heavy region comprising SEQ ID NO: B49;

[0070] a variable light chain region comprising SEQ ID NO: B59 and a variable heavy region comprising SEQ ID NO: B55;

[0071] a variable light chain region comprising SEQ ID NO: B67 and a variable heavy region comprising SEQ ID NO: B63; and

[0072] a variable light chain region comprising SEQ ID NO: B75 and a variable heavy region comprising SEQ ID NO: B71.

[0073] In some embodiments, the domain that binds CD3 (e.g., a CD3-targeted scFv) comprises:Attorney Docket No. 40056-0091 WO1 a VH comprising an amino acid sequence selected from SEQ ID NOs: C8, C24, C33, C41, C49, C59, C69, and C70, or a variant thereof having no more than 5 single amino acid substitutions, wherein the substitutions are not in the CDRs, and

[0074] a VL comprising an amino acid sequence selected from SEQ ID NOs: C4, C20, C29, C37, C45, C55, C62, and C74, or a variant thereof having no more than 5 single ammo acid substitutions, wherein the substitutions are not in the CDRs.

[0075] In various embodiments: the domain that binds CD3 (e.g., the CD3 targeted scFv) comprises any¬ one of the following:

[0076] a variable light chain region having CDR1, CDR2, and CDR3 regions comprising SEQ ID NOs: C1-C3 and a variable light chain region having CDR1, CDR2, and CDR3 regions comprising SEQ ID NOs: C5-C7;

[0077] a variable light chain region having CDR1, CDR2, and CDR3 regions comprising SEQ ID NOs: C9-C11 and a variable light chain region having CDR1, CDR2, and CDR3 regions comprising SEQ ID NOs: C13-C15;

[0078] a variable light chain region having CDR1, CDR2, and CDR3 regions comprising SEQ ID NOs: C17-C19 and a variable light chain region having CDR1, CDR2, and CDR3 regions comprising SEQ ID NOs: C21-C23;

[0079] a variable light chain region comprising SEQ ID NO: C4 and a variable heavy region comprising SEQ ID NO: C8;

[0080] a variable light chain region comprising SEQ ID NO: C12 and a variable heavy region comprising SEQ ID NO: C16; and

[0081] a variable light chain region comprising SEQ ID NO: C20 and a variable heavy region comprising SEQ ID NO: C24.

[0082] In some embodiments, a useful T cell engager (TCE; also called BiTE herein) comprises a GPC3 -targeting domain and a CD3-targeting domain. In some embodiments, the GPC 3 -targeting domain comprises a VH comprising a CDRl, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: B29, B30, and B31, respectively, and a VL comprising a CDRl, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: B32, B33, and B34, respectively. In some embodiments, the CD3 -targeting domain comprises a VH comprising a CDRl, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: C71, C72, and C73, respectively, and a VL comprising a CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: C75, C76, and C77, respectively.Attorney Docket No. 40056-0091 WO1 In some embodiments T cell engager (TCE; also called BiTE herein) comprising a GPC3-targeting domain and a CD3-targeting domain, wherein the GPC3 -targeting domain comprises:

[0083] a VH comprising an amino acid sequence selected from SEQ ID NOs: B8, B16, B26, B33, B41, B49, B55, B63, and B71, or a variant thereof having no more than 5 single amino acid substitutions, wherein the substitutions are not in the CDRs, and

[0084] a VL comprising an amino acid sequence selected from SEQ ID NOs: B4, B12, B20, B27, B37, B45, B53, B59, B67, and B75, or a variant thereof having no more than 5 single amino acid substitutions, wherein the substitutions are not in the CDRs; and

[0085] wherein the CD3-targeting domain comprises:

[0086] a VII comprising an ammo acid sequence selected from SEQ ID NOs: C8, C24, C33, C41, C49, C59, C69, and C70, or a variant thereof having no more than 5 single ammo acid substitutions, wherein the substitutions are not in the CDRs, and

[0087] a VL comprising an ammo acid sequence selected from SEQ ID NOs: C4, C20, C29, C37, C45, C55, C62, and C74, or a variant thereof having no more than 5 single amino acid substitutions, wherein the substitutions are not in the CDRs.

[0088] In some embodiments, the nucleotide sequence encoding GPC3 encodes an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 1-6 and 1B-6B.

[0089] In some embodiments, the methods comprise administering to the subject, any of the oncolytic viruses described herein; and, simultaneously or subsequently, administering to the subject a therapy, such as a TCE described herein (e.g., a GPC3 TCE). In some embodiments, the therapy (e.g., GPC3 CAR T cells, GPC3 NK cells, GPC3 TCE) is administered at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 26, 17, 18, 19, or 20 days after administration of the oncolytic virus. In some embodiments, the therapy (e.g., TCE, CART cells, CARNK cells) is administered at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks after administration of the oncolytic virus. In some embodiments, the therapy (e.g., TCE, CAR T cells, CAR NK cells) is administered at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 months after administration of the oncolytic virus.

[0090] In some embodiments, the solid tumor includes any one or more of: a sarcoma (e.g., tumors in a blood vessel, bone, fat tissue, ligament, lymph vessel, muscle or tendon), a carcinoma (e.g., tumors that form in epithelial cells), adrenocortical carcinoma, non-small cell lung carcinoma, gall bladder cancer, pancreatic cancer, prostate cancer, and urinary bladder cancer, gastric cancer, bone cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, endometrial cancer, esophageal cancer, skin cancer, lungAttorney Docket No. 40056-0091 WO1 cancer, ovarian cancer, testicular cancer, thyroid cancer, etc., a metastases of one or more of these cancers, or a subpopulation of one or more of these or other cancers. In some embodiments, the solid tumor includes any cancer cell expressing GPC3.

[0091] In some embodiments, the 5’ ITR comprises or consists of nucleotides (nt) 1 - 6,301 of SEQ ID NO: A1, and the 3’ ITR comprises or consists of nucleotides (nt) 190,103-196,397 of SEQ ID NO: A1. In some embodiments, the 5’ ITR comprises or consists of nucleotides (nt) 1 - 4,054 of SEQ ID NO: A2, and the 3’ ITR comprises or consists of nucleotides (nt) 185,351-189,404 of SEQ ID NO: A2, In some embodiments, the 5’ ITR comprises or consists of nucleotides (nt) 1 - 6,301 of SEQ ID NO: A3, and the 3’ ITR comprises or consists of nucleotides (nt) 190,103-196,397 of SEQ ID NO / X3 In some embodiments, the 5’ ITR comprises or consists of nucleotides (nt) 1 - 6,301 of SEQ ID NO: A4, and the 3’ ITR comprises or consists of nucleotides (nt) 190,103-196,397 of SEQ ID NO / X4 In some embodiments, the 5’ ITR comprises or consists of nucleotides (nt) 1 - 6,301 of SEQ ID NO: A5, and the 3’ ITR comprises or consists of nucleotides (nt) 191,595-198,422 of SEQ ID NO / X5

[0092] The oncolytic viruses as described herein include a transgene encoding a human GPC3 (e.g., GPC3-V2, e.g., SEQ ID NOs:l, IB, 2, 2B, 3, and 3B).

[0093] In some embodiments, the human glypican-3 (GPC3) comprises or consists of the amino acid sequence (or a sequence at least 90%, 95%, 97%, 98% or 99% identical to) of any one of SEQ ID NOs: 1-6 and 1B-6B. In some embodiments, the GPC3 comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 1-6 and 1B-6B, or a variant thereof with 1, 2, 3, 4, or 5 single amino acid modifications.

[0094] In some embodiments, the GPC3 comprises one or two point mutation that decreases GPC3 binding to Wnt (e.g., selected from F41E, L66A, W260R, Y264K, L268E, M269S, Y277A, Y408D, L421E, W423A, L428R, Y432A in GPC3-V2, e.g., ammo acids underlined:

[0095] MAGTVRTACLVVAMLLSLDFPGQAQPPPPPPDATCHQVRSFQRLQPGLKWVPETPVPGSDLQ VCLPKGPTCCSRKMEEKYQLTARLNMEQLLQSASMELKFLIIQNAAVFQEAFEIVVRHAKNYT NAMFKNNYPSLTPQAFEFVGEFFTDVSLYILGSDINVDDMVNELFDSLFPVIYTQLMNPGLPDS ALDINECLRGARRDLKVTGNFPKLIMTQVSKSLQVTRIFLQALNLGIEVINTTDHLKFSKDCGRM LTRMWYCSYCQGLMMVKPCGGYCNVVMQGCMAGVVEIDKYWREYILSLEELVNGMYRIYD MENVTLGLFSTIHDSIQYVQKNAGKLTTTIGKLCAHSQQRQYRSAYYPEDLFIDKKVLKVAHVE HEETLSSRRRELIQKLKSFISFYSALPGYICSHSPVAENDTLCWNGQELVERYSQKAARNGMKN QFNLHELKMKGPEPVSQIIDKIKHINIQLRTMSMPKGRVLDKNLDEEGFESGDCGDDEDECIGAttorney Docket No. 40056-0091 WO1 GSGDGMIKVKNQLRFLAELAYDLDVDDAPGNSQQATPKDNEISTFHNLGNyrHSPLKLLTSMAI SVVCFFFLVH (SEQ ID NO: 1).

[0096] In some embodiments, the GPC3 comprises or consists of MAGTVRTACLVVAMLLSLDFPGQAQPPPPPPDATCHQVRSEFQRLQPGLKWVPETPVPGSDLQV CLPKGPTCCSRKMEEKYQLTARLNMEQLLQSASMELKFLIIQNAAVFQEAFEIVVRHAKNYTNA MFKNNYPSLTPQAFEFVGEFFTDVSLYILGSDINVDDMVNELFDSLFPVIYTQLMNPGLPDSALD INECLRGARRDLKVFGNFPKLIMTQVSKSLQVTRIFLQALNLGIEVINTTDHLKFSKDCGRMLTR MWYCSYCQGLMMVKPCGGYCNVVMQGCMAGVVEIDKYWREYILSLEELVNGMYRIYDMEN VLLGLFSTIHDSIQYVQKNAGKLTTTIGKLCAHSQQRQYRSAYYPEDLFIDKKVLKVAHVEHEET LSSRRRELIQKLKSFISFYSALPGYICSHSPVAENDTLCWNGQELVERYSQKAARNGMKNQFNL HELKMKGPEPVVSQIIDKLKHINQLLRTMSMPKGRVLDKNLDEEGFESGDCGDDEDECIGGSSGD GMIKVKNQLRFLAELAYDLDVDDAPGNSQQATPKDNEISTFHNLGNVHSPLKLLTSMAISVVCF FFLVH SEQ ID NO: 2 (GPC3 F41E; aa 1-24 are the signal sequence) or the mature version thereof lacking the signal sequence (SEQ ID NO: 2B). In some embodiments, the GPC3 comprises or consists of an ammo acid sequence that comprises or consists of SEQ ID NO: 2 or 2B, or a variant thereof with 1, 2, 3, 4, or 5 single amino acid modifications (e.g., conservative amino acid substitutions).

[0097] In some embodiments, the GPC3 comprises or consists of MAGTVRTACEVVAMLLSLDFPGQAQPPPPPPDATCHQVRSEFQRLQPGLKWVPETPVPGSDLQV CLPKGPTCCSRKMEEKYQLTARLNAIEQLLQSASMELKFTIIQNAAVFQEAFEIVVRHAKNYTNA MFKNNYPSLIPQAFEFVGEFFTDVSLYILGSDINVDDMVNELFDSLFPVIYTQLMNPGLPDSALD INECLRGARRDLKVTGNFPKLIMTQVSKSLQVTRIFLQALNLGIEVINTTDHLKFSKDCGRMLTR MRYCSYCQGLMMVKPCGGYCNV\A1QGCMAGVWIDKYWREYILSLEELVNGMYRIYDMEN VTLGLFSTIHDSIQYVQKNAGKLTTTIGKLCAHSQQRQYRSAYYPEDLFIDKKVLKVAHVEHEET LSSRRRELIQKLKSFISFYSALPGYICSHSPVAENDTLCWNGQELVERYSQKAARNGMKNQFNL HELKMKGPEPWSQnDKLKHINQLLRTMSMPKGRVLDKNLDEEGFESGDCGDDEDECIGGSGD GMIKVKNQLRFLAELAYDLDVDDAPGNSQQATPKDNEISTFHNLGNVHSPLKLLTSMAISWCF FFLVH SEQ ID NO: 3 (GPC3 F41E W260R; aa 1-24 are the signal sequence) or the mature version thereof lacking the signal sequence (SEQ ID NO: 3B). In some embodiments, the GPC3 comprises or consists of an amino acid sequence that comprises or consists of SEQ ID NO: 3B, or a variant thereof with 1, 2, 3, 4, or 5 single amino acid modifications (e.g., conservative amino acid substitutions).Attorney Docket No. 40056-0091 WO1 In some embodiments, the GPC3 comprises or consists of

[0098] MAGT VRTACL WAMI. LSLDFPGQ AQPPPPPPD A TCHQVR SFFQRLQPGLKWVPETPVPGSDLQ VCLPKGPTCCSRKMEEKYQLTARLNl IEQLLQSASMELKFLnQNAAVFQEAFEIVVRHAKNYT NA KNNYPSLTPQAFEFVGEFFTDVSIYn. GSDINWDMA iLFDSLFP\lYTQLMNPGI. PDS ALDINECLRGARRDLKWGNFPKLE rrQVSKSLQVTRIFLQ ALNI. GIF. V1NTTDHI. KFSKDCGRM LTRMWYCSYCQGLMMVKPCGGYCNYVMQGCMAGVVEIDKYWREYILSLEFiLVNGMYRIYD MENVT. LGLFSTIHDSIQYAfQKNAGKI. TTTETEKKIWHFKYPIFFLCIGLDLQIGKLCAHSQQRQY RS A YYPEDLFI DKKVLK VAH VEI IEETLS SRRRELIQKLKSFISF YS ALPGYI OS HSP VAENDTLC WNGQELVERYSQKAA NGMKNQFNLHELKMKGPEPVVSQIIDKLKtlINQLLRTMSMPKGRVL DKNLDEEGFESGDCGDDEDECIGGSGDGMIKVKNQLRFLAELAYDLDVDDAPGNSQQATPKD NEIS TFHNLGNVHS PLKLLTS M AIS WCFF LVII (SEQ ID NO: 4; GPC3-V1; aa 1-24 are the signal sequence) or the mature version thereof lacking the signal sequence (SEQ ID NO: 4B). In some embodiments, the GPC3 comprises or consists of an amino acid sequence that comprises or consists of SEQ ID NO: 4 or 4B, or a variant thereof with 1, 2, 3, 4, or 5 single amino acid modifications (e.g., conservative amino acid substitutions).

[0099] In some embodiments, the GPC3 comprises or consists of MAGTVRTACLVVAMLLSLDFPGQAQPPPPPPDATCHQVRSFFQRLQPGLKWVPETPVPGSDLQV CLPKGPTCCSRKMEEKYQLTARLNMEQLLQSAKAFEIVVRFLWNYTNAWKNNYPSLTPQAFE FVGEFFTOVSLYILGSDLNVDDMVNELFDSLFPVIYTQLMNPGLPDSALDINECLRGARRDLKVF GNFPKLIMTQVSKSLQVTRIFLQALNLGIEV1NTTOHLKFSKDCGRMLTRMWYCSYCQGLMMV KI’CGGYCNVVMQGCMAGVVEIDKYWREYILSLEELVNGMYRIYDMENVLLGLFSIIHDSIQYV QKNAGKLTTTIGKLCAHSQQRQYRSAYYPEDLFIDKKVLKVAHVEHEETLSSRRRELIQKLKSFI SFYSALPGYICSHSPVAENDTLCWNGQELVERYSQKAARNGMKNQFNLHELKMKGPEPVVSQII DKLKHINQLLRTMSMPKGRVLDKNLDEEGFESGDCGDDEDECIGGSGDGMIKVKNQLRFLAEL AYDLDVDDAPGNSQQATPKDNEISTFHNLGNVHSPLKLLTSMAISVVCFFFLVH

[0100] (SEQ ID NO: 5; GPC3-V3; aa 1-24 are the signal sequence) or the mature version thereof lacking the signal sequence (SEQ ID NO: 5B). In some embodiments, the GPC3 comprises or consists of an ammo acid sequence that comprises or consists of SEQ ID NO: 5 or 5B, or a variant thereof with 1, 2, 3, 4, or 5 single amino acid modifications (e.g., conservative amino acid substitutions).

[0101] In some embodiments, the GPC3 comprises or consists of MAGTVRTACLVVAMLLSLDFPGQAQPPPPPPDATCHQVRSFFQRLQPGLKWVPETPVPEAFEIV

[0102] \T? HAKNYTNAWKNNYPSLTPQAFEFVGEFFTDVSIATI. GSDINVDDMVNELFDSLFPATYTQLAttorney Docket No. 40056-0091 WO1 MNPGLPDSALDINECLRGARRDLKVFGNFPKLIMTQVSKSLQVTRIFLQALNLGIEVINTTDHLK FSKDCGRMITRNIWYCSYCQGI rVKPCGGYCNVV GCMAGVVEroKYWRE lI SLEELV NGMYRIYDMENVLLGLFSTTHDSIQYVQKNAGKLTTTIGKLCAHSQQRQYRSAYYPEDLFIDKK VIXVAHVEHEETI SSRRRELIQKLKSFISFYSALPGYICSHSPVAENDTI. CWNGQELVERYSQKA ARNG] IKNQFNI., HELKMKGPEPWSQHDKLKHINQLLRAPGNSQQATPKDNEISTFHNIGNVHS PLKLLTSMAISWCFFFLVH

[0103] (SEQ ID NO: 6; GPC3-V4; aa 1 -24 are the signal sequence) or the mature version thereof lacking the signal sequence (SEQ ID NO: 6B). In some embodiments, the GPC3 comprises or consists of an amino acid sequence that comprises or consists of SEQ ID NO: 6 or 6B, or a variant thereof with 1, 2, 3, 4, or 5 single amino acid modifications (e.g., conservative ammo acid substitutions).

[0104] In some embodiments, a useful GPC3 can be proceeded by a signaling domain and / or a promoter. For example, a useful signaling domain can comprise the ammo acid sequence MAGTVRTACLVVAMLLSLDFPG A (SEQ ID NO: A7) or MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: A8), a variant thereof, or a different signaling domain known in the art. Thus, in some embodiments, an oncolytic virus described herein comprises a sequence comprising a nucleotide sequence encoding a human GPC3 operably linked to an expression control sequence (e.g., an early promoter).

[0105] In some embodiments, the GPC3 comprises an amino acid sequence that comprises or consists of any one of SEQ ID NOs: 1-6 and 1B-6B. In some embodiments, the GPC3 comprises or consists of an amino acid sequence that comprises or consists of SEQ ID NOs: 1-6 and 1B-6B, or a variant thereof with 1, 2, 3, 4, or 5 single ammo acid modifications. In some embodiments, the ammo acid modifications are amino acid substitutions (e.g., 1, 2, 3, 4, or 5 conservative amino acid substitutions). In some embodiments, expression of the GPC3 is operably linked to a promoter, optionally wherein the promoter is a synthetic early promoter.

[0106] Sequence identity for nucleotide and amino acid sequences are calculated using BLAST 2.0 with the default parameters. The percent sequence identity refers to a global alignment between the sequences.

[0107] In some embodiments, the recombinant oncolytic viruses described herein comprises a transgene, e.g., a transgene in an expression cassette, wherein the transgene encodes all or a portion of human GPC3 (e.g., Genbank Accession Nos: NP_004475.1, NP_001158089.1, NP_001158090.1, NP_001158091.1, XP_054182809.1, and XP_016884902.1; UmProt ID Nos: P51654-1, P51654-2, and P51654-3; SEQ ID NOs: 1-4) or a variant thereof that can be recognized by an antibody that bindsAttorney Docket No. 40056-0091 WO1 GPC3. In some embodiments, the portion of GPC3 encoded by a useful transgene is or includes a portion that can be expressed on the cell surface and can be recognized by an anti-GPC3 antibody.

[0108] In some embodiments, the recombinant oncolytic viruses described herein comprise a transgene that encodes a single protein (e.g., GPC3).

[0109] In some embodiments, the recombinant oncolytic viruses described herein do not include a transgene, e.g., a transgene in an expression cassette, wherein the transgene encodes all or a portion of human BCMA (e.g., Genbank Accession No: BAB60895.1, UniProt ID Nos: Q02223-1 and Q02223-2, the extracellular portion of BCMA, a truncated BCMA, BCMAt, or a variant of one or more thereof that can be recognized by an antibody that binds BCMA). In some embodiments, the recombinant oncolytic virus does not encode a BCMA, a portion of BCMA, or any portion of BCMA that comprises or consists of a BCMA portion that can be expressed on the cell surface and can be recognized by an anti-BCM A antibody.

[0110] In some embodiments, the recombinant oncolytic viruses described herein do not include a transgene, e.g., a transgene in an expression cassette, wherein the transgene encodes all or a portion of human CD19 (e.g., UniProt ID: P15391, the extracellular portion of CD19, or a truncated CD19, CD19t). In some embodiments, the recombinant oncolytic virus does not encode a CD 19, a portion of CD 19, or any portion of CD 19 that comprises or consists of a CD 19 portion that can be expressed on the cell surface and can be recognized by an anti-CD19 antibody. For example, in some embodiments, a recombinant oncolytic virus does not comprise a transgene encoding an amino acid sequence comprising or consisting of:

[0111] EPLVVKVEEGDNAVLQCLKGTSDGPTQQLTWSRESPLKPFLKLSLGLPGLGIHMRPLAI WLFIFNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSE GPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCVPPRDSLNQSLSQDLTMAPGSTLWLSCGVPPDS VSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMETGLLLPRATAQDAGKYYCHRGNLT MSFHLEITARPA'LWHWLLRTGGWKVSAVTLAYLIFCLCSLVGILHLQRALVLRRKR (SEQ ID NO: CD19)

[0112] In some embodiments, the recombinant oncolytic viruses described herein do not comprise a sequence that encodes or expresses any one or more of an AFP, a BCMA, a BCMAt, a CA125, a CD19, a CD19t, a CD20, a CD33, a CD22, a CD123, a CD30, a CD38, a CEA, a HER2, a GD2, a PSMA, CCL5, IL-12, anti-PD-1 antibody, a Claudin 18.2, a EpCAM, a GD2, a MSLN, an EGFR, an EGFRVUI, a Trop-2, a c-MET, a Nectin-4, a CD79b, a CCK4, a GPA33, a HLA-A2, a CLEC12A, a p-cadherin, a TD02, a MART-I, a MUCI, a Pmel 17, a MAGE-I, a TRP-1, a TRP-2, a NY-ESQ, a PSA, a CDK4, aAttorney Docket No. 40056-0091 WO1 BCA225, a CA 125, a MG7-Ag, a NY-CO-I, a RCAS 1, a SDCCAG16, a TAAL6, a TAG72, and any combination thereof.

[0113] Also described herein, inter alia, are oncolytic viruses expressing a human GPC3 (OV-GPC3), the nucleotide sequence of 0V-GPC3 comprising:

[0114] (a) an oncolytic virus nucleotide sequence; and

[0115] (b) a nucleotide sequence encoding a human GPC3;

[0116] wherein the oncolytic virus nucleotide sequence encodes at least 25, 50, 60, 70, 80, 90, 100, 110, 120, or 121 of SEQ ID NO: Z1, Z17, Z26, Z27, Z28, Z32, Z34, Z36, Z41, Z52, Z53, Z55, Z63, Z65, Z66, Z76, Z77, Z79, Z81, Z82, Z87, Z90, Z91, Z92, Z93, Z104, Z108, Zl 10, ZI 11, Zl 16, Zl 18, ZI20, Z123, Z128, Z129, Z130, Z138, Z140, ZI42, Z143, Z145, Z146, Z147, Z149, Z150, Zl 51, Z152, Z153, Z154, Z155, Z156, ZI57, Z158, Z160, Z161, Z165, Z168, Z169, ZI70, Z172, Z173, Z174, Z175, ZI76, Z177, Z178, Z181, Z182, Z186, Z192, Z193, ZI94, Z195, Z197, Z198, Z199, Z203, Z206, Z211, Z212, Z216, Z219, Z220, Z222, Z223, Z224, Z225, Z226, Z227, Z228, Z229, Z231, Z232, Z233, Z234, Z236, Z238, Z240, Z241, Z243, Z244, Z252, Z253, Z254, Z257, Z258, Z259, Z260, Z261, Z263, Z264, Z265, Z266, Z267, Z268, Z269, Z270, Z271, Z272, Z274, and Z275, or a variant of one or more thereof having 1, 2, 3, 4, or 5 single ammo acid substitutions (optionally wherein the amino acid substitutions are conservative and / or do not alter the function of the protein); and / or

[0117] wherein the OV-GPC3 nucleotide sequence is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to or has no more than 300 single nucleotide changes compared to:

[0118] i) nucleotides 6,302 - 190,102 of SEQ ID NO: Al, A3, or A4 over the entire length of nucleotides 6,302 - 190,102 of SEQ ID NO: Al, A3, or A4;

[0119] ii) nt 6,302 - 191,594 of SEQ ID NO: A5 over the entire length of nt 6,302 - 191,594 SEQ ID NO: A5;

[0120] iii) nucleotides 7,000-188,000 of SEQ ID NO: Al, A3, A4, or A5 over the entire length of nucleotides 7,000-188,000 of SEQ ID NO: Al, A3, A4, or A5;

[0121] iv) nucleotides 8,000-187,000 of SEQ ID NO: Al, A3, A4, or A5 over the entire length of nucleotides 8,000-187,000 SEQ ID NO: Al, A3, A4, or A5; or

[0122] v) nucleotides 10,000-185,000 of SEQ ID NO: A1, A3, A4, or A5 over the entire length of nucleotides 10,000-185,000 SEQ ID NO: Al, A3, A4, or A5.Attorney Docket No. 40056-0091 WO1 In some embodiments of any of the compositions or methods described herein, the OV-GPC3 nucleotide sequence is at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to:

[0123] i) SEQ ID NO: Al over the entire length of SEQ ID NO: Al;

[0124] ii) nt 6,302 - 190,102 of SEQ ID NO: Al over the entire length of nt 6,302 - 190,102 SEQ ID NO: Al;

[0125] iii) nucleotides 7,000-188,000 of SEQ ID NO: A 1 over the entire length of nucleotides 7,000-188,000 SEQ ID NO: Al;

[0126] iv) nucleotides 8,000-187,000 of SEQ ID NO: Al over the entire length of nucleotides 8,000-187,000 SEQ ID NO: Al;

[0127] v) nucleotides 10,000-185,000 of SEQ ID NO: Al over the entire length of nucleotides 10,000-185,000 SEQ ID NO: Al;

[0128] vi) SEQ ID NO: A3 over the entire length of SEQ ID NO: A3;

[0129] vii) nt 6,302 - 190,102 of SEQ ID NO: A3 over the entire length of nt 6,302 - 190,102 SEQ ID NO: A3;

[0130] viii) nucleotides 7,000-188,000 of SEQ ID NO: A3 over the entire length of nucleotides 7,000-188,000 SEQ ID NO: A3;

[0131] ix) nucleotides 8,000-187,000 of SEQ ID NO: A3 over the entire length of nucleotides 8,000-187,000 SEQ ID NO: A3;

[0132] x) nucleotides 10,000-185,000 of SEQ ID NO: A3 over the entire length of nucleotides 10,000-185,000 SEQ ID NO: A3;

[0133] xi) SEQ ID NO: A4 over the entire length of SEQ ID NO: A4;

[0134] xii) nt 6,302 - 190,102 of SEQ ID NO: A4 over the entire length of nt 6,302 - 190,102 SEQ ID NO: A4;

[0135] xiii) nucleotides 7,000-188,000 of SEQ ID NO: A4 over the entire length of nucleotides 7,000-188,000 SEQ ID NO: A4;

[0136] xiv) nucleotides 8,000-187,000 of SEQ ID NO: A4 over the entire length of nucleotides 8,000-187,000 SEQ ID NO: A4;

[0137] xv) nucleotides 10,000-185,000 of SEQ ID NO: A4 over the entire length of nucleotides 10,000-185,000 SEQ ID NO: A4; or

[0138] xvi) SEQ ID NO: A5 over the entire length of SEQ ID NO: A5;Attorney Docket No. 40056-0091 WO1 xvii) nt 6,302 - 191,594 of SEQ ID NO: A5 over the entire length of nt 6,302 - 191,594 SEQ ID NO: A5;

[0139] xviii) nucleotides 7,000-188,000 of SEQ ID NO: A5 over the entire length of nucleotides 7,000-188,000 SEQ ID NO: A5;

[0140] xix) nucleotides 8,000-187,000 of SEQ ID NO: A5 over the entire length of nucleotides 8,000-187,000 SEQ ID NO: A5; or

[0141] xx) nucleotides 10,000-185,000 of SEQ ID NO: A 1 over the entire length of nucleotides 10,000-185,000 SEQ ID NO: Al.

[0142] In some embodiments of any of the compositions or methods described herein, an OV-GPC3 comprises a nucleotide sequence comprising:

[0143] (a) an oncolytic virus nucleotide sequence; and

[0144] (b) a nucleotide sequence encoding a human GPC3;

[0145] wherein the oncolytic virus nucleotide sequence encodes at least 25, 50, 60, 70, 80, 90, 100, 110, 120, or 121 of SEQ ID NO: Z1, Z17, Z26, Z27, Z28, Z32, Z34, Z36, Z41, Z52, Z53, Z55, Z63, Z65, Z66, Z76, Z77, Z79, Z81, Z82, Z87, Z90, Z91, Z92, Z93, Z104, Z108, Zl 10, ZI 11, Zl 16, Z118, Z120, Z123, Z128, Z129, Z130, Z138, Z140, Z142, Z143, Z145, Z146, Z147, Z149, Z150, Z151, Z152, Z153, Z154, Z155, Z156, Z157, Z158, Z160, Z161, Z165, Z168, Z169, Z170, Z172, Z173, Z174, Z175, Z176, Z177, Z178, Z181, Z182, Z186, Z192, Z193, Z194, Z195, Z197, Z198, Z199, Z203, Z206, Z211, Z212, Z216, Z219, Z220, Z222, Z223, Z224, Z225, Z226, Z227, Z228, Z229, Z231, Z232, Z233, Z234, Z236, Z238, Z240, Z241, Z243, Z244, Z252, Z253, Z254, Z257, Z258, Z259, Z260, Z261, Z263, Z264, Z265, Z266, Z267, Z268, Z269, Z270, Z271, Z272, Z274, and Z275; and / or

[0146] wherein the OV-GPC3 nucleotide sequence comprises any one of:

[0147] i) nucleotides 6,302 - 190,102 of SEQ ID NO: Al, A3, or A4, or a variant thereof with up to 100, 200, or 300 single nucleotide substitutions;

[0148] ii) nt 6,302 - 191,594 of SEQ ID NO: A5 or a variant thereof with up to 100, 200, or 300 single nucleotide substitutions;

[0149] iii) nucleotides 7,000-188,000 of SEQ ID NO: Al, A3, A4, or A5, or a variant thereof with up to 100, 200, or 300 single nucleotide substitutions;

[0150] iv) nucleotides 8,000-187,000 of SEQ ID NO: Al, A3, A4, or A5, or a variant thereof with up to 100, 200, or 300 single nucleotide substitutions; or

[0151] v) nucleotides 10,000-185,000 of SEQ ID NO: Al, A3, A4, or A5, or a variant thereof with up to 100, 200, or 300 single nucleotide substitutions.Attorney Docket No. 40056-0091 WO1 In some embodiments of any of the compositions or methods described herein, the OV-GPC3 nucleotide sequence comprises nucleotides 6,302 - 191,594 of SEQ ID NO: Al or a variant thereof with up to 100 single nucleotide substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotide substitutions).

[0152] In some embodiments of any of the compositions or methods described herein, the OV-GPC3 nucleotide sequence comprises nucleotides 6,302 - 191,594 of SEQ ID NO: A3 or a variant thereof with up to 100 single nucleotide substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotide substitutions).

[0153] In some embodiments of any of the compositions or methods described herein, the OV-GPC3 nucleotide sequence comprises nucleotides 6,302 - 191,594 of SEQ ID NO: A4 or a variant thereof with up to 100 single nucleotide substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotide substitutions).

[0154] In some embodiments of any of the compositions or methods described herein, the OV-GPC3 nucleotide sequence comprises nucleotides 6,302 - 191,594 of SEQ ID NO: A5 or a variant thereof with up to 100 single nucleotide substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotide substitutions).

[0155] In some embodiments of any of the compositions or methods described herein, the OV-GPC3 nucleotide sequence comprises nucleotides 7,000-188,000 of SEQ ID NO: Al, A3, A4, or A5, or a variant thereof with up to 100 single nucleotide substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,Attorney Docket No. 40056-0091WO1 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotide substitutions).

[0156] In some embodiments of any of the compositions or methods described herein, the OV-GPC3 nucleotide sequence comprises nucleotides 8,000-187,000 of SEQ ID NO: Al, A3, A4, or A5, or a variant thereof with up to 100 single nucleotide substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotide substitutions).

[0157] In some embodiments of any of the compositions or methods described herein, the OV-GPC3 nucleotide sequence comprises nucleotides 9,000-186,000 of SEQ ID NO: A1, A3, A4, or A5, or a variant thereof with up to 100 single nucleotide substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotide substitutions).

[0158] In some embodiments of any of the compositions or methods described herein, any nucleotide modifications (e.g., nucleotide substitutions, nucleotide insertions) in the oncolytic virus nucleotide sequence do not change the ammo acid sequence of encoded proteins having an amino acid sequences SEQ ID NO: Z1, Z17, Z26, Z27, Z28, Z32, Z34, Z36, Z41, Z52, Z53, Z55, Z63, Z65, Z66, Z76, Z77, Z79, Z81, Z82, Z87, Z90, Z91, Z92, Z93, Z104, Z108, Z110, Z111, Z116, Z118, Z120, Z123, Z128, Z129, Z130, Z138, Z140, Z142, Z143, Z145, Z146, Z147, Z149, Z150, Z151, Z152, Z153, Z154, Z155, Z156, Z157, Z158, Z160, Z161, Z165, Z168, Z169, Z170, Z172, Z173, Z174, Z175, Z176, Z177, Z178, Z181, Z182, Z186, Z192, Z193, Z194, Z195, Z197, Z198, Z199, Z203, Z206, Z211, Z212, Z216, Z219, Z220, Z222, Z223, Z224, Z225, Z226, Z227, Z228, Z229, Z231, Z232, Z233, Z234, Z236, Z238, Z240, Z241, Z243, Z244, Z252, Z253, Z254, Z257, Z258, Z259, Z260, Z261, Z263, Z264, Z265, Z266, Z267, Z268, Z269, Z270, Z271, Z272, Z274, and Z275. In some embodiments of any of the compositions or methods described herein, any nucleotide modifications (e.g., nucleotide substitutions, nucleotide insertions) in the oncolytic virus nucleotide sequence do not change the function of the encoded proteins having an amino acid sequences SEQ ID NO: Z1, Z17, Z26, Z27, Z28, Z32, Z34, Z36, Z41, Z52, Z53, Z55, Z63, Z65, Z66, Z76, Z77, Z79, Z81, Z82, Z87, Z90, Z91, Z92, Z93, Z104, Z108, Z110, Z111,Attorney Docket No. 40056-0091 WO1 Z116, Z118, Z120, Z123, Z128, Z129, Z130, Z138, Z140, Z142, Z143, Z145, Z146, Z147, Z149, Z150, Z151, Z152, Z153, Z154, Z155, Z156, Z157, Z158, Z160, Z161, Z165, Z168, Z169, Z170, Z172, Z173, Z174, Z175, Z176, Z177, Z178, Z181, Z182, Z186, Z192, Z193, Z194, Z195, Z197, Z198, Z199, Z203, Z206, Z211, Z212, Z216, Z219, Z220, Z222, Z223, Z224, Z225, Z226, Z227, Z228, Z229, Z231, Z232, Z233, Z234, Z236, Z238, Z240, Z241, Z243, Z244, Z252, Z253, Z254, Z257, Z258, Z259, Z260, Z261, Z263, Z264, Z265, Z266, Z267, Z268, Z269, Z270, Z271, Z272, Z274, and Z275.

[0159] In some embodiments of any of the compositions or methods described herein, the OV-GPC3 does not encode any of an AFP, a BCMA, a BCMAt, a CA125, a CD19, a CD19t, a CD20, a CD33, a CD22, a GDI 23, a CD30, a CD38, a CEA, a HERZ, a GD2, a PSMA, a Claudin 18.2, a EpCAM, a GD2, a MSLN, an EGFR, an EGFRVIII, a Trop-2, a c-MET, a Nectin-4, a CD79b, a CCK4, a GPA33, a HLA-A2, a CLEC12A, a p-cadherin, a TDO2, a MART-I, a MUCI, a Pmel 17, a MAGE-I, a TRP-1, a TRP-2, a NY-ESQ, a PSA, a CDK4, a BCA225, a CA 125, a MG7-Ag, a NY-CO-I, a RCAS 1, a SDCCAG16, a TA / XL6, and a TAG72, and optionally functional variants of one or more thereof (e.g., a functional variant that would be recognized and bound by an antibody that binds the nonvariant version).

[0160] In some embodiments of any of the compositions or methods described herein, the GPC3 comprises an amino acid sequence that comprises or consists of SEQ ID NO: 1, 2, 3, 4, 5, 6, IB, 2B, 3B, 4B, 5B, or 6B; the nucleotide sequence encoding GPC3 is operably linked to a promoter.

[0161] In some embodiments of any of the compositions or methods described herein, the OV-GPC3 comprises a nucleotide sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to nucleotides 6,302 - 190,102 of SEQ ID NO: Al over the entire length of nucleotides 6,302 - 190,102 of SEQ ID NO: Al. In some embodiments of any of the compositions or methods described herein, the OV-GPC3 comprises a nucleotide sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to nucleotides 6,302 - 190,102 of SEQ ID NO: A3 over the entire length of nucleotides 6,302 - 190,102 of SEQ ID NO: A3. In some embodiments of any of the compositions or methods described herein, the OV-GPC3 comprises a nucleotide sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to nucleotides 6,302 - 190,102 of SEQ ID NO: A4 over the entire length of nucleotides 6,302 - 190,102 of SEQ ID NO: A4. In some embodiments of any of the compositions or methods described herein, the OV -GPC3 comprises a nucleotide sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to nucleotides 6,302-191,594 of SEQ ID NO: A5 over the entire length of nucleotides 6,302 - 191,594 of SEQ ID NO: A5.Attorney Docket No. 40056-0091 WO1 In some embodiments of any of the compositions or methods described herein, an 0V-GPC3 comprises nucleotides 10,000-185,000 of SEQ ID NO: Al, A3, A4, or A5, or a variant thereof with up to 100 nucleotide modifications (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 single nucleotide substitutions).

[0162] Also disclosed herein are T cell engagers (TCE, also called BiTE) comprising a GPC3 -targeting domain and a CD3-targeting domain. In some embodiments, the GPC3 -targeting domain comprises: a VII comprising an amino acid sequence selected from SEQ ID NOs: B8, Bl 6, B26, B33, B41, B49, B55, B63, and B71, or a variant thereof having no more than 5 single amino acid substitutions, wherein the substitutions are not in the CDRs, and

[0163] a VL comprising an amino acid sequence selected from SEQ ID NOs: B4, B12, B20, B27, B37, B45, B53, B59, B67, and B75, or a variant thereof having no more than 5 single amino acid substitutions, wherein the substitutions are not in the CDRs; and

[0164] wherein the CD3 -targeting domain comprises:

[0165] a VH comprising an amino acid sequence selected from SEQ ID NOs: C8, C24, C33, C41, C49, C59, C69, and C70, or a variant thereof having no more than 5 single amino acid substitutions, wherein the substitutions are not in the CDRs, and

[0166] a VL comprising an amino acid sequence selected from SEQ ID NOs: C4, C20, C29, C37, C45, C55, C62, and C74, or a variant thereof having no more than 5 single ammo acid substitutions, wherein the substitutions are not in the CDRs.

[0167] Any of the OV-GPC3 described herein can be administered to a subject (e.g., a subject having a cancer, e.g., a solid tumor). For example, methods of treating a subject having a cancer (e.g., a solid tumor) or methods of killing cancer cells in a subject having a cancer (e.g., a solid tumor) can comprise administering to the subject an effective amount of any one of the OV-GPC3 described herein. In some embodiments, the methods further comprise administering to the subject an effective amount of a therapy that targets GPC3 (e.g., a therapy that binds GPC3, e.g., a TCE or polypeptide comprising a GPC3 -targeting domain). In some embodiments, the therapy that binds to the GPC3 comprises an amino acid sequence selected from:Attorney Docket No. 40056-0091 WO1 a variable light chain region having CDR1, CDR2, and CDR3 regions comprising SEQ ID NOs: B1-B3 and a variable heavy chain region having CDR1, CDR2, and CDR3 regions comprising SEQ ID NOs: B5-B7;

[0168] a variable light chain region having CDR1, CDR2, and CDR3 regions comprising SEQ ID NOs: B9-B11 and a variable heavy chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B13-B15;

[0169] a region having CDR1, CDR2, and CDR3 comprising SEQ ID NOs: B17-B19;

[0170] a region having CDRl, CDR2, and CDR3 comprising SEQ ID NOs: B21-B23;

[0171] a variable light chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B32-B34 and a variable heavy chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B29-B31;

[0172] a variable light chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B38-B40 and a variable heavy chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B34-B36;

[0173] a variable light chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B46-B48 and a variable heavy chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B42-B44;

[0174] a variable light chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B46, B54, and B48 and a variable heavy chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B50-B52;

[0175] a variable light chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B60-B62 and a variable heavy chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B56-B58;

[0176] a variable light chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B68-B70 and a variable heavy chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B64-B66; and

[0177] a variable light chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B76-B78 and a variable heavy chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B72-B74

[0178] In some embodiments, the therapy that binds to the GPC3 comprises any one of:Attorney Docket No. 40056-0091 WO1 a variable light chain region comprising SEQ ID NO: B4 and a variable heavy region comprising SEQ ID NO: B8;

[0179] a variable light chain region comprising SEQ ID NO: B12 and a variable heavy region comprising SEQ ID NO: Bl 6;

[0180] a single domain comprising SEQ ID NO: B20;

[0181] an scFv comprising SEQ ID NO: B24;

[0182] a variable light chain region comprising SEQ ID NO: B27 and a variable heavy region comprising SEQ ID NO: B26;

[0183] an scFv comprising SEQ ID NO:28;

[0184] a variable light chain region comprising SEQ ID NO: B37 and a variable heavy region comprising SEQ ID NO: B33;

[0185] a variable light chain region comprising SEQ ID NO: B45 and a variable heavy region comprising SEQ ID NO: B41;

[0186] a variable light chain region comprising SEQ ID NO: B53 and a variable heavy region comprising SEQ ID NO: B49;

[0187] a variable light chain region comprising SEQ ID NO: B59 and a variable heavy region comprising SEQ ID NO: B55;

[0188] a variable light chain region comprising SEQ ID NO: B67 and a variable heavy region comprising SEQ ID NO: B63; and

[0189] a variable light chain region comprising SEQ ID NO: B75 and a variable heavy region comprising SEQ ID NO: B71.

[0190] Also described herein are methods of treating a solid tumor in a subject, the method comprising administering to the subject: an effective amount any OV-GPC3 described herein, and a means for targeting GPC3; thereby treating the solid tumor.

[0191] In various embodiments of any of the methods described herein, a solid tumor may be a carcinoma, adenocarcinoma, sarcoma, melanoma, mesothelioma, blastoma; a carcinoma or adenocarcinoma may for example be a bladder, a colon, a kidney, an ovary, a prostate, a lung, an uterus, a breast, or a prostate carcinoma or adenocarcinoma; a blastoma may for example be a neuroblastoma, a glioblastoma, or a retinoblastoma; the solid tumor is selected from the group consisting of prostate cancer (e.g,, prostate adenocarcinoma), lung cancer (e.g,, squamous cellular carcinoma), breast cancer (e.g., infiltrated ductal carcinoma), ovary cancer (e.g., serous papillary carcinoma), uterus cancer (e.g,, squamous cellular carcinoma), CNS cancer and brain cancer (e.g., glioma (such as brainstem glioma andAttorney Docket No. 40056-0091 WO1 mixed gliomas), glioblastoma (also known as glioblastoma multiforme), astrocytoma, CNS lymphoma, germinoma, medulloblastoma, Schwannoma craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, and brain metastases), colon cancer (e.g., colon adenocarcinoma or colon carcinoma), colorectal cancer, rectal cancer (e.g,, rectal adenocarcinoma), cancer of the striated muscle (e.g., rhabdomyosarcoma), thyroid cancer, testicular cancer, bladder cancer (e.g., bladder carcinoma), liver cancer, kidney cancer, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, lymphoid malignancy, pancreatic cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms’ tumor, cervical cancer, seminoma, melanoma, and metastases of one or more thereof.

[0192] A “nucleotide modification” refers to a nucleotide substitution, insertion, and / or deletion in a nucleic acid sequence. A “nucleotide substitution” refers to replacement of a nucleotide at a particular position in a parent nucleic acid sequence with another nucleotide.

[0193] An “amino acid modification” refers to an amino acid substitution, insertion, and / or deletion in a protein or peptide sequence. An “amino acid substitution” refers to replacement of an amino acid at a particular position in a parent peptide or protein sequence with another amino acid. A substitution can be made to change an amino acid in the resulting protein in a non-conservative manner (i.e., by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to another grouping) or in a conservative manner (i.e., by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to the same grouping). Such a conservative change generally leads to less change in the structure and function of the resulting protein. The following are examples of various groupings of amino acids: 1) Amino acids with nonpolar R groups: Alanine, Valine, Leucine, Isoleucine, Proline, Phenylalanine, Tryptophan, Methionine: 2) Amino acids with uncharged polar R groups: Glycine, Serine, Threonine, Cysteine, Tyrosine, Asparagine, Glutamine; 3) Amino acids with charged polar R groups (negatively charged at pH 6.0): Aspartic acid, Glutamic acid; 4) Basic amino acids (positively charged at pH 6.0): Lysine, Arginine, Histidine (at pH 6,0). Another grouping may be those amino acids with phenyl groups: Phenylalanine, Tryptophan, and Tyrosine.Attomey Docket No. 40056-0091 WO1 Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0194] Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

[0195] DESCRIPTION OF DRAWINGS FIG, I, A graphic depiction of the mechanism through which CF33-GPC3 could facilitate GPC3-CAR T mediated elimination of cancer cells.

[0196] FIGS. 2A-2D. OV mediated conversion of GPC3-negative cancer cells into GPC3-positive.

[0197] Cells were infected with indicated MOIs of CF33-GPC3. GPC3 expression was analyzed using flowcytometry at 24 h (FIG, 2A) and 48 h (FIG. 2C). Quantification of GPC3+ cells (% positive) have been plotted for expression at 24 h (FIG. 2B) and at 48 h (FIG.2D).

[0198] FIGS. 3A-3B. Immunofluorescence-based detection of virus-encoded GPC3. Detection of virus-encoded GPC3 through immunofluorescence in the TNBC cells MDAMB468 (FIG.3A) and in the gpc3-negative liver cancer cells HEPG2GPC3-KO(FIG.3B). Cells were stained for GPC3 first and then stained for virus. Cells were stained for DAPI before acquiring images. Scale bar in FIG. 3A and the upper panel of FIG. 3B is 400 pm, whereas the scale bar on confocal images in the lower panel in FIG. 3B is 50 pm. Confocal images were acquired at 40x magnification. Colors: green-GPC3, red-virus, blue-nucleus.

[0199] FIGS. 4A-4E. Activation of CAR-T cells by virus-encoded GPC3. HEPG2GPC3-KOcells were either mock infected (MOI 0) or infected with CF33-GPC3 at indicated MOIs. Mock CAR or GPC3-CAR T cells were cocultured with the infected cells for 24 hours. PE-conjugated anti-CD107 antibody was added to the well at the time of adding CAR T cells. Cells were stained for surface activation markers using APC / CY7-conjugated CD69 antibody and PerCp / Cy5.5-conjugated CD137 (4-1BB). The cells were then fixed-permeabilized before staining for the intracellular activation marker IFNy. An AF488-conjugated IFNy antibody was used. Staining for CD107 (FIG.4A), CD69 (FIG.4B), CD137 (FIG. 4C) and IFNy (FIG.4D) are shown. Regular HEPG2 (highly positive for GPC3) was also co-Attorney Docket No. 40056-0091 WO1 cultured with mock CAR or GPC3-CAR T and used as positive control for each of the markers. (FIG.

[0200] 4E) Quantification of activation markers on GPC3-CAR and mock CAR cells were performed and compared.

[0201] FIGS. 5A-5B. Activation of CAR-T cells by virus-encoded GPC3 in cells. MDAMB-468 cells (FIG. 5A) and SK-HEPP1 cells (FIG. 5B) were infected with CF33-GPC3 at an MOI 0.1 and cocultured with mock CAR or GPC3-CAR at 1:1 for 24 hours. Levels of expression markers were analyzed on the CAR T cells after 24 h of co-culture.

[0202] FIGS. 6A-6C. Virus-encoded GPC3 facilitates killing of cancer cells by GPC3-CAR T cells:

[0203] Cancer cells were infected with the virus (CF33-GPC3) at indicated MOI in a 96-well round bottom plate. 6 h post-infection mock CAR or GPC3-CAR T cells were added to the wells at E: T of 1:1. The plates were incubated and cell survival was determined at 24, 48 and 72 h using a luciferase-based assay for MDAMB-468-Luc cells (FIG.6A) and SNU16-Luc cells (FIG.6B) or LDH-release assay for HEPG2GPC3’KOcells (FIG.6C). Stats: 2-Way ANOVA; *===p<0.05, **=== / K0.01, ***- <0.005, ****===^<0.001

[0204] FIGS. 7A-7C. CF33-GPC3 robustly converts GPC3-negative tumors into GPC3 in vivo in a human xenograft model. Liver cancer cell line (HEPG2GPC3-KO) was used to generate subcutaneous tumor in NSG mice. Mice were injected with virus either through I T or I. V. route. Mice were euthanized on day 3 or 7 post-treatment and tumors were harvested. Tumors were stained for GPC3 using immunohistochemical technique. (FIG. 7A) tumor section from mice treated with PBS or virus through l. T route. (FIG. 7B) tumor section from mice treated with PBS or virus through I. V. route.

[0205] (FIG. 7C) An array of 9 tumor sections from 9 different liver cancer patients was used as a positive control for GPC3 staining.

[0206] FIG. 8. Combination of CF33-GPC3 with GPC3-CAR T results in higher anti-tumor efficacy. Liver cancer cell line (HEPG2GPC3-KO) was used to generate subcutaneous tumor in NSG mice. Mice were injected with a single injection of virus (1E6 pfu / mouse) and a single injection of GPC3-CAR T or mock CAR T (5E6 cells / mouse). CAR T cells were injected 7 days after virus injection. Stats: unpaired t-test with Welch’s correction comparing 2 curves at a time. *= / ?<0.05.

[0207] FIG. 9. A graphic depiction of the CF33 constructs encoding hNIS together with full-length or truncated GPC3 variant# 1 (“VI”). The GPC3 cassette is controlled by a promoter (here, a synthetic early promoter (pSE)), The full-length cDNAfor GPC3-V1 (Variant 1) was inserted at the F14.5L locus of CF33-hNIS virus to generate CF33-hNIS-GPC3-Vl (top panel), A truncated GPC3 (GPC3t) with onlyAttorney Docket No. 40056-0091 WO1 the c-terminal domain (CTD) of GPC3, 669 bp long, was inserted at the F14.5L locus of CF33-hNIS virus to generate CF33-hNIS-GPC3t-Vl (bottom panel).

[0208] FIG. 10. Comparison of GPC3 expression in DAMB-468 cells infected with CF33-hNIS-GPC3-V1 (top panel) or CF33-hNIS-GPC3t-V1 (bottom panel), 24 hours post-infection.

[0209] FIGS. 11A-11B. Comparison of GPC3 expression (left panel) and MFI (right panel) in U251 cells infected with CF33-hNIS-GPC3-Vl (FIG. 11A) or CF33-hNIS-GPC3t-V1 (FIG. 11B), 24 h postinfection. Stats: One-Way ANOVA; *= <0.05,**= <0,01 and ***=p<0.005.

[0210] FIG. 12. Cancer cells (U251 ) were infected with the CF33-hNIS-GPC3-Vl (left panel) or CF33-hNIS-GPC3t-Vl (right panel) at MOI 1 in a 96-well round bottom plate. 6 h post-infection, mock CAR or GPC3-C / XR T cells were added to the wells at E: T of 1:1. The plates were incubated and cell survival was determined at 24 h. Stats: One-Way ANOVA; **=yp<0.01 and ***=== ><0.005.

[0211] FIGS. 13A-13B. Activation of CAR-T cells by virus-encoded GPC3. U25I cells were infected with CF33-hNIS-GPC3-Vl (FIG. 13A) or CF33-hNIS-GPC3t (FIG. 13B) at an MOI 1 and co-cultured with mock CAR (third panel) or GPC3-CAR (fourth panel) at 1: 1 for 24 hours. Cells were harvested 24 h later and stained with live / dead Zombie dye, APC-conj ugated anti-CD45, PE-conjugated anti-IFNy and APC-Cy7-conjugated anti-CD69 antibodies. IFNy as well as CD69 expression was measured on T cells (CD45+ cells).

[0212] FIG. 14. Comparing CF33-GPC3-V1 and CF33-GPC3-V2. A graphic depicting CF33 constructs that encode full length GPC3 variant 1 (VI) or variant 2 (V2) as single transgene from the J2R locus. The cartoon depicts two CF33 constructs encoding full-length GPC3 variant 1, CF33-GPC3-VI (top panel), and variant 2, CF33- -GPC3t-V2 (bottom panel). The GPC3 expression is controlled by a promoter (here, a synthetic early promoter (pSE)).

[0213] FIG. 15. Comparison of GPC3 levels in MDAMB-468 cells after infection with CF33 encoding VI or V2 of GPC3, CF33-GPC3-V1 (fourth panel) and CF33-GPC3t-V2 (fifth panel), respectively.

[0214] FIG. 16. Killing of cancer cells by the oncolytic viruses (OVs) and in combination with GPC3-CAR-T therapy. Cancer cells (MDAMB468-Luc) were infected with the viruses at indicated MOIs in a 96-well round bottom plate. 6 h post-infection mock CAR or GPC3-CAR T cells were added to the wells at E: T of 1: 1. The plates were incubated and cell survival was determined at 48 h using luciferase based assay. Stats: Two-Way ANOVA; ****=p<0.001.

[0215] FIG. 17. A graphic depiction of the CF33 constructs encoding truncated versions of GPC3-V2. The two CF33 constructs: CF33-GPC3-V2-ΔN500 (also called CF33-GPC3-AN500), encoding a GPC3 with 500 bp deleted from the N-terminal end (top panel), and CF33-GPC3-V2-ΔC500 (also calledAttorney Docket No. 40056-0091 WO1 CF33-GPC3-AC500), encoding a GPC3 with 500 bp deleted from the C -terminal end (bottom panel). The GPC3 expression is controlled by a promoter (here, a synthetic early promoter (pSE)).

[0216] FIGS. 18A-18B. Comparison of GPC3 expression from virus encoding GPC3 with C-terminal truncation, CF33-GPC3-AC500 (FIG. 18A), or GPC3 with N terminal truncation, CF33-GPC3-AN500 (FIG. 18B)

[0217] FIGS. 19A-19B. MDAMB-468 cells infected with CF33-GPC3-AN500 at MOIs 0.025 or 0.1 were incubated with mock CAR (top panels) or GPC3-C AR (bottom panels) at 1: 1. 24 h later, cells were harvested and stained for CD45, CD69, followed by permeabilization of cells and staining for IFNy. Data show detection of activation markers IFNy (FIG. 19A) and CD69 (FIG. 19B) on the T cells.

[0218] FIG.20. A graphic depiction of CF33 constructs encoding mutated versions of full length GPC3-V2. The two CF33 constructs: CF33-GPC3-F41E, encoding a GPC3 with a single point mutation F4IE (top panel), and CF33-GPC3-F41E-W260R, encoding a GPC3 with two point mutations F41E-W260R (bottom panel). GPC3 expression is controlled by a promoter (here, a synthetic early promoter (pSE)).

[0219] FIGS. 21A-21C. Comparison of GPC3 expression from CF33-GPC3-V2 and OVs encoding mutant GPC3-V2: CF33-GPC3-F41E-W260R and CF33-GPC3-F41E-W260R. GPC3 levels at 24 h (FIG. 21A) or 48 h (FIG. 21B) post-infection. Quantification of GPC3+ cells and comparison between the viruses FIG. 21C).

[0220] FIG. 22. Activation of CAR-T cells by virus-encoded wild-type or mutated GPC3.

[0221] MDAMB468 cells were either mock infected (MOI 0) or infected with CF33-GPC3-V2 (bottom row) or CF33-GPC3-F41E (top row) or CF33-GPC3-G41E-W260R (middle row) at indicated MOIs. Mock CAR (left side panels) or GPC3-CAR T cells (right side panels) were cocultured with the infected cells for 24 hours. PE-conjugated anti-CD107 antibody was added to the well at the time of adding CAR T cells. Cells were stained for surface activation markers using APC / CY7-conjugated CD69 antibody and PerCp / Cy5.5-conjugated CD137 (4-1BB). The cells were then fixed-permeabilized before staining for the intracellular activation marker IFNy. An BUV395-conjugated IFNy antibody was used.

[0222] FIGS. 23A-23D. Quantification of activation markers induced in / on CAR-T cells by virus-encoded wild-type or mutated GPC3. MDAMB-468 with CF33-GPC3 or CF33-GPC3-F41E or CF33- GPC3-G41E-W260R at MOIs 0.025 or 0.1 and co-cultured with GPC3-CAR T cells at effector Target (E: T) ratio of 1:1 for 24 hours. After the 24 incubation, cells were stained for T-cell activation markers (CD69, CD 137, CD 107 and IFNy), The CD 107 antibody was added to the cells at the start of co-culture. After harvesting the cells at 24 h, cells were first stained for activation markers present on cell surface (CD69 & CD137). Next, the cells were fixed and permeabilized before staining for intracellularAttorney Docket No. 40056-0091 WO1 activation marker, interferon-y (IFN-y). Data show percentage of CD69+T cells (FIG. 23 A), CD 107' T cells (FIG. 23B), CD137 T cells (FIG. 23C) and IFNγ+T cells (FIG.23D) on the T cells.

[0223] FIG. 24. Killing of cancer cells by the oncolytic viruses (OVs) and in combination with GPC3-CAR-T therapy. Cancer cells (MDAMB468-Luc) were infected with the viruses at indicated MOIs in a 96-well round bottom plate. 6 h post-infection mock CAR or GPC3-CAR T cells were added to the wells at E: T of 1: 1. The plates were incubated and cell survival was determined at 48 h using luciferase based assay. Stats: Two-Way ANOVA; ****=p<0.001.

[0224] FIGS. 25A-25D. Virus-encoded GPC3 facilitates killing of cancer cells by GPC3-CAR T cells: Cancer cells were infected with the virus (CF33-hNIS-GPC3) at indicated MOI in a 96-well round bottom plate. 6 h post- infection mock CAR or GPC3-CAR T cells were added to the wells at E: T of 1: 1. The plates were incubated and cell survival was determined at 24, 48 and 72 h using a luciferase-based assay for MDAMB-468-Luc cells (FIG. 25 A) and SNU16-Luc cells (FIG.25B) or LDH-release assay for HEPG2GPC3-KOcells (FIG.25C) and AsPCl cells (FIG.25D).

[0225] FIG.26. OV mediated conversion of GPC3-negative cancer cells into GPC3-positive. Sk-Hepl (human liver cancer cells), AsPCl -Luc (human pancreatic cancer cell line), MDA-MB231 and MDAMB468 (human triple negative breast cancer cell lines) were infected with indicated MOIs of CF33-GPC3-V2. GPC3 expression was analyzed at 24 h post-infection using flowcytometry. The graphs show7quantification of GPC3+ cells (% positive) at 24 h (right panel), 48 h (middle panel), and 72 hours (left panel).

[0226] FIG. 27. OV mediated conversion of GPC3-negative cancer ceils into GPC3-positive. Sk-Hepl (human liver cancer cells), AsPCl -Luc (human pancreatic cancer cell line), MDA-MB231 and MDAMB468 (human triple negative breast cancer cell lines) were infected with the virus at different MOIs of CF33-GPC3-V2. GPC3 expression was analyzed at 24 h post-infection using flowcytometry.

[0227] FIG. 28. Detection of virus-encoded GPC3 in SK-HEP-1 cells. Human liver cancer cells (SK-Hep-1) were infected with CF33ΔTK or CF33-GPC3-V2 virus at an MOI of 0.1 in a 8-well chamber slide. 24 h post-infection, cells were fixed using 4%PFA and stained with anti-CF33 virus (rabbit) and anti-GPC3 (mouse) primary antibodies followed by staining with PE-conjugated anti-rabbit secondary and Alexafluor-488 (FITC)-conjugated anti-mouse secondary. Images were acquired at 10X using fluorescent microscope. Scale bar=400 pM.

[0228] FIG. 29. Detection of virus-encoded GPC3 in MDA-MB-231 cells. Human TNBC cells (MDA-MB231) were infected with CF33ΔTK or CF33-GPC3-V2 virus at an MOI of 0.1 in a 8-well chamber slide. 24 h post-infection, cells were fixed using 4%PFA and stained with anti-CF33 virusAttorney Docket No. 40056-0091 WO1 (rabbit) and anti-GPC3 (mouse) primary antibodies followed by staining with PE-conjugated anti-rabbit secondary and Al exafluor-488 (FITC)-conjugated anti-mouse secondary. Images were acquired at 1 OX using fluorescent microscope. Scale bar=400 pM.

[0229] FIG. 30. GPC3 copy number in un-infected and CF33-GPC3 infected cells 24 h postinfection. Cells were plated m a 96-well plate and infected with mock (PBS) or CF33-GPC3-V2 (encoding variant 2 of GPC3). The data is based on two independent experiments: one with single well per sample and one with three wells per samples (samples were in triplicates). Plates were incubated overnight and the next day, cells were harvested using 10 mM ED TA and stained for GPC3 using AF488-conjugated and anti- GPC3 antibody (NBP2-47762AF488 (clone: gpc3 / 863); Novus). For quantification of GPC3 copy number, Quantum beads pre-labelled with Al exa-fluor-488 (Bangs Laboratories, Cat#488) were used according to manufacturer’s instruction and calculations were performed using manufacturer-provided template called QuickCal. BD Fortessa was used for running samples. Data presented as Mean of means plus SEM from two independent experiments as discussed above.

[0230] FIG. 31. CAR-T activation in presence of virus (in SK-Hep-1 cells, 24 h post-treatment). Cancer cells were infected with the virus (CF33-GPC3-V2) at MOI 0.1, in a 96-well round bottom plate.

[0231] 6h post-infection Mock CAR or GPC3-CAR cells were added to the wells at E: T of 1: 1. Cells were analyzed at 24 hour and activation markers on T cells were determined.

[0232] FIG. 32. GPC3-targeted CAR-T cells are activated in presence of CF33-GPC3 virus. Cancer cells (Sk-Hepl (human liver cancer cells; left graph), MDA-MB-468 (human triple negative breast cancer cells; middle graph), and AsPCl-Luc (human pancreatic cancer cells; right graph) were infected with CF33-GPC3 at MOI 0.1, in a 96-well round bottom plate. 6-h post-infection Mock CAR or GPC3- CAR cells were added to the wells at E: T of 1: 1. Cells were analyzed at 24 hours, and the relative expression of each activation marker: CD69, CD 107, and IFNy is shown in the bar graphs.

[0233] FIG. 33 provides the amino acid sequence of the GPC3xCD3 TCE (SEQ ID NO: 11).

[0234] FIG. 34 provides the nucleotide sequence of an OV-GPC3 (SEQ ID NO: Al). The sequences for the promoter and the sequence encoding human GPC3 (“GPC3”), are underlined and identified. The remnants of the J2R gene are also identified.

[0235] FIG. 35 provides the nucleotide sequence for the parent strain OV (CF33; SEQ ID NO: A2). The portion of the J2R gene that is deleted for insertion of GPC3 encoding sequence is in bold, italics and underlined. The portion of the JR2 that are not deleted flank the deleted portion and are in bold and shaded. The 5’ ITR is a nt 1-6,815 and the 3’ ITR is a nt 188,162 - 194,971Attorney Docket No. 40056-0091 WO1 FIG. 36 provides the nucleotide sequence of an 0V-GPC3 F41E (SEQ ID NO: A3; also called CF33-GPC3-F41E). The sequences for the promoter and the sequence encoding human GPC3 (“GPC3”), are underlined and identified on part 28. The remnants of the J2R gene are also identified, FIG. 37 provides the nucleotide sequence of an OV-GPC3-F41E-W260R (SEQ ID NO: A4; also called CF33-GPC3-F41E-W260R). The sequences for the promoter and the sequence encoding human GPC3 (“GPC3”), are underlined and identified on part 28, The remnants of the J2R gene are also identified.

[0236] FIG. 38 provides the nucleotide sequence of an OV-hNIS-GPC3 (SEQ ID NO: A5; also called CF33-hNIS-GPC3-Vl). The sequences for the promoter and the sequence encoding human GPC3 Variant 1 (GPC3 VI), are underlined and identified on part 15. The remnants of the F14.5L gene are also identified.

[0237] FIGS. 39A-39B. GPC3xCD3 TCE mediates killing of GPC3+ ceils. (A) GPC3 + IIEPG2 cells or T cells were incubated with Alexa-Fluor-647-labelled TCE at 1 pg / ml for 30 minutes, cells were washed and binding of TCE to these cells was analyzed by F / XCS. (B) Luciferase-encoding MDA-MB-468 cells were infected with CF33-GPC3-V2 at different MOIs (0, 0.025 and 0.1) in a 96-well plate and added freshly isolated T cells (from 2 different healthy donors) in presence or absence of 5 ug / ml TCE. Cell survival was measured 48 hours post-treatment using luciferase-based assay. Stat: 2Way-ANOVA.

[0238] FIG. 40. GPC3-encoding oncolytic virus enhance CAR-T spread within the tumors.

[0239] HEPG2-GPC3-KO cells injected in NSG mice to generate tumors. Tumors were treated I T. with CF33-GPC3 (1E6 pfu, single injection). 1 week after virus-injection, Mock CAR or GPC3-CAR T cells (from Eureka) were Injected I. T. (5E6 cells / mouse, single injection). Mice were euthanized 2 weeks after CAR-t injection (3 weeks from virus injection) at end point when the mice appeared sick. Tumors were formalin fixed, paraffin embedded and sections were stained for vaccinia (abeam, x2000 dilution), GPC3 (Genetex, 1:50), and CD3 (Ventana).

[0240] FIG. 41. Combination of CF33-GPC3-V2 with GPC3-CAR improves therapeutic efficacy when delivered systemically. The triple negative breast cancer cell line MDA-MB-231 was used to generate bilateral orthotopic tumors (2 tumors per mouse) in NSG mice. When tumors were ~50 mm3 in volume, mice were injected with 1E6 pfu of the oncolytic virus CF33-GPC3-V2 through tail vein. Mice were injected with mock CAR or GPC3-CAR T cells (5E6 cell / mouse) through tail vein on day 5 post¬ virus treatment. Tumor volume was measured twice weekly.

[0241] FIGS. 42A-42D. Heterogeneous expression of GPC3 in HCC. (FIG. 42A) Heatmap of the top 20 differentially expressed genes in TCGA-HCC shows GPC3 upregulation in HCC samples comparedAttorney Docket No. 40056-0091 WO1 to normal tissues. (FIG. 42B) GPC3 transcript levels (normalized counts) in TCGA-HCC reveal subsets of tumors with low or absent expression (n=374 for HCC and 50 for Normal). (FIG. 42C) Proteomic data from 165 paired HCC and adjacent liver samples (NCI proteomics data; PDC000198) show variable GPC3 protein abundance. Protein abundance is expressed as log2ratio of the samples compared to a reference. Statistical comparison was performed using student’s / -test (FIG. 42D) GPC3 staining on biopsy samples from 9 HCC patients (MTB80).

[0242] FIGS. 43A-43E. OV-mediated robust surface expression of GPC3 in vitro (FIG. 43A) Flow cytometric analysis of GPC3 expression in SK-Hep1 and HepG2GPC3-KOcells infected with CF33-GPC3. (FIG. 43B) GPC3 expression on cell surface of SK-Hep1 and HepG2GPC3-KOcells at indicated MOIs and time points. Data presented as mean±SEM from 3 independent experiments. (FIG. 43C) Quantification of surface GPC3 molecules using pre-labelled calibration beads. Data presented as mean±SD. (FIG. 43D) Immunofluorescence analysis confirmed GPC3 expression only in CF33-GPC3-infected SK-Hep1 cells, with viral infection evident in both CF33-GPC3 and CF33ΔJ2R infected cells. Scale bar= 400 μm. (FIG. 43E) Comparison of growth kinetics of CF33-GPC3 and CF33AJ2R. The oncolytic virus CF33- GPC3 has similar growth kinetics as the control virus CF33AJ2R in HepG2 cells. Statistical analysis was performed using 2way ANOVA; NS= not significant.

[0243] FIG. 44. OV-mediated GPC3 delivery to GPC3-negative tumors in vivo.

[0244] NSG mice bearing subcutaneous HepG2GPC3-KOtumors were treated with CF33-GPC3 via IT or IV injection (1E06 PFU / tumor for IT and 2E06 PFU / mouse for IV). Tumors harvested on days 3 and 7 were assessed by 1HC for GPC3 expression. Each tumor section is from an individual mouse (n=l for PBS and n=3 for other treatments). Scale bar=2.5 mm.

[0245] FIGS. 45A-45D. OV-delivered GPC3 activates ECT204 cells and enables them to kill cancer cells in vitro: (FIGS. 45A-45B) Activation of ECT204 by OV-delivered GPC3. HepG2GPC3’KOcells were infected with OV (CF33-GPC3) at varying MOIs and co-cultured with mock T cells or ECT204 T cells at a 1:1 ratio for 24-48 h. (FIG. 45A) T cell activation (CD 137, CD69) and activity (CD107, IFNy) were assessed by flow cytometry. (FIG. 45B) OV-infected cancer cells induced MOI- dependent activation of ECT204 cells. (FIGS. 45C-45D) Cytotoxicity assays using HepG2GPC3‘KOand SK-Hepl cells showed significantly greater killing with OV plus ECT204 T cells compared to OV alone or OV plus mock T cells in HepG2GPC3“KO(FIG. 45C) and SK-Hepl (FIG. 45D) cells at 48 hours post¬ infection. Data presented as meaniSD. Statistical comparison was performed using 2way ANOVA; *= / ?<0.05; **= <0.01; ***=p<0.005; ****=p<0.001; ns=not significant. All experiments have been repeated 3 times with similar results.Attorney Docket No. 40056-0091 WO1 FIGS. 46A-46D. Combination of IT delivered OV and ECT204 results in improved antitumor efficacy in HepG2GPC3 KOxenograft model. (FIG. 46A) NSG mice bearing subcutaneous HepG2GPC3“KOtumors were treated as depicted (n=7 mice for ECT204, n=8 for all other groups). (FIG.

[0246] 46B) Tumor volumes were measured twice weekly and average tumor volume±SEM for each group was plotted and compared. Statistical analysis was performed using 2way ANOVA comparing all treatment groups at each time point. P values on the graph indicate differences between OV + mock vs OV + ECT204. (FIG. 46C) Tumors were harvested at the end of the study and IHC was performed to visualize virus infected cells, GPC3 expression and T cells distribution. Scale bar= 250 μm. (FIG. 46D) CD3 + cells (T cells) were counted from 3 tumors / group (3 positive fields / tumor counted) and compared among the treatment groups. Statistical analysis using one-way ANOVA; **==p<0.01.

[0247] FIGS. 47A-47B. Combination of IV delivered O V and ECT204 results in improved anti¬ tumor efficacy in HepG2GPC3-KOxenograft model. (FIG. 47A) Mice (n=10 for OV + ECT204 and n=7 for all other groups) were treated as in FIG. 46A except that all treatments were given through IV route. Tumor volumes were measured twice weekly and average tumor volume-fcSEM for each group was plotted and compared. Statistical analysis was performed using 2way ANOVA comparing all treatment groups at each time point. P values on the graph indicate differences between OV 4- mock vs OV 4- ECT204; *=p<0.05. (FIG. 47B) H& E staining of tumors at study endpoint. There were minimal necrotic regions in the PBS or ECT204 treated tumors, tumors treated with OV plus mock T cells displayed increase in necrotic area whereas the tumors treated with the combination of OV and ECT204 were mostly necrotic (necrotic regions have been highlighted with blue line). Scale bar= 2.5 mm.

[0248] FIGS. 48A-48I. GPC3-targeted BiTE facilitates T cell activation and cytotoxicity against tumor cells expressing virally delivered GPC3. (FIG. 48A) Binding of Alexa-647-conjugated BiTE to GPC3 on HepG2 cells and to CD3 on primary human T cells was confirmed by flow cytometry’. (FIG. 48B) HepG2GPC3-KOcells were infected with OV (CF33-GPC3) at an MOI of 0.1 and co-cultured with human T cells (1: 1) in the presence or absence of GPC3 BiTE (at 5 pM concentration).

[0249] Representative images taken at 48 hours post-infection, show higher killing of OV -infected cancer cells by T cells in presence of BiTE. (FIG. 48C) Cytotoxicity assay was performed as in (FIG. 48B) using T cells from 3 healthy donors and cell survival was compared 48 hours post-treatment. Data presented as mean±SD. Statistical analysis using one-way ANOVA; *=p<0.05, **=p<0.01, ***=p<0.005, ****= / ?<0.001, ns= not significant. (FIGS. 48D-48E) T cells activation markers (CD137 & CD107) were analyzed after the co-culture of T cells with mock-infected or OV-infected cancer cells in presence or absence of BiTE. (FIGS. 48F-48G) T cell activation-associated cytokines (IL-2 and IFNy) wereAttorney Docket No. 40056-0091 WO1 measured in the supernatant of the co-culture, using ELISA. (FIG.48H) Twenty-four hours after co¬ culture, cells were analyzed by flowcytometry to determine proportion of GPC3+ cancer cells, BiTE treatment led to marked reduction in GPC3+ cells. (FIG.481) Pooled data from T cells of 3 independent donors, presented as meaniSEM. For FIGS.48D, 48E, 48F, 48G & 481 statistical comparison was performed using 2way ANOVA; *= <0.05, **=p<0.01, ***=p<0.005, ****= <0,001.

[0250] FIGS. 49A-49E. Codrituzumab (Cdzb) binds virus-encoded GPC3 and enhances NK cell- mediated cytotoxicity. (FIG.49 A) HepG2GPC3-KOcells were infected with CF33-GPC3 at indicated MOIs for 18 hours, incubated with fluorescently labeled Cdzb, and analyzed by flow cytometry. (FIG.

[0251] 49B) Immunofluorescence shows selective Cdzb binding to OV-infected cells. (FIG. 49C) NK cells from two healthy donors were co-cultured with OV-infected target cells in the presence or absence of Cdzb (5 |iM). Cell viability was assessed 48 h later. Cdzb significantly enhanced NK cell -mediated killing of infected cells. Statistical analysis using one-way ANOVA; *yp<005, **==p<0.01, ***::p<0.005, * * * *===p<0.001, NS:::not significant. (FIGS.49D-49E) In a separate experiment, activation of NK cells co-cultured with target cells infected with CF33-GPC3 or control virus (CF33AJ2R) was assessed by surface markers (CD107, CD69). Increased activation was observed only in the presence of CF33-GPC3, which appeared to be dependent on the dose of OV. Statistical comparison in (FIG. 49E) was performed using 2way ANOVA; *=p< Q. O5, **=p<0.01, ***=p<0.005, ****=p<0.001, ns= not significant.

[0252] DETAILED DESCRIPTION

[0253] Oncolytic Viruses and Therapies

[0254] Useful oncolytic viruses (OV) expressing a glypican-3 (GPC3), include an OV expressing a GPC3 (OV-GPC3) comprising a nucleotide sequence comprising:

[0255] (i) nucleotides 6,302 - 80,395 of SEQ ID NO: Al, A3, or A4 (or 8,000 - 80,300 of SEQ ID NO: Al, A3, or A4) having no more than 300 single nucleotide modifications (e.g., modifications that do not change the amino acid sequence of the encoded proteins);

[0256] (ii) a nucleotide sequence encoding a human GPC3; and

[0257] (in) nucleotides 82,356 - 190,102 of SEQ ID NO: Al, A3, or A4 (or 82,400 - 189,000 of SEQ ID NO: Al, A3, or A4) having no more than 300 single nucleotide modifications (e.g., modifications that do not change the amino acid sequence of the encoded proteins).Attorney Docket No. 40056-0091 WO1 In some embodiments, the OV-GPC3 nucleotide sequence comprises:

[0258] (i) nucleotides 6,302 - 80,395 of SEQ ID NO: A1, A3, or A4 (or 8,000 - 80,300 of SEQ ID NO: A1, A3, or A4) having no more than 250, 200, 150, 100, 75, 50, or 30 single nucleotide modifications (e.g., nucleotide substitutions), wherein the modifications do not change the amino acid sequence of the encoded proteins; and

[0259] (ii) nucleotides 82,356 - 190,102 of SEQ ID NO: A1, A3, or A4 (or 82,400 - 189,000 of SEQ ID NO: A1, A3, or A4) having no more than 250, 200, 150, 100, 75, 50, or 30 single nucleotide modifications (e.g., nucleotide substitutions), wherein the modifications do not change the amino acid sequence of the encoded proteins. In some embodiments, the nucleotide sequence comprises nucleotides 6,302 - 80,395 of SEQ ID NO: A1, A3, or A4 and nucleotides 82,356 - 190,102 of SEQ ID NO: A1, A3, or A4. In some embodiments, the nucleotide sequence comprises nucleotides 8,000 - 80,300 of SEQ ID NO: A1, A3, or A4 and nucleotides 82,400 - 189,000 of SEQ ID NO: A1, A3, or A4. In some embodiments, the nucleotide sequence comprises nucleotides 9,000 - 80,000 of SEQ ID NO: A1, A3, or A4 and nucleotides 83,000 - 187,000 of SEQ ID NO: A1, A3, or A4.

[0260] In some embodiments, the OV-GPC3 comprises a nucleotide sequence comprising:

[0261] (i) nucleotides 6,302 - 82,231 of SEQ ID NO: A5 (or 8,000 - 82,200 of SEQ ID NO: A5) having no more than 500, 450, 400, 350, 300, 250, 200, 150, 100, 75, 50, or 30 single nucleotide modifications (e.g., nucleotide substitutions), and optionally wherein the modifications do not change the amino acid sequence of the encoded proteins;

[0262] (ii) a nucleotide sequence encoding a human GPC3; and

[0263] (iii) nucleotides 84,381 - 191,594 of SEQ ID NO: A5 (or 82,400 - 189,000 of SEQ ID NO: A5) having no more than 300 single nucleotide modifications (e.g., nucleotide substitutions), and optionally wherein the modifications do not change the amino acid sequence of the encoded proteins.

[0264] In some embodiments, the nucleotide sequence comprises nucleotides 6,302 - 82,231 of SEQ ID NO: A5 and nucleotides 84,381 - 191,594. In some embodiments, the nucleotide sequence comprises nucleotides 8,000 - 81,300 of SEQ ID NO: A5 and nucleotides 86,000 - 189,000 of SEQ ID NO: A5. In some embodiments, the nucleotide sequence comprises nucleotides 9,000 - 80,000 of SEQ ID NO: A5 and nucleotides 87,000 - 187,000 of SEQ ID NO: A5.

[0265] In various embodiments: the GPC3 comprises an amino acid sequence selected from SEQ ID NOs: 1-6 and 1B-6B; the GPC3 comprises at least one or at least two amino acid substitutions selected from F41E, L66A, W260R, Y264K, L268E, M269S, Y277A, Y408D, L421E, W423A, L428R,Attorney Docket No. 40056-0091 WO1 Y432A using SEQ ID NO: 1 as a reference sequence (e.g., F41E corresponds to F17E in SEQ ID NO: IB, F41E in SEQ ID NO:4, and Fl 7E in SEQ ID NO:5B); the GPC3 comprises at least one amino acid substitution selected from F41E, L66A, W260R, Y264K, L268E, M269S, Y277A, Y408D, L421E, W423A, L428R, Y432A using SEQ ID NO: 1 as a reference sequence (e.g., F41E corresponds to F17E in SEQ ID NO:1B, F41E in SEQ ID NO:4, and F17E in SEQ ID NO:5B); the GPC3 comprises two amino acid substitutions selected from F41E, L66A, W260R, Y264K, L268E, M269S, Y277A, Y408D, L421E, W423A, L428R, Y432A using SEQ ID NO: 1 as a reference sequence (e.g., F41E corresponds to F17E in SEQ ID NO: IB, F41E in SEQ ID NO:4, and F17E m SEQ ID NO:5B); the two amino acid substitutions are F41E and W260R; the GPC3 comprises the amino acid sequence of SEQ ID NO: 3 or 3B.

[0266] A human GPC3 (e.g., GPC3-V2) can comprise or consist of the amino acid sequence (including a signal sequence):

[0267] MAGTVRTACLVVAMLLSLDFPGQAQPPPPPPDATCHQVRSFFQRLQPGLKWVPETPVPGSDLQVCLPKG PTCCSRKMEEKYQLTARLNMEQLLQSASMELKFLIIQNAAVFQEAFEIWRHAKNYTNAMFKNNYPSLT PQAFEFVGEFFTDVSLYILGSDINVDDMVNELFDSLFPVIYTQLMNPGLPDSALDINECLRGARRDLKVFGNFPKLIMTQVSKSLQVTRIFLQALNLGIEVINTTDHLKFSKDCGRMLTRMWYCSYCQGLMMVKPCGGY CNVVMQGCMAGVVEIDKYWREYILSLEELVNGMYRIYDMENVLLGLFSTIHDSIQYVQKNAGKLTTTIG KLCAHSQQRQYRSAYYPEDLFIDKKVLKVAHVEHEETLSSRRRELIQKLKSFISFYSALPGYICSHSPVAE NDTLCWNGQELVERYSQKAARNGMKNQFNLHELKMKGPEPVVSQIIDKLKHINQLLRTMSMPKGRVL DKNLDEEGFESGDCGDDEDECIGGSGDGMIKVKNQLRFLAELAYDLDVDDAPGNSQQATPKDNEISTFH NLGNVHSPLKLLTSMAISVVCFFFLVH (SEQ ID NO:1).

[0268] Ahuman GPC3 (e.g., GPC3-V2) can comprise or consist of the amino acid sequence (without a signal sequence):

[0269] QPPPPPPDATCHQVRSFFQRLQPGLKWVPETPVPGSDLQVCLPKGPTCCSRKMEEKYQLTARLNMEQLL QSASMELKFLIIQNAAVFQEAFEIVVRIIAKNYTNAMFKNNYPSLTPQAFEFVGEFFTDVSLYILGSDINVD DMV’NELFDSLFPVIYTQLMNPGLPDSALDINECLRGARRDLKVFGNFPKLIMTQVSKSLQVTRIFLQALN LGIEVINTTDHLKFSKDCGRMLTRMWYCSYCQGLMMVKPCGGYCNVVxMQGCMAGWEIDKYWREYI LSLEELVNGMYRIYDMENVLLGLFSTIHDSIQYVQKNAGKLTTTIGKLCAHSQQRQYRSAYYPEDLFIDK KVLKVAHVEHEETLSSRRRELIQKLKSFISFYSALPGYICSHSPVAENDTLCWNGQELVERYSQKAARNG MKNQFNLHELKMKGPEPVVSQIIDKLKHINQLLRTMSMPKGRVLDKNLDEEGFESGDCGDDEDECIGGS GDGMIKVKNQIRFLAELAYDLDVDDAPGNSQQATPKDNEISTFHNLGNVHSPLKLLTSMAISVVCFFFL VH (SEQ ID NO: IB).

[0270] In some embodiments, the OV-GPC3 further comprises a promoter that is operably linked to the sequence encoding the human GPC3Attorney Docket No. 40056-0091 WO1 In various embodiments of any of the methods or compositions described herein: the nucleotide sequence encoding GPC3 is inserted into a noncoding region of SEQ ID NO: Al, A2, A3, A4, or A5; the nucleotide sequence encoding GPC3 encodes any of GPC3-V1, GPC3-V2, GPC3-V3, GPC3-V4 (e.g., nucleotide sequence of any one of NM_001164617; NM_004484; NM_001164618; NM_001164619, SEQ ID NOs:l-6, and SEQ ID NOs: 1B-6B); the nucleotide sequence encoding the GPC3 comprises one or two point mutations that decrease or eliminate GPC3 binding to Wnt (e.g., selected from: F41E, L66A, W260R, Y264K, L268E, M269S, Y277A, Y408D, L421E, W423 A, L428R, Y432A in GPC3-V2); the nucleotide sequence encoding GPC3 does not encode the entirety of the GPC3 (e.g., is a truncated GPC3, GPC3t); the GPC3 comprises or consists of an amino acid sequence identical to SEQ ID NO: 1 (or a variant thereof having 1, 2, 3, 4, or 5 single amino acid modifications (e.g., conservative ammo acid substitutions, one or more substitutions selected from: F41E, L66A, W260R, Y264K, L268E, M269S, Y277A, Y408D, L421E, W423A, L428R, Y432Ain GPC3-V2)); the GPC3 comprises or consists of an amino acid sequence identical to SEQ ID NO: 2, or a variant thereof having 1, 2, 3, 4, or 5 single amino acid modifications (e.g., conservative ammo acid substitutions, one or more substitutions selected from: F41E, L66A, W260R, Y264K, L268E, M269S, Y277A, Y408D, L421E, W423A, L428R, Y432A in GPC3-V2); the GPC3 comprises or consists of an amino acid sequence identical to SEQ ID NO: 3, or a variant thereof having 1, 2, 3, 4, or 5 single amino acid modifications (e.g., conservative amino acid substitutions, one or more substitutions selected from: F41E, L66A, W260R, Y264K, L268E, M269S, Y277A, Y408D, L421E, W423A, L428R, Y432A in GPC3-V2); the GPC3 comprises or consists of an amino acid sequence identical to SEQ ID NO: 4, or a variant thereof having 1, 2, 3, 4, or 5 single amino acid modifications (e.g., conservative ammo acid substitutions, one or more substitutions selected from: F41E, L66A, W260R, Y264K, L268E, M269S, Y277A, Y408D, L421E, W423A, L428R, Y432A in GPC3-V2); the GPC3 comprises or consists of an amino acid sequence identical to SEQ ID NO: 1B (or a variant thereof having 1, 2, 3, 4, or 5 single amino acid modifications (e.g., conservative ammo acid substitutions, one or more substitutions selected from: F41E, L66A, W260R, Y264K, L268E, M269S, Y277A, Y408D, L421E, W423A, L428R, Y432A m GPC3-V2)); the GPC3 comprises or consists of an amino acid sequence identical to SEQ ID NO: 2B, or a variant thereof having 1, 2, 3, 4, or 5 single amino acid modifications (e.g., conservative amino acid substitutions, one or more substitutions selected from: F41E, L66A, W260R, Y264K, L268E, M269S, Y277A, Y408D, L421E, W423 A, L428R, Y432A in GPC3-V2); the GPC3 comprises or consists of an amino acid sequence identical to SEQ ID NO: 3B, or a variant thereof having 1, 2, 3, 4, or 5 single ammo acid modifications (e.g., conservative amino acid substitutions, one or more substitutions selectedAttorney Docket No. 40056-0091 WO1 from: F41E, L66A, W260R, Y264K, L268E, M269S, Y277A, Y408D, L421E, W423A, L428R, Y432A in GPC3-V2); the GPC3 comprises or consists of an ammo acid sequence identical to SEQ ID NO: 4B, or a variant thereof having 1, 2, 3, 4, or 5 single amino acid modifications (e.g., conservative ammo acid substitutions, one or more substitutions selected from: F41E, L66A, W260R, Y264K, L268E, M269S, Y277A, Y408D, L421E, W423 A, L428R, Y432A in GPC3-V2); the nucleotide sequence encoding GPC3 is operably linked to a promoter, e.g,, a synthetic early promoter.

[0271] In various embodiments of any of the methods or compositions described herein: OV-GPC3 does not encode functional thymidine kinase; the OV-GPC3 comprises a nucleotide sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO: Al over the entire length of SEQ ID NO: Al; the OV-GPC3 comprises a nucleotide sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO: Al, but lacks the 5’ ITR sequence and the 3’ ITR sequence of SEQ ID NO: / XI (i.e., lacks nucleotides 1 - 6,301 and 190,103 - 196,397 of SEQ ID NO: Al; i.e., nucleotides 6,302-190,102 of SEQ ID NO: Al); the OV-GPC3 comprises a nucleotide sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO: A3 over the entire length of SEQ ID NO: A3; the OV-GPC3 comprises a nucleotide sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO: A3, but lacks the 5’ ITR sequence and the 3’ ITR sequence of SEQ ID NO: A3 (i.e., lacks nucleotides 1 - 6,301 and 190,103 - 196,397 of SEQ ID NO: A3; i.e., nucleotides 6,302-190,102 of SEQ ID NO: A3); the OV-GPC3 comprises a nucleotide sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO: A4 over the entire length of SEQ ID NO: A4; the OV-GPC3 comprises a nucleotide sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO: A4, but lacks the 5’ ITR sequence and the 3’ ITR sequence of SEQ ID NO: A4 (i.e., lacks nucleotides 1 - 6,301 and 190,103 - 196,397 of SEQ ID NO: A4; i.e., nucleotides 6,302-190,102 of SEQ ID NO: A4); the OV-GPC3 comprises a nucleotide sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO: A5 over the entire length of SEQ ID NO: A5; the OV-GPC3 comprises a nucleotide sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO: A5, but lacks the 5’ ITR sequence and the 3’ ITR sequence of SEQ ID NO: A5 (i.e., lacks nucleotides 1 - 6,301 and 191,595 -198,422 of SEQ ID NO: A5; i.e., nucleotides 6,302-191,421 of SEQ ID NO: A5); the oncolytic virus nucleotide sequence comprises a nucleotide sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100% identical to: a) SEQ ID NO: A2 over the entire length of SEQ ID NO: A2 without the JR2 sequence of SEQ ID NO: A2 (i.e., as identified in FIG. 35; (i.e., not includingAttorney Docket No. 40056-0091 WO1 nucleotides 77682-78084 of SEQ ID NO: A2); or b) SEQ ID NO: A2 over the entire length of SEQ ID NO: A2 but not including the JR2 gene sequence, the 5’ ITR sequence and the 3’ ITR sequence of SEQ ID NO: A2 (i.e,, not including nucleotides 77682-78084, nucleotides 1-4054, and nucleotides 185351-189404 of SEQ ID NO: A2); the oncolytic virus nucleotide sequence comprises at least 25, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140 or 150 of the ORF in Table 4 and encodes at least 25, 50, 60, 70, 80, 90, 100, 110, 120, or 121 of SEQ ID NOs: Z1, Z17, Z26, Z27, Z28, Z32, Z34, Z36, Z41, Z52, Z53, Z55, Z63, Z65, Z66, Z76, Z77, Z79, Z81, Z82, Z87, Z90, Z91, Z92, Z93, Z104, Zl 08, Zl 10, Z111, Zl 16, Zl 18, Z120, Z123, Z128, Z129, Z130, Z138, Z140, Z142, Z143, Z145, Z146, Z147, Z149, Z150, Z151, Z152, Z153, Z154, Z155, Z156, Z157, Z158, Z160, Z161, Z165, Z168, Z169, Z170, Z172, Z173, Z174, Z175, Z176, Z177, Z178, Z181, Z182, Z186, Z192, Z193, Z194, Z195, Z197, Z198, Z199, Z203, Z206, Z211, Z212, Z216, Z219, Z220, Z222, Z223, Z224, Z225, Z226, Z227, Z228, Z229, Z231, Z232, Z233, Z234, Z236, Z238, Z240, Z241, Z243, Z244, Z252, Z253, Z254, Z257, Z258, Z259, Z260, Z261, Z263, Z264, Z265, Z266, Z267, Z268, Z269, Z270, Z271, Z272, Z274, and Z275; the oncolytic virus nucleotide sequence has no modifications in the regions encoding at least 25, 50, 60, 70, 80, 90, 100, 110, 120, or 121 of SEQ ID NOs: Z1, Z17, Z26, Z27, Z28, Z32, Z34, Z36, Z41, Z52, Z53, Z55, Z63, Z65, Z66, Z76, Z77, Z79, Z81, Z82, Z87, Z90, Z91, Z92, Z93, Z104, Z108, Z110, Z111, Z116, Z118, Z120, Z123, Z128, Z129, Z130, Z138, Z140, Z142, Z143, Z145, Z146, Z147, Z149, Z150, Z151, Z152, Z153, Z154, Z155, Z156, Z157, Z158, Z160, Z161, Z165, Z168, Z169, Z170, Z172, Z173, Z174, Z175, Z176, Z177, Z178, Z181, Z182, Z186, Z192, Z193, Z194, Z195, Z197, Z198, Z199, Z203, Z206, Z211, Z212, Z216, Z219, Z220, Z222, Z223, Z224, Z225, Z226, Z227, Z228, Z229, Z231, Z232, Z233, Z234, Z236, Z238, Z240, Z241, Z243, Z244, Z252, Z253, Z254, Z257, Z258, Z259, Z260, Z261, Z263, Z264, Z265, Z266, Z267, Z268, Z269, Z270, Z271, Z272, Z274, and Z275, or all of the open reading frame (ORF) in Table 4; the oncolytic virus nucleotide sequence comprises modifications within the regions encoding at least 25, 50, 60, 70, 80, 90, 100, 110, 120, or 121 of SEQ ID NO: Z1, Z17, Z26, Z27, Z28, Z32, Z34, Z36, Z41, Z52, Z53, Z55, Z63, Z65, Z66, Z76, Z77, Z79, Z81, Z82, Z87, Z90, Z91, Z92, Z93, Z104, Z108, Z110, Z111, Z116, Z118, Z120, Z123, Z128, Z129, Z130, Z138, Z140, Z142, Z143, Z145, Z146, Z147, Z149, Z150, Z151, Z152, Z153, Z154, Z155, Z156, Z157, Z158, Z160, Z161, Z165, Z168, Z169, Z170, Z172, Z173, Z174, Z175, Z176, Z177, Z178, Z181, Z182, Z186, Z192, Z193, Z194, Z195, Z197, Z198, Z199, Z203, Z206, Z211, Z212, Z216, Z219, Z220, Z222, Z223, Z224, Z225, Z226, Z227, Z228, Z229, Z231, Z232, Z233, Z234, Z236, Z238, Z240, Z241, Z243, Z244, Z252, Z253, Z254, Z257, Z258, Z259, Z260, Z261, Z263, Z264, Z265, Z266, Z267, Z268, Z269, Z270, Z271, Z272, Z274, and Z275, or any ORF identified in Table 4, that do not change the amino acid sequence of the encodedAttorney Docket No. 40056-0091 WO1 protein; the oncolytic virus nucleotide sequence comprises the regions encoding SEQ ID NOs: Z1, Z17, Z26, Z27, Z28, Z32, Z34, Z36, Z41, Z52, Z53, Z55, Z63, Z65, Z66, Z76, Z77, Z79, Z81, Z82, Z87, Z90, Z91, Z92, Z93, Z104, Z108, Z110, Z111, Z116, Z118, Z120, Z123, Z128, Z129, Z130, Z138, Z140, Z142, Z143, Z145, Z146, Z147, Z149, Z150, Zl 51, Z152, Z153, Z154, Z155, Z156, Z157, Z158, Z160, Z161, Z165, Z168, Z169, Z170, Z172, Z173, Z174, Z175, Z176, Z177, Z178, Z181, Z182, Z186, Z192, Z193, Z194, Z195, Z197, Z198, Z199, Z203, Z206, Z211, Z212, Z216, Z219, Z220, Z222, Z223, Z224, Z225, Z226, Z227, Z228, Z229, Z231, Z232, Z233, Z234, Z236, Z238, Z240, Z241, Z243, Z244, Z252, Z253, Z254, Z257, Z258, Z259, Z260, Z261, Z263, Z264, Z265, Z266, Z267, Z268, Z269, Z270, Z271, Z272, Z274, and Z275 and at least 130, 140, 150, 160, 170, 180, 190, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, or 275 of the ORF in Table 4; the oncolytic virus nucleotide sequence comprises at least 25, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140 or 150 of the ORF in Table 4 or all of the ORF in Table 4; the oncolytic virus nucleotide sequence comprises a nucleotide sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or 100% identical to: SEQ ID NO: A2 over the entire length of SEQ ID NO: A2, excluding all or a portion of the JR2 gene sequence (nt 77,603-78-137 of SEQ ID NO: A2) and excluding the ITR sequences, and comprises nucleotide sequences that encode at least 25, 50, 60, 70, 80, 90, 100, 110, 120, or 121 of SEQ ID NOs: Z1, Z17, Z26, Z27, Z28, Z32, Z34, Z36, Z41, Z52, Z53, Z55, Z63, Z65, Z66, Z76, Z77, Z79, Z81, Z82, Z87, Z90, Z91, Z92, Z93, Z104, Z108, Z110, Z111, Z116, Z118, Z120, Z123, Z128, Z129, Z130, Z138, Z140, Z142, Z143, Z145, Z146, Z147, Z149, Z150, Z151, Z152, Z153, Z154, Z155, Z156, Z157, Z158, Z160, Z161, Z165, Z168, Z169, Z170, Z172, Z173, Z174, Z175, Z176, Z177, Z178, Z181, Z182, Z186, Z192, Z193, Z194, Z195, Z197, Z198, Z199, Z203, Z206, Z211, Z212, Z216, Z219, Z220, Z222, Z223, Z224, Z225, Z226, Z227, Z228, Z229, Z231, Z232, Z233, Z234, Z236, Z238, Z240, Z241, Z243, Z244, Z252, Z253, Z254, Z257, Z258, Z259, Z260, Z261, Z263, Z264, Z265, Z266, Z267, Z268, Z269, Z270, Z271, Z272, Z274, and Z275, or a variant of one or more thereof having 1, 2, 3, 4, or 5 single amino acid substitutions. In some embodiments, the single amino acid substitutions are conservative and / or do not alter the function of the protein.

[0272] In various embodiments of any of the methods or compositions described herein: the OV-GPC3 comprises a nucleotide sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99% or 100% identical to: SEQ ID NO: Al over the entire length of SEQ ID NO: Al, and encodes at least 25, 50, 60, 70, 80, 90, 100, 110, 120, or 121 proteins having the ammo acid sequences of SEQ ID NO: Z1, Z17, Z26, Z27, Z28, Z32, Z34, Z36, Z41, Z52, Z53, Z55, Z63, Z65, Z66, Z76, Z77, Z79, Z81, Z82, Z87, Z90, Z91, Z92, Z93, Z104, Z108, Z110, Z111, Z116, Z118, Z120, Z123, Z128, Z129, Z130,Attorney Docket No. 40056-0091 WO1 Z138, Z140, Z142, Z143, Z145, Z146, Z147, Z149, Z150, Z151, Z152, Z153, Z154, Z155, Z156, Z157, Z158, Z160, Z161, Z165, Z168, Z169, Z170, Z172, Z173, Z174, Z175, Z176, Z177, Z178, Z181, Z182, Z186, Z192, Z193, Z194, Z195, Z197, Z198, Z199, Z203, Z206, Z211, Z212, Z216, Z219, Z220, Z222, Z223, Z224, Z225, Z226, Z227, Z228, Z229, Z231, Z232, Z233, Z234, Z236, Z238, Z240, Z241, Z243, Z244, Z252, Z253, Z254, Z257, Z258, Z259, Z260, Z261, Z263, Z264, Z265, Z266, Z267, Z268, Z269, Z270, Z271, Z272, Z274, and Z275, or a variant of one or more thereof having 1, 2, 3, 4, or 5 single ammo acid substitutions; the single amino acid substitutions are conservative and / or do not alter the function of the protein; the OV-GPC3 comprises a nucleotide sequence: a) that is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100% identical to: SEQ ID NO: Al over the entire length of SEQ ID NO: Al, but lacks the 5’ ITR sequence and the 3’ ITR sequence of SEQ ID NO: Al; and b) encodes at least 25, 50, 60, 70, 80, 90, 100, 110, 120, or 121 proteins having the ammo acid sequences of SEQ ID NO: Z1, Z17, Z26, Z27, Z28, Z32, Z34, Z36, Z41, Z52, Z53, Z55, Z63, Z65, Z66, Z76, Z77, Z79, Z81, Z82, Z87, Z90, Z91, Z92, Z93, Z104, Z108, ZI10, Z111, ZI16, Z118, Z120, Z123, ZI28, Z129, ZI30, Z138, Z140, Z142, Z143, Z145, Z146, ZI47, Z149, Z150, Z151, Z152, Z153, Z154, Z155, Z156, Z157, Z158, Z160, Z161, ZI65, Z168, Z169, Z170, Z172, Z173, Z174, ZI75, Z176, Z177, Z178, Z181, Z182, Z186, Z192, Z193, Z194, Z195, Z197, Z198, Z199, Z203, Z206, Z211, Z212, Z216, Z219, Z220, Z222, Z223, Z224, Z225, Z226, Z227, Z228, Z229, Z231, Z232, Z233, Z234, Z236, Z238, Z240, Z241, Z243, Z244, Z252, Z253, Z254, Z257, Z258, Z259, Z260, Z261, Z263, Z264, Z265, Z266, Z267, Z268, Z269, Z270, Z271, Z272, Z274, and Z275, or a variant of one or more thereof having 1, 2, 3, 4, or 5 single amino acid substitutions; the amino acid substitutions are conservative and / or do not alter the function of the protein; the oncolytic virus sequence is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to nucleotides 6,302 - 190,102 of SEQ ID NO: Al over the entire length of nucleotides 6,302 - 190,102 of SEQ ID NO: Al; the oncolytic virus sequence is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to nucleotides 7,000-188,000 of SEQ ID NO: A1 over the entire length of nucleotides 7,000-188,000 SEQ ID NO: Al; the oncolytic virus sequence is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to nucleotides 8,000-187,000 of SEQ ID NO: Al over the entire length of nucleotides 8,000-187,000 SEQ ID NO: Al; the oncolytic virus sequence is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to nucleotides 10,000-185,000 of SEQ ID NO: Al over the entire length of nucleotides 10,000-185,000 SEQ ID NO: Al; the oncolytic virus sequence is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to nucleotides 10,001-184,901 ofAttorney Docket No. 40056-0091 WO1 SEQ ID NO: Al over the entire length of the oncolytic virus sequence is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99,6%, 99.7%, 99.8%, 99.9%, 99,99%, or 100% identical to nucleotides 10,001- 184,901 of SEQ ID NO: Al; the oncolytic virus sequence is at. least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to nucleotides 6,302 - 190,102 of SEQ ID NO: A3 over the entire length of nucleotides 6,302 - 190,102 of SEQ ID NO: A3; the oncolytic virus sequence is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to nucleotides 7,000-188,000 of SEQ ID NO: A3 over the entire length of nucleotides 7,000-188,000 SEQ ID NO:; X3; the oncolytic virus sequence is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to nucleotides 8,000-187,000 of SEQ ID NO: A3 over the entire length of nucleotides 8,000-187,000 SEQ ID NO: A3; the oncolytic virus sequence is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to nucleotides 10,000-185,000 of SEQ ID NO: A3 over the entire length of nucleotides 10,000-185,000 SEQ ID NO: A3; the oncolytic virus sequence is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to nucleotides 10,001-184,901 of SEQ ID NO: A3 over the entire length of the oncolytic virus sequence is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to nucleotides 10,001- 184,901 of SEQ ID NO: A3; the oncolytic virus sequence is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to nucleotides 6,302 - 190,102 of SEQ ID NO: A4 over the entire length of nucleotides 6,302 - 190,102 of SEQ ID NO: A4; the oncolytic virus sequence is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to nucleotides 7,000-188,000 of SEQ ID NO: A4 over the entire length of nucleotides 7,000-188,000 SEQ ID NO: A4; the oncolytic virus sequence is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to nucleotides 8,000-187,000 of SEQ ID NO: A4 over the entire length of nucleotides 8,000-187,000 SEQ ID NO: A4; the oncolytic virus sequence is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to nucleotides 10,000-185,000 of SEQ ID NO: A4 over the entire length of nucleotides 10,000-185,000 SEQ ID NO: A4; the oncolytic virus sequence is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to nucleotides 10,001-184,901 of SEQ ID NO: A4 over the entire length of the oncolytic virus sequence is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to nucleotides 10,001- 184,901 of SEQ ID NO: A4; the oncolytic virus sequence is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to nucleotides 6,302 - 191,594 of SEQAttorney Docket No. 40056-0091 WO1 ID NO: A5 over the entire length of nucleotides 6,302 - 191,594 of SEQ ID NO: A5; the oncolytic virus sequence is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to nucleotides 7,000-190,000 of SEQ ID NO: A5 over the entire length of nucleotides 7,000-190,000 SEQ ID NO: A5; the oncolytic virus sequence is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to nucleotides 8,000-187,000 of SEQ ID NO: A5 over the entire length of nucleotides 8,000-187,000 SEQ ID NO: A5; the oncolytic virus sequence is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99,99%, or 100% identical to nucleotides 10,000-185,000 of SEQ ID NO: A5 over the entire length of nucleotides 10,000-185,000 SEQ ID NO: A5; the oncolytic virus sequence is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to nucleotides 10,001-184,901 of SEQ ID NO: A5 over the entire length of the oncolytic virus sequence is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to nucleotides 10,001-184,901 of SEQ ID NO: A5; the OV-GPC3 is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100% identical to: SEQ ID NO: A2 over the entire length of SEQ ID NO: A2 except that the nucleotide sequence encoding GPC3 and a promoter sequence for expressing GPC3 replaces at least 10 contiguous nucleotides of the JR2 gene sequence; and at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 250, 275, 400, 425, 450, 475, or 500 nucleotides (nts) of the J2R gene sequence have been deleted.

[0273] Also described herein, inter aha, are oncolytic viruses expressing a human GPC3 (OV-GPC3), the nucleotide sequence of OV-GPC3 comprising:

[0274] (a) an oncolytic virus nucleotide sequence; and

[0275] (b) a nucleotide sequence encoding a human GPC3;

[0276] wherein the oncolytic virus nucleotide sequence encodes at least 25, 50, 60, 70, 80, 90, 100, 110, 120, or 121 of SEQ ID NO: Z1, Z17, Z26, Z27, Z28, Z32, Z34, Z36, Z41, Z52, Z53, Z55, Z63, Z65, Z66, Z76, Z77, Z79, Z81, Z82, Z87, Z90, Z91, Z92, Z93, Z104, Z108, Z110, Z111, Z116, Z118, Z120, Z123, Z128, Z129, Z130, Z138, Z140, Z142, Z143, Z145, Z146, Z147, Z149, Z150, Z151, Z152, Z153, Z154, Z155, Z156, Z157, Z158, Z160, Z161, Z165, Z168, Z169, Z170, Z172, Z173, Z174, Z175, Z176, Z177, Z178, Z181, Z182, Z186, Z192, Z193, Z194, Z195, Z197, Z198, Z199, Z203, Z206, Z211, Z212, Z216, Z219, Z220, Z222, Z223, Z224, Z225, Z226, Z227, Z228, Z229, Z231, Z232, Z233, Z234, Z236, Z238, Z240, Z241, Z243, Z244, Z252, Z253, Z254, Z257, Z258, Z259, Z260, Z261, Z263, Z264, Z265, Z266, Z267, Z268, Z269, Z270, Z271, Z272, Z274, and Z275, or a variant of one or more thereof havingAttorney Docket No. 40056-0091 WO1 1, 2, 3, 4, or 5 single amino acid substitutions (optionally wherein the amino acid substitutions are conservative and / or do not alter the function of the protein); and / or

[0277] wherein the 0V-GPC3 nucleotide sequence is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to or has no more than 300 single nucleotide changes compared to:

[0278] i) nucleotides 6,302 - 190,102 of SEQ ID NO: A1, A3, or A4 over the entire length of nucleotides 6,302 - 190,102 of SEQ ID NO: Al, A3, or A4;

[0279] ii) nt 6,302 - 191,594 of SEQ ID NO: A5 over the entire length of nt 6,302 - 191,594 SEQ ID NO: A5;

[0280] iii) nucleotides 7,000-188,000 of SEQ ID NO: Al, A3, A4, or A5 over the entire length of nucleotides 7,000-188,000 of SEQ ID NO: Al, A3, A4, or A5;

[0281] iv) nucleotides 8,000-187,000 of SEQ ID NO: A1, A3, A4, or A5 over the entire length of nucleotides 8,000-187,000 SEQ ID NO: Al, A3, A4, or A5; or

[0282] v) nucleotides 10,000-185,000 of SEQ ID NO: A1, A3, A4, or A5 over the entire length of nucleotides 10,000-185,000 SEQ ID NO: Al, A3, A4, or A5.

[0283] In some embodiments of any of the compositions or methods described herein, the OV-GPC3 nucleotide sequence is at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to:

[0284] i) SEQ ID NO: Al over the entire length of SEQ ID NO: Al;

[0285] ii) nt 6,302 - 190,102 of SEQ ID NO: Al over the entire length of nt 6,302 - 190,102 SEQ ID NO: Al;

[0286] iii) nucleotides 7,000-188,000 of SEQ ID NO: A1 over the entire length of nucleotides 7,000-188,000 SEQ ID NO: Al;

[0287] iv) nucleotides 8,000-187,000 of SEQ ID NO: Al over the entire length of nucleotides 8,000-187,000 SEQ ID NO: Al;

[0288] v) nucleotides 10,000-185,000 of SEQ ID NO: Al over the entire length of nucleotides 10,000-185,000 SEQ ID NO: Al;

[0289] vi) SEQ ID NO: A3 over the entire length of SEQ ID NO: A3;

[0290] vii) nt 6,302 - 190,102 of SEQ ID NO: A3 over the entire length of nt 6,302 - 190,102 SEQ ID NO: A3;

[0291] viii) nucleotides 7,000-188,000 of SEQ ID NO: A3 over the entire length of nucleotides 7,000-188,000 SEQ ID NO: A3;Attorney Docket No. 40056-0091 WO1 ix) nucleotides 8,000-187,000 of SEQ ID NO: A3 over the entire length of nucleotides 8,000-187,000 SEQ ID NO: A3;

[0292] x) nucleotides 10,000-185,000 of SEQ ID NO: A3 over the entire length of nucleotides 10,000-185,000 SEQ ID NO: A3;

[0293] xi) SEQ ID NO: A4 over the entire length of SEQ ID NO: A4;

[0294] xii) nt 6,302 - 190,102 of SEQ ID NO: A4 over the entire length of nt 6,302 - 190,102 SEQ ID NO: A4;

[0295] xin) nucleotides 7,000-188,000 of SEQ ID NO: A4 over the entire length of nucleotides 7,000-188,000 SEQ ID NO: A4;

[0296] xiv) nucleotides 8,000-187,000 of SEQ ID NO: A4 over the entire length of nucleotides 8,000-187,000 SEQ ID NO: A4;

[0297] xv) nucleotides 10,000-185,000 of SEQ ID NO: A4 over the entire length of nucleotides 10,000-185,000 SEQ ID NO: A4; or

[0298] xvi) SEQ ID NO: A5 over the entire length of SEQ ID NO: A5;

[0299] xvii) nt 6,302 - 191,594 of SEQ ID NO: A5 over the entire length of nt 6,302 - 191,594 SEQ ID NO: A5;

[0300] xviii) nucleotides 7,000-188,000 of SEQ ID NO: A5 over the entire length of nucleotides 7,000- 188,000 SEQ ID NO: A5;

[0301] xix) nucleotides 8,000-187,000 of SEQ ID NO: A5 over the entire length of nucleotides 8,000-187,000 SEQ ID NO: A5; or

[0302] xx) nucleotides 10,000-185,000 of SEQ ID NO: A1 over the entire length of nucleotides 10,000-185,000 SEQ ID NO: Al.

[0303] In some embodiments of any of the compositions or methods described herein, an OV-GPC3 comprises a nucleotide sequence comprising:

[0304] (a) an oncolytic virus nucleotide sequence; and

[0305] (b) a nucleotide sequence encoding a human GPC3;

[0306] wherein the oncolytic virus nucleotide sequence encodes at least 25, 50, 60, 70, 80, 90, 100, 110, 120, or 121 of SEQ ID NO: Z1, Z17, Z26, Z27, Z28, Z32, Z34, Z36, Z41, Z52, Z53, Z55, Z63, Z65, Z66, Z76, Z77, Z79, Z81, Z82, Z87, Z90, Z91, Z92, Z93, Z104, Z108, Z110, Z111, Z116, Z118, Z120, Z123, Z128, Z129, Z130, Z138, Z140, Z142, Z143, Z145, Z146, Z147, Z149, Z150, Z151, Z152, Z153, Z154, Z155, Z156, Z157, Z158, Z160, Z161, Z165, Z168, Z169, Z170, Z172, Z173, Z174, Z175, Z176, Z177, Z178, Z181, Z182, Z186, Z192, Z193, Z194, Z195, Z197, Z198, Z199, Z203, Z206, Z211, Z212,Attorney Docket No. 40056-0091 WO1 Z216, Z219, Z220, Z222, Z223, Z224, Z225, Z226, Z227, Z228, Z229, Z231, Z232, Z233, Z234, Z236, Z238, Z240, Z241, Z243, Z244, Z252, Z253, Z254, Z257, Z258, Z259, Z260, Z261, Z263, Z264, Z265, Z266, Z267, Z268, Z269, Z270, Z271, Z272, Z274, and Z275; and / or

[0307] wherein the OV-GPC3 nucleotide sequence comprises any one of:

[0308] i) nucleotides 6,302 - 190,102 of SEQ ID NO: A1, A3, or A4, or a variant thereof with up to 100, 200, or 300 single nucleotide substitutions;

[0309] ii) nt 6,302 - 191,594 of SEQ ID NO: A5 or a variant thereof with up to 100, 200, or 300 single nucleotide substitutions;

[0310] iii) nucleotides 7,000-188,000 of SEQ ID NO: Al, A3, A4, or A5, or a variant thereof with up to 100, 200, or 300 single nucleotide substitutions;

[0311] iv) nucleotides 8,000-187,000 of SEQ ID NO: Al, A3, A4, or A5, or a variant thereof with up to 100, 200, or 300 single nucleotide substitutions; or

[0312] v) nucleotides 10,000-185,000 of SEQ ID NO: Al, A3, A4, or A5, or a variant thereof with up to 100, 200, or 300 single nucleotide substitutions.

[0313] In some embodiments of any of the compositions or methods described herein, the OV-GPC3 nucleotide sequence comprises nucleotides 6,302 - 191,594 of SEQ ID NO: A1 or a variant thereof with up to 100 single nucleotide substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotide substitutions).

[0314] In some embodiments of any of the compositions or methods described herein, the OV-GPC3 nucleotide sequence comprises nucleotides 6,302 - 191,594 of SEQ ID NO: A3 or a variant thereof with up to 100 single nucleotide substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotide substitutions).

[0315] In some embodiments of any of the compositions or methods described herein, the OV-GPC3 nucleotide sequence comprises nucleotides 6,302 - 191,594 of SEQ ID NO: A4 or a variant thereof with up to 100 single nucleotide substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,Attorney Docket No. 40056-0091 WO1 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotide substitutions).

[0316] In some embodiments of any of the compositions or methods described herein, the OV-GPC3 nucleotide sequence comprises nucleotides 6,302 - 191,594 of SEQ ID NO: A5 or a variant thereof with up to 100 single nucleotide substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotide substitutions).

[0317] In some embodiments of any of the compositions or methods described herein, the OV-GPC3 nucleotide sequence comprises nucleotides 7,000-188,000 of SEQ ID NO: Al, A3, A4, or A5, or a variant thereof with up to 100 single nucleotide substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotide substitutions).

[0318] In some embodiments of any of the compositions or methods described herein, the OV-GPC3 nucleotide sequence comprises nucleotides 8,000-187,000 of SEQ ID NO: A1, A3, A4, or A5, or a variant thereof with up to 100 single nucleotide substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotide substitutions).

[0319] In some embodiments of any of the compositions or methods described herein, the OV-GPC3 nucleotide sequence comprises nucleotides 9,000-186,000 of SEQ ID NO: A1, A3, A4, or A5, or a variant thereof with up to 100 single nucleotide substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotide substitutions).Attorney Docket No. 40056-0091 WO1 In some embodiments of any of the compositions or methods described herein, any nucleotide modifications (e.g., nucleotide substitutions, nucleotide insertions) in the oncolytic virus nucleotide sequence do not change the amino acid sequence of encoded proteins having an amino acid sequences SEQ ID NO: Z1, Z17, Z26, Z27, Z28, Z32, Z34, Z36, Z41, Z52, Z53, Z55, Z63, Z65, Z66, Z76, Z77, Z79, Z81, Z82, Z87, Z90, Z91, Z92, Z93, Z104, Z108, Z110, Z111, Z116, Z118, Z120, Z123, Z128, ZA 29, Z 30, Z138, Z140, Z142, Z143, Z145, Z146, Z147, Z149, Z150, Z151, Z152, ZA 53, Z154, Z155, Z156, Z157, Z158, Z160, Z161, Z165, Z168, Z169, Z170, Z172, Z173, Z174, Z175, Z176, Z177, Z178, Z181, Z182, Z186, Z192, Z193, Z194, Z195, Z197, Z198, Z199, Z203, Z206, Z211, Z212, Z216, Z219, Z220, Z222, Z223, Z224, Z225, Z226, Z227, Z228, Z229, Z231, Z232, Z233, Z234, Z236, Z238, Z240, Z241, Z243, Z244, Z252, Z253, Z254, Z257, Z258, Z259, Z260, Z261, Z263, Z264, Z265, Z266, Z267, Z268, Z269, Z270, Z271, Z272, Z274, and Z275. In some embodiments of any of the compositions or methods described herein, any nucleotide modifications (e.g., nucleotide substitutions, nucleotide insertions) in the oncolytic virus nucleotide sequence do not change the function of the encoded proteins having an ammo acid sequences SEQ ID NO: Z1, Z17, Z26, Z27, Z28, Z32, Z34, Z36, Z41, Z52, Z53, Z55, Z63, Z65, Z66, Z76, Z77, Z79, Z81, Z82, Z87, Z90, Z91, Z92, Z93, Z104, Z108, Z110, Z111, Z116, Z118, Z120, Z123, Z128, Z129, Z130, Z138, Z140, Z142, Z143, Z145, Z146, Z147, Z149, Z150, Z151, Z152, Z153, Z154, Z155, Z156, Z157, Z158, Z160, Z161, Z165, Z168, Z169, Z170, Z172, Z173, Z174, Z175, Z176, Z177, Z178, Z181, Z182, Z186, Z192, Z193, Z194, Z195, Z197, Z198, Z199, Z203, Z206, Z211, Z212, Z216, Z219, Z220, Z222, Z223, Z224, Z225, Z226, Z227, Z228, Z229, Z231, Z232, Z233, Z234, Z236, Z238, Z240, Z241, Z243, Z244, Z252, Z253, Z254, Z257, Z258, Z259, Z260, Z261, Z263, Z264, Z265, Z266, Z267, Z268, Z269, Z270, Z271, Z272, Z274, and Z275.

[0320] In some embodiments of any of the compositions or methods described herein, the OV-GPC3 does not encode any of an AFP, a BCMA, a BCMAt, a CA125, a CD19, a CD19t, a CD20, a CD33, a CD22, a CD123, a CD30, a CD38, a CEA, a HER2, a GD2, a PSMA, a Claudm 18.2, a EpCAM, a GD2, a MSLN, an EGFR, an EGFRVIII, a Trop-2, a c-MET, a Nectin-4, a CD79b, a CCK4, a GPA33, a HLA-A2, a CLEC12A, a p-cadherm, a TD02, a MART-I, a MUCI, a Pmel 17, a MAGE-I, a TRP-1, a TRP-2, a NY-ESQ, a PSA, a CDK4, a BCA225, a CA 125, a MG7-Ag, a NY-CO-I, a RCAS 1, a SDCCAG16, a TAAL6, and a TAG72, and optionally functional variants of one or more thereof (e.g., a functional variant that would be recognized and bound by an antibody that binds the nonvariant version).

[0321] In some embodiments of any of the compositi ons or methods described herein, the GPC3 comprises an amino acid sequence that comprises or consists of SEQ ID NO: 1, 2, 3, 4, 5, 6, IB, 2B, 3B, 4B, 5B, or 6B. In various embodiments of any of the methods or compositions described herein, theAttorney Docket No. 40056-0091 WO1 nucleotide sequence encoding GPC3 is inserted into a noncoding region of any OV-GPC3 described herein (e.g., SEQ ID NO: Al, A2, A3, A4, or A5).

[0322] In some embodiments of any of the compositions or methods described herein, the OV-GPC3 comprises a nucleotide sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to nucleotides 6,302 - 190,102 of SEQ ID NO: Al over the entire length of nucleotides 6,302 - 190,102 of SEQ ID NO: Al. In some embodiments of any of the compositions or methods described herein, the OV-GPC3 comprises a nucleotide sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to nucleotides 6,302 - 190,102 of SEQ ID NO: A3 over the entire length of nucleotides 6,302 - 190,102 of SEQ ID NO: A3. In some embodiments of any of the compositions or methods described herein, the OV-GPC3 comprises a nucleotide sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to nucleotides 6,302 - 190,102 of SEQ ID NO: A4 over the entire length of nucleotides 6,302 - 190,102 of SEQ ID NO: A4. In some embodiments of any of the compositions or methods described herein, the OV-GPC3 comprises a nucleotide sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to nucleotides 6,302-191,594 of SEQ ID NO: A5 over the entire length of nucleotides 6,302 - 191,594 of SEQ ID NO: A5.

[0323] In some embodiments of any of the compositions or methods described herein, an OV-GPC3 comprises nucleotides 10,000-185,000 of SEQ ID NO: Al, A3, A4, or A5, or a variant thereof with up to 100 nucleotide modifications (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 single nucleotide substitutions).

[0324] Also described herein are methods of treating a subject having a solid tumor, the method comprising administering to the subject: an effective amount any OV-GPC3 described herein. In some embodiments, the methods further comprise a means for targeting GPC3 (e.g., administering an effective amount of a GPC3-targeted therapy described herein, e.g., the below section).

[0325] In various embodiments, a solid tumor may be a carcinoma, adenocarcinoma, sarcoma, melanoma, mesothelioma, blastoma; a carcinoma or adenocarcinoma may for example be a bladder, a colon, a kidney, an ovary, a prostate, a lung, an uterus, a breast, or a prostate carcinoma or adenocarcinoma; a blastoma may for example be a neuroblastoma, a glioblastoma, or a retinoblastoma;Attorney Docket No. 40056-0091 WO1 the solid tumor is selected from the group consisting of prostate cancer (e.g., prostate adenocarcinoma), lung cancer (e.g,, squamous cellular carcinoma), breast cancer (e.g., infiltrated ductal carcinoma), ovary cancer (e.g,, serous papillary carcinoma), uterus cancer (e.g., squamous cellular carcinoma), CNS cancer and brain cancer (e.g., glioma (such as brainstem glioma and mixed gliomas), glioblastoma (also known as glioblastoma multiforme), astrocytoma, CNS lymphoma, germinoma, medulloblastoma, Schwannoma craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, and brain metastases), colon cancer (e.g., colon adenocarcinoma or colon carcinoma), colorectal cancer, rectal cancer (e.g., rectal adenocarcinoma), cancer of the striated muscle (e.g., rhabdomyosarcoma), thyroid cancer, testicular cancer, bladder cancer (e.g., bladder carcinoma), liver cancer, kidney cancer, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, lymphoid malignancy, pancreatic cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, seminoma, melanoma, and metastases of one or more thereof.

[0326] GPC3-targeted Therapies

[0327] Useful GPC3 -targeting therapies can comprise an scFv that binds GPC3. A useful therapy can be, for example, a TCE, a population of immune cells expressing a receptor that binds GPC3 e g., a GPC3 CAR (e.g., GPC3 CAR T cells or GPC3 CAR NK cells), an anti-GPC3 antibody or antibody fragment, an anti-GPC3 antibody or antibody fragment linked to a chemotherapeutic agent, a functional variant of each, and combinations thereof.

[0328] 1. Bispecific T cell Engagers (TCEs) and Antibodies

[0329] The methods described herein can include the use of a GPC3-targeted therapy such as a TCE (e.g., a therapeutic agent that binds to CDS and to GPC3; e.g., ERY-974) or an antibody or antibody fragment (e.g,, BlenRep / GSK2857916),

[0330] There are a number of GPC3-targeted therapies (e.g., CAR, TCEs, and antibodies) known in the art that can be used in any of the methods described herein. For example, useful GPC3 TCEs includeAttorney Docket No. 40056-0091 WO1 ERY-974 and ABP-110. Useful TCEs can comprise a humanized immunoglobulin G4 (IgG4) bispecific GPC3-directed cluster of differentiation (CD3) T-cell engager. The TCE can utilize an IgG4 PAA scaffold containing Pro mutations (S229P in the anti-GPC3 mAb and S233P in the anti-CD3 mAb, according to sequential numbering of amino acids) that stabilize the hinge region and Ala / Ala mutations (F235A, L236A in the anti-GPC3 mAb and F239A, L240A in anti-CD3 mAb, according to sequential numbering of amino acids) that suppress FcyR binding. The resulting bispecific antibody comprises Fc substitutions, with the GPC3-binding arm and CD3-binding arm each comprising Pro / Ala / Ala substitutions at amino acid positions 228 / 234 / 235, respectively (according to EU index numbering); and the CD3-binding arm also comprising F405L and R409K substitutions (according to EU index numbering).

[0331] Useful therapies described herein (e.g., a TCE, GPC3 CAR immune cells, GPC3 antibody drug conjugates) can be administered in single or repeated doses. Useful therapies described herein can be administered via intradermal, subcutaneous, intravenous, transdermal, intraperitoneal, intramuscular, pulmonary, and / or parenteral administration. Additional information on administering a TCE, Ab, and / or antibody-drug conjugate known in the art and can be found, for example, in US 2022 / 0041742 Al, WO 2024 / 044545, WO 2024 / 082051, and WO 2024 / 044548.

[0332] Examples of additional GPC3 TCE and antibody constructs can include any one or more of the therapeutic agents listed in Table Al.

[0333] Table Al: GPC3- targeting Therapies

[0334] Name Organization / Company Clinical Stage

[0335] Phase I

[0336] ERY-974 Chugai

[0337] (NCT02748837)

[0338] ABP-110 AbPro Preclinical

[0339] GC33

[0340] Phase 1

[0341] RG-7686 Chugai

[0342] (NCT00746317)

[0343] (Codrituzumab)

[0344] Phase II

[0345] ECT204 Eureka Therapeutics

[0346] (NCT04864054)

[0347] HN3

[0348] hYP7

[0349]

[0350] Attorney Docket No. 40056-0091 WO1

[0351] Phase I

[0352] National Cancer Institute

[0353] hYP7 CAR T Cells (NCT0500389;

[0354] (NCI)

[0355] )

[0356] Zhejiang University /

[0357] CT017 CAR T cells Phase I (NCT03980288)

[0358] CARsgen Therapeutics

[0359] C-CAR031 Zhejiang University Phase II (NCT05155189)

[0360] GLYCAR T cells Baylor College of Medicine Phase I (NCT02905188)

[0361] Chinese PLA General Phase I / II

[0362] GPC3-CAR T cells

[0363] Hospital (NCT06641453)

[0364]

[0365] Adapted from Table 4 of Nie, S. et al, (2020) “Biology drives the discovery of bispecific antibodies as innovative therapeutics” Antibody Therapeutics, 3(1): 18-62, Additional therapies are also known in the art (e.g., to Anti-Glypican 3 antibody [SP86](ab95363), ECT-204, Anti-CD3 / MUC1 -armed-cytokine induced killer cells, GPC-3298306, ERY-974, MDX-1414, B010-A, HLX-63, LQ-102, GSK2857916, BMS-986182, and BMS-986183 (an antibody-drug conjugate (ADC) of the anti-GPC3 antibody BMS-986182 (also known as GPC3.1 (BMS) or 4A6 (Medarex)) conjugated to a tubulysin drug moiety), A TCE as described herein includes bispecific antibody constructs, which are recombinant protein constructs made from two flexibly linked antibody-derived binding domains. One binding domain of a GPC3-TCE is specific for GPC3, GPC3t, or a variant thereof (e.g,, a functional variant); the second binding domain is specific for CD3 or a variant thereof (e.g., a functional variant). The TCEs and antibody constructs disclosed herein can be prepared by methods known in the art, for example, by methods disclosed in US 7,919,086, WO 2008 / 119657 and WO 2017 / 134140. TCE constructs are uniquely suited to transiently connect T cells with target cells and, at the same time, potently activate the inherent cytolytic potential of T cells against target cells.

[0366] An increased half-life is generally useful in in vivo applications of TCEs and immunoglobulins in general, especially antibodies and antibody fragments of small size. Some approaches described in the art to achieve such effect comprise the fusion of the TCE or bispecific antibody construct to larger proteins, which preferably do not interfere with the therapeutic effect of the TCE or bispecific antibody construct. Examples for such further developments of bispecific T cell engagers comprise bispecific Fc- molecules e.g., described in US 2014 / 0302037, US 2014 / 0308285, WO 2014 / 144722, WO 2014 / 151910, WO 2015 / 048272, WO 2017 / 031104, WO 2018 / 052503, WO 2018 / 204907, WO 2020 / 072306, WO 2021 / 222578, US 2021 / 0403587 Al, and US 2023 / 0272102 Al.Attorney Docket No. 40056-0091 WO1 In some embodiments, a useful TCE can be a bispecific antibody construct that further comprises a half-life extending (HLE) moiety (e.g., a scFc domain, a heteroFc domain, or an albumin binding domain). In some embodiments, the N-terminus or the C-terminus of the HLE domain is connected to the TCE (e.g., the portion that binds CD3, e.g., the VH or VL of a CD3-targeted scFv). In some embodiments, the HLE domain is connected to the bispecific antibody construct via a linker.

[0367] Useful TCE include a BiTE; in some case, the BiTE further comprises a third domain comprising two polypeptide monomers, each comprises a hinge, a CH2 and a CH3 domain, wherein the two polypeptide monomers are linked to each other via a peptide linker. In some embodiments, the third domain comprises in an amino to carboxyl order hinge-CH2-CH3- linker-hinge-CH2-CH3. In some embodiments, the third domain is a half-life extended (HLE) domain.

[0368] Useful TCEs and antibodies comprise a domain that targets GPC3 (e.g., an scFv comprising sequences in Table A2). A useful TCE includes a domain that binds GPC3 and a domain that binds CD3 The domain that binds GPC3 is a GPC3 -targeted scFv and the domain that binds CD 3 is a CD3-targeted scFv.

[0369] For example, a useful therapy can comprise Codrituzuinab (also known as GC33, RG-7686, etc.) comprises 4 subunits:

[0370] Subunit 1:

[0371] QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAY SQKFKGRVITTADKSTSTAYMELSSLTSEDTAVYYCTRFYSYTYWGQGTLVTVSSASIKGPSV FPLAPS SKS TSGGTAALGCLVKDYFPEP V TVSWNSGALTSGVHTFPA VLQS SGLY SLSS VVTVPS SSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAI’ELLGGPSVFLFPPKI’KDTLMISR TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPAl’IEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK SEQ ID NO:12

[0372] Subunit 2:

[0373] QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAY SQKFKGRVTLTADKSTSTAYMELSSLTSEDTAVYYCTRFYSYTYWGQGTLVTVSSASTKGPSV FPLAPSSKSTSGGTAALGCLVKDYFPEPVWSyWSGALTSGWTFPAVTQSSGLYSLSSWTVTS SSLGTQTYICNWHKPSNTKWKKWLKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR TPEVTCytytyT)VSHEDPEVKFNWYVDG ATiINAKTKPREEQYNSTYRVVSVITVI., HQD^ NGAttorney Docket No. 40056-0091 WO1 KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK SEQ ID NO: 13

[0374] Subunit 3:

[0375] DVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNRNTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQL KSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 14

[0376] Subunit 4:

[0377] DVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNRNTYLHWYLQKPGQSPQLLIYKVSNRFSGVPD RFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQL KSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 15A

[0378] Another useful therapy can comprise the GPC3xCD3 I CE depicted in FIG. 33:

[0379] QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYS QKFKGRVTLTADKSTSTAYMELSSLTSEDTAVYYCTRFYSYTYWGQGTLVTVSSGGGGSGGGGS GGGGSDVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNRNTYLHWYLQKPGQSPQLLIYKVSNRF SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIKGGGGSQVQLQQS GAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKAT LTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGG GSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFR GSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEIK (SEQ ID NO: 11).

[0380] The TCE in FIG. 33 includes:

[0381] an scFv targeted to GPC3 having a VH domain having the sequence:

[0382] QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADKSTSTAYMELSSLTSEDTAVYYCTRFYSYTYWGQGTLVTVSS (CDRs, 1, 2 and 3 underlined; SEQ ID NO: B26; VH CDR1 SEQ ID NO: B29; VH CDR2 SEQ ID NO: B30; VH CDR3 SEQ ID NO: B31) and a VL domain having the sequence:

[0383] DVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNRNTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDAttorney Docket No. 40056-0091 WO1 RFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIK (CDRs, 1, 2 and 3 underlined; SEQ ID NO: B27; VL CDR1 SEQ ID NO: B32; VL CDR2 SEQ ID NO: B33; VL CDR3 SEQ ID NO: B34)

[0384] linked to a scFv targeted to CD3 having a VH domain having the sequence:

[0385] QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSS (CDRs, 1, 2 and 3 underlined; SEQ ID NO: C70; VH CDR1 SEQ ID NO: C71; VH CDR2 SEQ ID NO: C72; VH CDR3 SEQ ID NO: C73) and a VL domain having the sequence:

[0386] QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEIK (CDRs, 1, 2 and 3 underlined; SEQ ID NO: C74; VL CDR1 SEQ ID NO: C75; VL CDR2 SEQ ID NO: C76; VL CDR3 SEQ ID NO: C77).

[0387] a. GPC3-Targeting Domains

[0388] A GPC3-targeted scFv can include:

[0389] (a) a heavy chain variable region (VH) comprising a VH complementarity determining region (CDR)1 comprising or consisting of a sequence that is identical to a VH CDRI amino acid sequence set forth in Table A2; a VH CDR2 comprising or consisting of a sequence that is identical to a VH CDR2 amino acid sequence set forth in Table A2; and a VH CDR3 comprising or consisting of a sequence that is identical to a VH CDR3 amino acid sequence set forth in Table A2; and

[0390] (b) a light chain variable region (VL) comprising a VL CDRI comprising or consisting of a sequence that is identical to a VL CDRI amino acid sequence set forth in Table A2; a VL CDR2 comprising or consisting of a sequence that is identical to a VL CDR2 amino acid sequence set forth in Table A2; and a VL CDR3 comprising or consisting of a sequence that is identical to a VL CDR3 ammo acid sequence set forth in Table A2.

[0391] A GPC3-targeted scFv includes a VH comprising or consisting of an amino acid sequence that is identical to a VH amino acid sequence set forth in Table A2, or a variant thereof having 1, 2, 3, 4, or 5 single amino acid modifications (e g., substitutions), wherein the modifications are not in the CDR regions (also as set forth in Table A2); and a VL comprising or consisting of an amino acid sequence that is identical to a VL amino acid sequence set forth in Table A2, or a variant thereof having 1, 2, 3, 4, or 5 single ammo acid modifications (e.g., substitutions), wherein the modifications are not in the CDR regions (also as set forth in Table A2).

[0392] A useful GPC3-targeted scFv comprises any one of the following:Attorney Docket No. 40056-0091 WO1 a variable light chain region having CDR1, CDR2, and CDR3 regions comprising SEQ ID NOs: B1-B3 and a variable heavy chain region having CDR1, CDR2, and CDR3 regions comprising SEQ ID NOs: B5-B7;

[0393] a variable light chain region having CDR1, CDR2, and CDR3 regions comprising SEQ ID NOs: B9-B11 and a variable heavy chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B13-B15;

[0394] a single-domain antibody having CDR1, CDR2, and CDR3 regions comprising SEQ ID NOs: B17-B19;

[0395] a single-domain antibody having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B21-B23;

[0396] a variable light chain region comprising SEQ ID NO: B4 and a variable heavy region comprising SEQ ID NO: B8;

[0397] a variable light chain region comprising SEQ ID NO: B12 and a variable heavy region comprising SEQ ID NO: Bl 6;

[0398] an scFv comprising SEQ ID NO: B24;

[0399] a variable light chain region comprising SEQ ID NO: B27 and a variable heavy region comprising SEQ ID NO: B26; and

[0400] an scFv comprising SEQ ID NO: B28.

[0401] TABLE A2: GPC3-Binding Sequences

[0402] GPC3 VL1 CDR1 GGNNIGSKSV H (SEQ ID NO: B1)

[0403] GPC3 VL1 CDR2 DDSDRPS (SEQ ID NO: B2)

[0404] GPC3 VL1 CDR3 QVWDSSSDHV V (SEQ ID NO: B3)

[0405] SYVLTQPPSV SVAPGQTARI TCGGNNIGSK SVHWYQQPPG QAPVVVVYDD SDRPSGIPER GPC3 VL1

[0406] FSGSNSGNTA TLTISRVEAG DEAVYYCQVW DSSSDHWFG GGTKLTVLGQ P (SEQ ID NO: B4)

[0407] GPC3 VH1 CDR1 SGSYFWG (SEQ ID NO: B5)

[0408] GPC3 VH1 CDR2 SIYYSGITYY NPSLKS (SEQ ID NO: B6)

[0409] GPC3 VH1 CDR3 HDGAVAGLFD Y (SEQ ID NO: B7)

[0410]

[0411] Attorney Docket No. 40056-0091 WO1

[0412] QLQLQESGPG LVKPSETLSL TCTVSGGS1S

[0413] SGSYFWGWIR QPPGKGLEWI GSIYYSGITY GPC3 VH1 YNPSLKSRVT ISVDTSKNQF SLKLSSVTAA

[0414] DTAVYYCARH DGAVAGLFDY WGQGTLVTVS

[0415] S (SEQ ID NO: B8) DIVMTQSPDSIAVSI. GERATINCKSSOSIXYSSNOKNYLA WYOQKPGOPPKLLIYWASSRESGVPDRFSGSGSGTDFTLT GPC3 VL2

[0416] ISSLOAEDVAVYYCOOYYNYPIirFGOGTKLEIK

[0417] hYP7

[0418] (SEQ ID NO: B12; VL CDR1 SEQ ID NO: B9; VL CDR2 SEQ

[0419] ID NO: B10; VL CDR3 SEQ ID NO: B11) EVQLVESGGGLVQPGGSLRLSCAASGFTFN KNAMNWVROAPGKGLEWVGRIRNKTNNYAT GPC3 VH2 YYADSVKARFTISRDDSKNSLYLOMNSLKT

[0420] hYP7 EDTAVYYCVAGNSFAYWGOGILVTVSA

[0421] (SEQ ID NO: BI 6; VH CDRI SEQ ID NO: BI3; VH CDR2

[0422] SEQ ID NO: B14; VH CDR3 SEQ ID NO: B15) DIVMTOSPDSLAVSLGERATINCKSSOSLLYSSNQKNYLA WYQQKPGQPP KLLIYWASSR ESGVPDRFSG SGSGTDFTLT ISSLQAEDVA VYYCOO YYNY PLIFGQGTKL EIKRSRGGGG GPC3 scFv

[0423] SGGGGSGGGG SLEMAEVQLV ESGGGLVQPG

[0424] hYP7

[0425] GSLRLSCAAS GFTFNKNAMN WVRQAPGKGL EWVGRIRNKT NNYATYYADS VKARFTISRD DSKNSLYLQM NSLKTEDTAV YYCVAGNSFA YVVGQGTLVTV SA (SEQ ID NO: B24)

[0426]

[0427] Attorney Docket No. 40056-0091 WO1

[0428] QVQLVQSGGG LVQPGGSLRL SCAAS YFDFD SYEMSWVRQA PGKGLEWIGS I YHSGSTYYN PSLKSRVTIS RDNSKNTLYL QMNTLRAEDT ATYYCARVNM DRFDYWGQGT LV TVSS

[0429] (SEQ ID NO: B20;

[0430] HN3

[0431] HN3 CDR1 SEQ ID NO: B17 (aa 31- 35); HN3 CDR2 SEQ ID

[0432] NO: B18 (aa 50-65); HN3 CDR3 SEQ ID NO: B19 (aa 96-105);

[0433] OR HN3 CDR1 SEQ ID NO: B21 (aa 26-33); HN3 CDR2 SEQ ID

[0434] NO: B22 (aa 51-57); HN3 CDR3 SEQ ID NO: B23 (aa 96-105)) QVOLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVR OAPGOGLEWMGALDPKTGDTAYSQKFKGRVTLTADKSTS TAYMELSSLTSEDTAVYYCTRFYSYTYWGOGTLVTVSS GPC3 VH3

[0435] (SEQ ID NO: B26)

[0436] (VH CDR1 SEQ ID NO: B29; VH CDR2 SEQ ID NO: B30; VH

[0437] CDR3 SEP ID NO: B31) DVVMTOSPLSLPVTPGEPASISCRSSOSLVHSNRNTYLHW YLOKPGOSPOLL. IYKVSNRFSGVPDRFSGSGSGTDFTLKIS RVEAEDVGVYYCSQNTHVPPTFGOGTKLEIK (SEQ ID

[0438] GPC3 VL3

[0439] NO: B27)

[0440] (VL CDRl SEQ ID NO: B32; VL CDR2 SEQ ID NO: B33; VL

[0441] CDR3 SEQ ID NO: B34) QVQLVQSGAEVKKPGASWVSCKASGYTFTDYEMHWX’R QAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADKSTS TAYMELSSLTSEDTAVYYCTRFYSYTYWGQGTLVTVSSG GPC3 scFv GGGSGGGGSGGGGSDWMTQSPLSLPVTPGEPASISCRSS QSLVHSNRNTYLHWYLQKPGQSPQLLIYKVSNRFSGVPD RFSGSGSGTDFTF. KISRVEAEDVGVYYCSQNTHVPPTFGQ GTKLEIK (SEQ ID NO: B28)

[0442]

[0443] Attorney Docket No. 40056-0091 WO1

[0444] OVOLVESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVRQ APGKGLEWVSVIYSGGSSTYYADSVKGRFTISRDNSKNTL YLQMNSLRAEDTAV Y Y C ARTSYLNHGDYWGOGTLVTVS GPC3 VH4

[0445] S (SEQ ID NO B33)

[0446] (VH CDR1 SEQ ID NO: B34; VH CDR2 SEQ ID NO: B35; VH

[0447] CDR3 SEQ ID NO: B36) OSVLTOPPSVSAAPGORVTISCSGTRSNIGSDYVSWYOHL PGTAPKLLVYGDNI. RPSGIPDRFS ASKSGTS ATLGITGLQT GDEADYYCGTWDYTLNGVVFGGGTKLTVLG (SEQ ID

[0448] GPC3 VL4

[0449] NO: B37)

[0450] (VL CDRl SEQ ID NO: B38; VL CDR2 SEQ ID NO: B39; VL

[0451] CDR3 SEQ ID NO: B40) OVQLVOSGAEVKKPGASVKVSCKASGYTFTSYYMHWVR OAPGOGLEWMGIINPSGGSTSYAOKFQGRVTMTRDTSTS TVYMELSSLRSEDTAVYYCARWHGGPYDYWGOGTI-yTV

[0452] GPC3 VI 15

[0453] SS (SEQ ID NO: B41)

[0454] (VH CDRl SEQ ID NO: B42; VH CDR2 SEQ ID NO: B43; VH

[0455] CDR3 SEQ ID NO: B44)

[0456] QPVLTQPPS VS VAPGKTARI TCGGNNIGSKS VHWYQQKPG QAI’VLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDE ADYYCOVWDSSSDHYVFGTGTKVTVLG (SEQ ID NO:

[0457] GPC3 VL5

[0458] B45)

[0459] (VL CDRl SEQ ID NO: B46; VLCDR2 SEQ ID NO: B47; VL

[0460] CDR3 SEQ ID NO: B48) OVOLVOSGADVRKPGASVKVSCKASGYTFASHGISWVR OAPGOGLEWLGWISPYTGNTNYAOKFOGRVTMATDTSTS TAYMELRSLRSDDTAIYYCARGKRTLASCFDYWGOGTLV GPC3 VH6

[0461] TVSS (SEQ ID NO: B49)

[0462] (VH CDRl SEQ ID NO: B50; VH CDR2 SEQ ID NO: B51; VH

[0463] CDR3 SEQ ID NO: B52)

[0464]

[0465] Attorney Docket No. 40056-0091 WO1

[0466] QSVLTOPPSVSVAPGKTARITCGGNNIGSKSVHWYOQKPG QAPVLVVYDDSDRPSGIPERFSGSNSGNTATLT1SRVEAGD

[0467] E AD YYCO V WD S S SDHVFGTGTKVTVLG (SEQ ID NO:

[0468] GPC3 VI.,6

[0469] B53)

[0470] (VL CDRl SEQ ID NO: B46; VL CDR2 SEQ ID NO: B54; VL

[0471] CDR3 SEQ ID NO: B48) QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWGEINHSGSTNYNPSLKSRVTISVDTSKNQFSLELSSVTAADTAVYYCARGYGGRFDYWGQGTLVTVSS GPC3 VH7

[0472] (SEQ ID NO: B55)

[0473] (VH CDRl SEQ ID NO: B56; VH CDR2 SEQ ID NO: B57; VH

[0474] CDR3 SEQ ID NO: B58) OPVLTOPPSASGTPGORVTISCSGSSSNIGSNNXIWYQOLP GAAPKLLIYSNHRRPSGVPDRFSGSRSGTSASLAISGLQSE DEADYYCAAWDDSLDGYLFGTGTKVTVLG (SEQ ID NO:

[0475] GPC3 VL7

[0476] B59)

[0477] (VL CDRl SEQ ID NO: B60; VL CDR2 SEQ ID NO: B61; VL

[0478] CDR3 SEQ ID NO: B62) OMOLVOSGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRO APGKGLEWVSYISSSGSTI YYADSVKGRFTISRDNAKNSL YLQMN SLRAEDI AVY YCARASDLYGD WGQGTLV TVS S GPC3 W

[0479] (SEQ ID NO: B63)

[0480] (VH CDRl SEQ ID NO: B64; ATI CDR2 SEQ ID NO: B65; VH

[0481] CDR3 SEQ ID NO: B66) OSVLTOPPSVSGTPGORVnSCPGSTSNIGTNTVNWYOQFP GTAPKLLIYSNNQRPSGVPDRFSGSKSGTSASLAISGLQSE DEADYYCAAWDDSLNGVVFGGGTKLTVLG (SEQ ID NO:

[0482] GPC3 VL8

[0483] B67)

[0484] (VL CDRl SEQ ID NO: B68; VL CDR2 SEQ ID NO: B69; VL

[0485] CDR3 SEQ ID NO: B70)

[0486]

[0487] Attorney Docket No. 40056-0091 WO1

[0488] OVOLVOSGAEVKKPGASVTVSCKASGYRFSNYGVSWVR OAPGOGLEWMGWISGSNGNTNYAQKFLGRVTMTTDTST TTAYMELSSLRSDDTAVYYCARGNRRYYSPIIDPWGOGTL GPC3 VH9

[0489] VTVSS (SEQ ID NO: B71)

[0490] (VH CDRl SEQ ID NO: B72; VH CDR2 SEQ ID NO: B73; VH

[0491] CDR3 SEQ ID NO: B74) OAVLTOPPSVSGTPGORVTISCSGSSSNFGSNTVHWYOOV PGTAPKLLIFSNTQRPSEIPDRFSGSKSGTSASLAISGLQSE DEADYYCAAWDDSLTGVVFGGGTKLTVLG (SEQ ID NO:

[0492] GPC3 VL9

[0493] B75)

[0494] (VL CDRl SEQ ID NO: B76; VL CDR2 SEQ ID NO: B77; VL

[0495] CDR3 SEQ ID NO: B78)

[0496]

[0497] The hYP7 VL domain CDR1, CDR2 and CDR3 sequences respectively include amino acids 24-40, 56-62, and 95-103 of SEQ ID NO: Bl 2, The hYP7 VL domain CDR1, CDR2 and CDR3 sequences respectively include amino acids 27-38, 56-58, and 95-103 of SEQ ID NO: B12.

[0498] The hYP7 VH domain CDRl, CDR2 and CDR3 sequences respectively include amino acids 31 -35, 50-68, and 101-106 of SEQ ID NO: B16. The hYP7 VH domain CDRl, CDR2 and CDR3 sequences respectively include amino acids 26-33, 51-60, and 99-106 of SEQ ID NO: B16,

[0499] Any GPC3-targeting domain discussed herein can comprise:

[0500] a VH comprising an amino acid sequence selected from SEQ ID NOs: B8, Bl 6, B26, B33, B41, B49, B55, B63, and B71, or a variant thereof having no more than 5 single amino acid substitutions, wherein the substitutions are not in the CDRs, and

[0501] a VL comprising an ammo acid sequence selected from SEQ ID NOs: B4, Bl 2, B20, B27, B37, B45, B53, B59, B67, and B75, or a variant thereof having no more than 5 single ammo acid substitutions, wherein the substitutions are not in the CDRs.

[0502] In some embodiments, the domain that binds GPC3 (e.g., a GPC3 -targeted scFv or any GPC3-targeting domain described herein) comprises any one of the following:

[0503] a variable light chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B1-B3 and a variable heavy chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B5-B7;Attorney Docket No. 40056-0091 WO1 a variable light chain region having CDR1, CDR2, and CDR3 regions comprising SEQ ID NOs: B9-B11 and a variable heavy chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B13-B15;

[0504] a single domain having CDRl, CDR2, and CDR3 comprising SEQ ID NOs: B17-B19;

[0505] a single domain having CDRl, CDR2, and CDR3 comprising SEQ ID NOs: B21-B23;

[0506] a variable light chain region having CDR1, CDR2, and CDR3 regions comprising SEQ ID NOs: B32-B34 and a variable heavy chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B29-B3I;

[0507] a variable light chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B38-B40 and a variable heavy chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B34-B36;

[0508] a variable light chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B46-B48 and a variable heavy chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B42-B44;

[0509] a variable light chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B46, B54, and B48 and a variable heavy chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B50-B52;

[0510] a variable light chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B60-B62 and a variable heavy chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B56-B58;

[0511] a variable light chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B68-B70 and a variable heavy chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B64-B66; and

[0512] a variable light chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B76-B78 and a variable heavy chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B72-B74.

[0513] Additional GPC3 binding sequences are known m the art, e.g., in WO 2024 / 082051, WO 2023 / 215738, WO 2021 / 226321, US 12,264,190, US 11,447,564, and as described throughout the present application.

[0514] b, CD3-Targeting Domains

[0515] ACD3-targeted domain (e.g., a CD3-targeted scFv) can include:Attorney Docket No. 40056-0091 WO1 (a) a heavy chain variable region (VH) comprising a VH complementarity determining region (CDR)l comprising or consisting of a sequence that is identical to a VH CDRl amino acid sequence set forth in Table A3; a VH CDR2 comprising or consisting of a sequence that i identical to a VH CDR2 amino acid sequence set forth in Table A3; and a VH CDR3 comprising or consisting of a sequence that is identical to a VH CDR3 amino acid sequence set forth in Table A3; and

[0516] (b) a light chain variable region (VL) comprising a VL CDRl comprising or consisting of a sequence that is identical to a VL CDRl amino acid sequence set forth in Table A3; a VL CDR2 comprising or consisting of a sequence that is identical to a VL CDR2 amino acid sequence set forth in Table A3; and a VL CDR3 comprising or consisting of a sequence that is identical to a VL CDR3 ammo acid sequence set forth in Table A3.

[0517] A CD3-targeted scFv can include: a VH comprising or consisting of a sequence that is identical to a VH amino acid sequence set forth in Table A3, or a variant thereof having 1, 2, 3, 4, or 5 single ammo acid modifications (e.g., substitutions), wherein the ammo acid modifications are not in a CDR (also as set forth in Table A3); and a VL comprising or consisting of a sequence that is identical to a VL ammo acid sequence set forth in Table A3, or a variant thereof having 1, 2, 3, 4, or 5 single amino acid modifications (e.g., substitutions), wherein the amino acid modifications are not in a CDR (also as set forth m Table A3).

[0518] The CD3-targeted scFv can include any one of the following:

[0519] a variable light chain region having CDR1, CDR2, and CDR3 regions comprising SEQ ID NOs: C1-C3 and a variable light chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: C5-C7;

[0520] a variable light chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: C9-C11 and a variable light chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: C13-C15;

[0521] a variable light chain region having CDR1, CDR2, and CDR3 regions comprising SEQ ID NOs: C17-C19 and a variable light chain region having CDR1, CDR2, and CDR3 regions comprising SEQ ID NOs: C21-C23;

[0522] a variable light chain region comprising SEQ ID NO: C4 and a variable heavy region comprising SEQ ID NO: C8;

[0523] a variable light chain region comprising SEQ ID NO: Cl 2 and a variable heavy region comprising SEQ ID NO: Cl 6; andAttorney Docket No. 40056-0091 WO1 a variable light chain region comprising SEQ ID NO: C20 and a variable heavy region comprising SEQ ID NO: C24,

[0524] In some embodiments, the domain that binds CD3 (e.g., a CD3-targeted domain, eg. a CD3-targeting scFv) comprises:

[0525] a VH comprising an amino acid sequence selected from SEQ ID NOs: C8, C24, C33, C41, C49, C59, C69, and C70, or a variant thereof having no more than 5 single amino acid modifications (e.g., 1 or 2 single amino acid substitutions), wherein the modifications are not in the CDRs, and

[0526] a VL comprising an amino acid sequence selected from SEQ ID NOs: C4, C20, C29, C37, C45, C55, C62, and C74, or a variant thereof having no more than 5 single ammo acid modifications (e.g., I or 2 single amino acid substitutions), wherein the modifications are not in the CDRs.

[0527] TABLE A3: CD3-Binding Sequences

[0528] CD 3 VL1 CDR1 RASSSVSYMN (SEQ ID NO: C1)

[0529] CD 3 VL1 CDR2 DTSKVAS (SEQ ID NO: C2)

[0530] CD3 VL1 CDR3 QQWSSNPLT (SEQ ID NO: C3)

[0531] VDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQ KSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSME CD3 VL1

[0532] AED AAT Y YCQQW S SNPLTFGAG TKLELK

[0533] (SEQ ID NO: C4)

[0534] CD3 VH1 CDR1 RYTMH (SEQ ID NO: C5)

[0535] CD3 VH1 CDR2 YINPSRGYTNYNQKFKD (SEQ ID NO: C6)

[0536] CD3 VH1 CDR3 YYDDHYCLDY (SEQ ID NO: C7)

[0537] DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVK QRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKS S ST CD3 VIII

[0538] AYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLT VSS (SEQ ID NO: C8)

[0539] CD3 VL2 CDR1 RSSTGAVTTSNYAN (SEQ ID NO: C9)

[0540] CD3 VL2 CDR2 GTNKRAP (SEQ ID NO: C10)

[0541] CD3 VL2 CDR3 ALWYSNLWV (SEQ ID NO: C11)

[0542]

[0543] Attorney Docket No. 40056-0091 WO1

[0544] QTVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWVQQ KPGQAPRGLI GGTNKRAPGT PARFSGSLLG GKAALTLSGV QPEDEAEYYC ALWYSNLWVF GGGTKLTVLG QPKAAPSVTL CD3 LC2 (VL2)

[0545] FPPSSEELQA NKATLVCLIS DFYPGAVTVA WKADSSPVKA GVETTTPSKQ SNNKYAASSY LSLTPEQWKS HRSYSCQVTH EGSTVEKTVA PTECS (SEQ

[0546] ID NO: Cl 2)

[0547] CD 3 VH2 CDR1 TYAMN (SEQ ID NO: C13)

[0548] CD3 VH2 CDR2 RIRSKYNNYATYYAASVKG (SEQ ID NO: C14)

[0549] CD3 VH2 CDR3 HGNFGNSYVSWFAY (SEQ ID NO: C15)

[0550] EVQLVESGGG LVQPGGSLRL SCAASGFTFN TYAMNWVRQA PGKGLEWVAR IRSKYNNYAT YYAASVKGRF TISRDDSKNS LYLQMNSLKT EDTAVYYCAR HGNFGNSYVS WFAYWGQGTL VTVSSASTKG PSVFPLAPCS RSTSESTAAL GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA VLQSSGLYSL SSWTVPSSS LGTKTYTCNV DHKPSNTKVD KRVESKYGPP CD3 HC2 (VH2)

[0551] CPPCPAPEAA GGPSVFEFPP KPKDTLMISR TPEVTCVVVD VSQEDPEVQF NWYVDGVEVH NAKTKPREEQ FNSTYRWSV LTVLHQDWLN GKEYKCKVSN KGLPSSIEKT ISKAKGQPRE PQVYTLPPSQEEMIKNQVSL TCi;v4<(}FYPSDIAVEWESNGQPENNYKTTPPV[.. DSDGSFL.

[0552] LYSKLTVDKSRWQEGN\'rFSCS\rMIIEALIINIIYTQKSLSLS

[0553] L GK (SEQ ID NO: Cl 6)

[0554] CD3 VL3 CDR1 RSSTGAVTTS NYAN (SEQ ID NO: C17)

[0555] CD3 VL3 CDR2 GTNKRAP (SEQ ID NO: C18)

[0556] CD3 VL3 CDR3 ALWYSNLWV (SEQ ID NO: C19)

[0557] QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWVQQ KPGQAPRGLI GGTNKRAPWT CD3 VL3

[0558] PARFSGSLLG DKAALTLSGA QPEDEAEYFC ALWYSNLWVF GGGTKLTVL (SEQ ID NO: C20)

[0559]

[0560] Attorney Docket No. 40056-0091 WO1 CD3 VH3 CDR1 TYAMN (SEQ ID NO: C21)

[0561] CD3 VH3 CDR2 RIRSKYNNYA TYYADSVKD (SEQ ID NO: C22)

[0562] CD3 VH3 CDR3 HGNFGNSYVS WFAY (SEQ ID NO: C23)

[0563] EVQLVESGGG LVQPGGSLKL SCAASGFTFN TYAMNWV RQA SGKGLEWVGR IRSKYNNYATYYADSVKDRF TISRD CD3 VH3

[0564] DSKST LYLQMNSLKT EDTAVYYCVR HGNFGNSYVS WF AYWGQGTL VTVSS (SEQ ID NO: C24)

[0565] EVQLVESGGG LVQPGGSLKL SCAASGFTFN TYAMNWV RQA SGKGLEWVGR IRSKYNNYAT YYADSVKDRF TISRDDSKST LYLQMNSLKT EDTAVYYC VR HGNFGNSYVS WFAYWGQGTL CD3 scFv 3 VTVSSGGGGS GGGGSGGGGS GGGGSQAWT QEPSLTVS

[0566] PG GTVTLTCRSS TGAVTTSNYA NWVQQKPGQA PRGLIGGTNK RAPWTPARFS GSLLGDK AAL TLSGAQPEDE AEYFCALWYS NLWVFGGGTK LTVL (SEQ ID NO: C25)

[0567] CD 3 VL4 CDR1 RSSTGAVTTS NYAN (SEQ ID NO: C26)

[0568] CD3 VL4 CDR2 GTNKRAP (SEQ ID NO: C27)

[0569] CD3 VL4 CDR3 ALWYSNLWV (SEQ ID NO: C28)

[0570] QTWTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWFQQ KPGQAPRGLIGGTNKRAPGVPARFSGSIDGGKAALTLSGV CD3 VL4

[0571] QPEDEAEYYCAIAVYSNIAAATGGGTKWiIK (SEQ ID NO:

[0572] C29)

[0573] CD3 VH4 CDR1 TYAMN (SEQ ID NO: C30)

[0574] CD3 VH4 CDR2 RIRSKYNNYA TYYADSVKD (SEQ ID NO: C31)

[0575] CD3 VH4 CDR3 HGNFGNSYVS WAAY (SEQ ID NO: C32)

[0576] QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVR QAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDS CD3 VH4

[0577] KNTLYLQMNSLRAEDTAVYYCARFIGNFGNSYVSWAAYW GQGTLVTVSS (SEQ ID NO: C33)

[0578] CD 3 VL5 CDR1 GSSTGAVTSGNYPN (SEQ ID NO: C34)

[0579] CD3 VL5 CDR2 GTKFLAP (SEQ ID NO: C35)

[0580]

[0581] Attorney Docket No. 40056-0091 WO1 CD 3 VL5 CDR3 VLWYSNRWV (SEQ ID NO: C36)

[0582] QTVVTQEPSLWSPGGTVTLTCGSSTGAVTSGNYPNWVQ QKPGQAPRGIJGGTKFIAPGTPARFSGSLLGGKAALTLSG CD3 VL5

[0583] VQPEI)EAEYYCVLWYSNRWVFGGGTKIJTVL (SEQ ID

[0584] NO: C37)

[0585] CD3 VH5 CDR1 KYAMN (SEQ ID NO: C38)

[0586] CD3 VH5 CDR2 RIRSKYNNYA TYYADSVKD (SEQ ID NO: C39)

[0587] CD3 VH5 CDR3 HGNFGNSYIS YWAY (SEQ ID NO: C40)

[0588] EVQIATtSGGGLVQPGGSLKLSCAASGFTFNKYAMNWyTR QAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDS CD3 VH5

[0589] KNTAYLQMNNLKTEDTAVYYCVRIIGNFGNSYISYWAYW GQGTLVTVSS (SEQ ID NO: C41)

[0590] QTWTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWVOQ KPGQAPRGLI GGTNKRAPGT CD3 VL6 PARFSGSLLG GKAALTLSGV QPEDEAEYYC

[0591] Teclistamab ALWYSNLWVF GGGTKLTVLG QP

[0592] (SEQ ID NO: C45; VL CDR1 SEQ ID NO: C42; VLCDR2 SEQ

[0593] ID NO: C43; VL CDR3 SEQ ID NO: C44)

[0594] EVQLVESGGG LVQPGGSLRL SCAASGFTFN TYAMNWVROA PGKGLEWVAR IRSKYNNYAT YYAASVKGRF TISRDDSKNS LYLQMNSLKT CD3 VH6

[0595] EDTAVYYCAR HGNFGNSYVS WFAYWGOGTL

[0596] Teclistamab

[0597] VTVSS

[0598] (SEQ ID NO: C49; VH CDR1 SEQ ID NO: C46; W CDR2

[0599] SEQ ID NO C47; VH CDR3 SEQ ID NO C48)

[0600]

[0601] Attorney Docket No. 40056-0091 WO1

[0602] QTVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWV

[0603] 00 KPGQAPRGLI GGTNKRAPGT

[0604] PARFSGSLLG GKAALTLSGV QPEDEAEYYC ALWYSNLW CD3 LC6 (VL6) VF GGGTKLTVLG QPKAAPSVTL

[0605] Teclistamab FPPSSEELQA NKATLVCLIS DFYPGAVTVA WKADSSPVK

[0606] A GVETTTPSKQ SNNKYAASSY LSLTPEQWKS HRSYSCQVTH EGS TVEKTVA PTECS

[0607] (SEQ ID NO: C50)

[0608] EVQLVESGGG LVQPGGSLRL SCAASGFTFN TYAMNWVRQA PGKGLEWVAR IRSKYNNYAT YYAASVKGRF TISRDDSKNS LYLQMNSLKT EDTAVYYC AR I IGNFGNS YVS WFAYWGQGTL VTVSSASTKG PSVFPLAPCS RSTSESTAAL GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA VLQSSGLYSL SSVVTVPSSS LGTKTYTCNV CD3 HC6 (VH6) DHKPSNTKVD KRVESKYGPP CPPCPAPEAA

[0609] Teclistamab GGPSVFLFPP KI’KDTLMISR TPEVTCVVVD

[0610] VSQEDPEVQF ^WYVDGVEVH M AKTKPREEQ

[0611] FNSTYRVVS V LTVLHQDWLN GKEYKCKVSN KGLPSSIEKT ISKAKGQPRE PQVYTLPPSQ EEMTKNQVSL TCLVKGFYPS DIAVEWESNG QPENN YKTTP PVLDSDGSFL LYSKLTVDKS RWQEGNVFSC S VMHEALHNH YTQKSLSLSL GK

[0612] (SEQIDNO C51)

[0613] DIVMTQSPDS LAVSLGERAT INCKSSQSLF NVRSRKNYLA WYQQKPGQPP KLLISWASTR CD3 VL7 ESGVPDRFSG SGSGTDFTLT ISSLQAEDVA

[0614] Elranatamab VYYCKQSYDL FTFGSGTKLE IK (SEQ ID NO: C55; VL

[0615] CDR1 SEQ ID NO: C52; VL CDR2 SEQ ID NO: C53; VL CDR3

[0616] SEQ ID NO: C54)

[0617]

[0618] Attorney Docket No. 40056-0091 WO1

[0619] EVQLVESGGG LVQPGGSLRL SCAASGFTFS DYYMTWVRO A PGKGLEWVAF IRNRARGYTS CD3 VH7 DHNPSVKGRF TISRDNAKNS LYLQMNSLRA

[0620] Elranatamab ED TAV YYCAR DRPSYYVLDY WGQGTTVTVS

[0621] S (SEQ ID NO: C59; VH CDR1 SEQ ID NO C56; VH CDR2

[0622] SEQ ID NO C57; VH CDR3 SEQ ID NO C58)

[0623] DIVMTQSPDS LAVSLGERAT INCKSSOSLF NVRSRKNYL A WYQQKPGQPP KLLISWASTR ESGVPDRFSG SGSGTDFTLT ISSLQAEDVA VYYCKOSYD CD3 LC7 (VL7) I. FTFGSGTKLE IKRTVAAPSV

[0624] Elranatamab FIFPPSDEQL KSGTASVVCL LNNFYPREAK VQWKVDNA

[0625] LQ SGNSQESVTE QDSKDSTYSL SSTLTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGE

[0626] C (SEQ ID N(): C60)

[0627] EVQLVESGGG LVQPGGSLRL SCAASGFTFS DYYMTWVROA PGKGLEWVAF IRNRARGYTS DHNPSVKGRF TISRDNAKNS LYLQMNSLRA EDTAVYYCAR DRPSYYVLDY WGQGTTVTVS SASTKGPSVF PLAPCSRSTS ESTA ALGCLV KDYFPEPVTV SWNSGALTSG VHTFPAVTQS SGLYSLSSW TVPSSNFGTQ TYTCNVDHKP CD3 HC7 (VH7)

[0628] SNTKVDKTVE RKCRVRCPRC PAPPVAGPSV

[0629] Elranatamab

[0630] FLFPPKPKDT LMISRTPEVT CVVVAVSHED PEVQFNWYVD GVEVHNAKTK PREEQFNSTF RVWSVLTVVT I QDWLNGKEYK CKVSNKGLPS SIEKTISKTK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPAILDS DGSFFLYSRL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK (SEQ ID NO: C61)

[0631]

[0632] Attorney Docket No. 40056-0091 WO1

[0633] EIVMTQSPAT LSVSPGERAT LSCRASQSVS SNLAWYQQKP GOAPRLL1YG ASTRATGIPA

[0634] CD3 VL8 RFSGSGSGTE FTLT1SSLQS EDFAVYYCQQ

[0635] TNB-383B YNNWPWTFGO GTKVE1K (SEQ ID NO: C65; VL CDR1

[0636] SEQ ID NO: C62; VL CDR2 SEQ ID NO: C63; VL CDR3 SEQ ID NO C64)

[0637] EVQLVESGGG LVQPGRSLRL SCAASGFTFD DYAMHWVROA PGKGLEWVSG ISWNSGSIGY CD3 VH8 ADSVKGRFTI SRDNAKNSLY LQMNSLRAED

[0638] TNB-383B TALYYCAKDS RGYGDYRLGG AYWGOGTLVT

[0639] VSS (SEQ ID NO: C69; VH CDR1 SEQ ID NO: C66; VII

[0640] CDR2 SEQ ID NO: C67; VI I CDR3 SEQ ID NO: C68) OVOLOOSGAELARPGASVKMSCKASGYTFTRYTMHWV KQRPGOGLEWIGYINPSRGYTNYNQKFKDKATLTTDKS S S TA¥K4OLSSL. TST: DSAVYY(: ARYYDDH¥CL. DYW(}Q(}TTL. T

[0641] CD 3 VH9 (FIG. 33)

[0642] VS (SEQ ID NO: C70)

[0643] (VH CDR 1 SEQ ID NO: C71; VH CDR2 SEQ ID NO: C72; VH

[0644] CDR3 SEQ ID NO: C73) QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKS GTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEA CD3 VL9 (FIG. 33) EDAATYYCQQWSSNPFTFGSGTK LEIK (SEQ ID NO: C74)

[0645] (VL CDR1 SEQ ID NO C75; VL CDR2 SEQ ID NO: C76; VL

[0646] CDR3 SEQ ID NO: C77) QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWV KQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSS TAYM QLS SLTSEDS AVYYC ARY YDDHYCLD Y WGQGTTLT CD3 scFv 9 VSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMT CSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHF RGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGT KLEIK (SEQ ID NO: C78)

[0647]

[0648] Attorney Docket No. 40056-0091 WO1 Additional CD 3 binding sequences are known in the art, e.g., in WO 2013 / 072415, US 2015 / 0376287 Al, WO 2017 / 031104, WO 2017 / 134134, WO 2018 / 052503, WO 2018 / 119215, WO 2019 / 075378, WO 2019 / 164891, WO 2020 / 018820, US 2021 / 0403587 Al, US 2022 / 0041742 Al, US 20220041742 Al, WO 2022 / 006316, WO 2023 / 056391, US 2023 / 0272102 Al, US 2023 / 0257473 Al, US 20230398147 Al, US 2024 / 0018235 Al, and as described throughout.

[0649] 2 GPC3 Cell-Based Therapies

[0650] A useful therapy m any of the methods described herein is a population of autologous or allogeneic GPC3-targeted human immune cells (e.g., macrophages, NK cells, NKT cells, T cells, subpopulations of one or more thereof, and combinations thereof). In some cases, any of the methods described herein comprise administering to the subject an effective amount of a population of immune cells (e.g., human T cells or human NK cells) expressing a CAR or harboring a nucleic acid encoding a CAR, wherein the CAR binds to GPC3 In some cases, the methods described herein comprise the use of a population of immune cells expressing a GPC3-targeted receptor, such as a CAR comprising a GPC3 -targeting domain. In some embodiments, the GPC3 -targeting therapy comprises a population of immune cells, wherein the immune cells express a GPC3 -targeting domain on the cell surface. The GPC3 -targeting domain can include the sequences listed in Table A2, those described throughout, and those known in the art.

[0651] In some cases, the methods described herein comprise administering a population of autologous or allogeneic human immune cells (e.g., macrophages, NK cells, NKT cells, T cells, subpopulations of one or more thereof, and combinations thereof) targeted to GPC3. In some cases, useful autologous or allogenic T cells comprise central memory’ T cells (TCM cells) or a combination of central memory’ T cells, naive T cells, and stem central memory cells (i.e., the T cells are TCM / SCM / Ncells). In some cases, the population of T cells includes both CD4+ cells and CD8+ cells. In some cases, the immune cells express a CAR or are transduced by a nucleic acid encoding a CAR (e.g., a GPC3 CAR).

[0652] In general, a GPC3 CAR includes a GPC3-targeted domain (e.g., a GPC3 scFv), a spacer domain, a transmembrane domain, one or more co-stimulatory domains, and a CD3zeta cytoplasmic domain.

[0653] For example, a useful CAR can comprise the following domains: GPC3 scFv-IgG4(HL-CH3)-CD4tm-41 BB-Zeta.

[0654] A GPC3 CAR can comprise or consist of the ammo acid sequence:Attorney Docket No. 40056-0091 WO1 MLLLVTSLLLCELPHPAFLLIPOVOLVOSGAEVKKPGASVKVSCKASGYTFTDYEMH WVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADKSTSTAYMELSSLTSEDTAVYY CTRFYSYTYWGQGTLVTVSSGGGGSGGGGSGGGGSDVVMTQSPLSLPVTPGEPASISCRSS QSLVHSNRNTYLHWYEQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAED VGVYYCRQNTHVPPTFGOGTKEEIKESKYGPPCPPCPGGGSSGGGSGGOPREPOVYTEPPSQE EAITKNOVSLTCLVKGFYPSDIAYTAVESNGQPENNYKTTPPVI.. DSDGSFFLYSRLTVDKSRWOEG NVFSCSVMHEALHNHYTQKSLSLSLGKMALIVI> GGVAGLLLFIGLGIFFKRGRKKLLYIFKQPFM RPVOTTQEEDGCSCRFPEEEEGGCELGGGRVKFSRSADAPAYQOGQNQLYNELNLGRREEYDV LDKRRGRDPEMGGKPRRKNPOEGLYNELOKDKMAEAYSEIGMKGERRRGKGFIDGLYOGLSTA TKDTYDALHMQALPPR (SEQ ID NO: D1), or a variant thereof have 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ammo acid modifications (e.g., substitutions), wherein the ammo acid modifications are not in the GPC3 targeting domain (e.g., the GPC3 scFv or the CDRs of the GPC3 scFv). The amino acids in SEQ ID NO: DI are underlined above to indicate the domain as in Signal sequence-GPC3scFv-IgG4(IIL-CFI3)-CD4tm-41BB-L-Zeta.

[0655] A useful CAR can comprise the following domains: GPC3scFv-IgG4(IIL-CH3)-CD28tm-CD28gg-Zeta. A GPC3 CAR can comprise or consist of the amino acid sequence:

[0656] MLLLVTSLLLCELPHPAFLLIPQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMH WVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADKSTSTAYMELSSLTSEDTAVYY CTRFYSYTYWGQGTLVTVSSGGGGSGGGGSGGGGSDVVMTQSPLSLPVTPGEPASISCRSS QSLVHSNRNTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAED VGVYYCSONTHVPPTFGOGTKLEIKESKYGPPCPPCPGGGSSGGGSGGQPREPQVYTLPPSQE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEG NVFSCSVMHEALHNHYTOKSLSLSLGKMFWVLVVVGGVLACYSLLVTVAFnFWVRSKRSRGG HSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSGGGRVKFSRSADAPAYQQGQNQLYNELNLG RREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDG LYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: D2), or a variant thereof have 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., substitutions), wherein the amino acid modifications are not in the GPC3 targeting domain (e.g., the GPC3 scFv or the CDRs of the GPC3 scFv). The amino acids in SEQ ID NO: D2 above are underlined to indicate the domain as in Signal sequence-GPC3scFv-IgG4(FR-CH3)-CD28tm-CD28gg-L-Zeta.

[0657] A CAR can comprise any GPC3 targeting domain described herein (e.g., a GPC3 targeted scFv or functional variant, thereof) or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g.,Attorney Docket No. 40056-0091 WO1 substitutions); a transmembrane domain selected from: a CD4 transmembrane domain or variant thereof having 1-5 (e.g., 1 or 2) ammo acid modifications (e.g., substitutions), a CD8 transmembrane domain or variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions), a CD28 transmembrane domain or a variant thereof having 1 -5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions), and a CD3ζ transmembrane domain or a variant thereof having 1-5 (e.g,, 1 or 2) ammo acid modifications (e.g., substitutions); a costimulatory domain (e.g., a CD28 co-stimulatory domain or a variant thereof having 1- 5 (e.g., 1 or 2) ammo acid modifications (e.g., substitutions); or a 4-1 BB costimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions); or both a CD28 co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions) and a 4-1 BB co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions); and a CD3ζ signaling domain or a variant thereof having 1-5 (e.g., 1 or 2) ammo acid modifications.

[0658] Other suitable CAR and immune cells (e.g., CAR T cells and caTCR-T cells) targeted to GPC3 are described in US11447564, hereby incorporated by reference.

[0659] (a) Extracellular Binding Domain (e.g., GPC3 targeting domain)

[0660] A useful GPC3-targeted receptor, such as a CAR, includes a GPC3 -targeting domain (e.g., a GPC3 scFv). In some embodiments, the GPC3 -targeting domain can include any of the sequences and domains listed in A2. In some embodiments, the GPC 3 -targeting domain comprises a GPC3 scFv. In some embodiments, the GPC3 scFv can comprise or consist of the amino acid sequence:

[0661] DIVMTQSPDS LAVSLGERAT INCKSSQSLL YSSNQKNYLA WYQQKPGQPP KLLIYWASSR ESGVPDRFSG SGSGTDFTLT ISSLQAEDVA VYYCQQYYNY PLTFGOGTKL EIKRSRGGGG SGGGGSGGGG SLEMAEVQLV ESGGGLVQPG GSLRLSCAAS GFTFNKNAMN WVRQAPGKGL EWVGRIRNKT NNYATYYADS VKARFTISRD DSKNSLYLQM NSLKTEDTAV YYCVAGNSFA YWGQGTLVTV SA (SEQ ID NO: D3), or a variant thereof having 1, 2, 3, 4, or 5 single amino acid modifications (e.g., substitutions), wherein the amino acid modifications are not in a CDR (underlined).

[0662] A GPC3 scFv can comprise or consist of the amino acid sequence:

[0663] QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADKSTSTAYMELSSLTSEDTAVYYCTRFYSYTYWGQGTLVTVSSGGGGSGGGGSGGGGSDVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNRNTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIK (SEQ ID NO: B28),Attorney Docket No. 40056-0091 WO1 or a variant thereof having 1, 2, 3, 4, or 5 single amino acid modifications (e.g., substitutions), wherein the amino acid modifications are not in a CDR (underlined),

[0664] A GPC3 scFv can comprise a light chain variable domain as set forth in table A2, or a variant thereof with 1, 2, 3, 4, or 5 single ammo acid substitutions, wherein the ammo acid substitutions are not in the light chain CDRs 1-3 (underlined); and

[0665] a heavy chain variable domain as set forth in table A2, or a variant thereof with 1, 2, 3, 4, or 5 single ammo acid substitutions, wherein the amino acid substitutions are not in heavy chain CDRs 1-3 (underlined).

[0666] A GPC3 -targeting domain (e.g., a GPC scFv) can comprise:

[0667] a variable light chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: BI -B3 and a variable heavy chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B5-B7;

[0668] a variable light chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B9-B11 and a variable heavy chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B13-B15;

[0669] a single-domain having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B17-B19; a single-domain having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B21-B23; a variable light chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B32-B34 and a variable heavy chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B29-B31;

[0670] a variable light chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B38-B40 and a variable heavy chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B34-B36;

[0671] a variable light chain region having CDRl, CDR2, and CDRS regions comprising SEQ ID NOs: B46-B48 and a variable heavy chain region having CDRl, CDR2, and CDRS regions comprising SEQ ID NOs: B42-B44;

[0672] a variable light chain region having CDRl, CDR2, and CDRS regions comprising SEQ ID NOs: B46, B54, and B48 and a variable heavy chain region having CDRl, CDR2, and CDRS regions comprising SEQ ID NOs: B50-B52;

[0673] a variable light chain region having CDRl, CDR2, and CDRS regions comprising SEQ ID NOs: B60-B62 and a variable heavy chain region having CDRl, CDR2, and CDRS regions comprising SEQ ID NOs: B56-B58;Attorney Docket No. 40056-0091 WO1 a variable light chain region having CDR1, CDR2, and CDR3 regions comprising SEQ ID NOs: B68-B70 and a variable heavy chain region having CDR1, CDR2, and CDR3 regions comprising SEQ ID NOs: B64-B66;

[0674] a variable light chain region having CDR1, CDR2, and CDR3 regions comprising SEQ ID NOs: B76-B78 and a variable heavy chain region having CDR1, CDR2, and CDR3 regions comprising SEQ ID NOs: B72-B74;

[0675] a variable light chain region comprising SEQ ID NO: B4 and a variable heavy region comprising SEQ ID NO: B8;

[0676] a variable light chain region comprising SEQ ID NO: B12 and a variable heavy region comprising SEQ ID NO: Bl 6;

[0677] a single domain comprising SEQ ID NO: B20;

[0678] an scFv comprising SEQ ID NO: B24;

[0679] a variable light chain region comprising SEQ ID NO: B27 and a variable heavy region comprising SEQ ID NO: B26;

[0680] an scFv comprising SEQ ID NO:28;

[0681] a variable light chain region comprising SEQ ID NO: B37 and a variable heavy region comprising SEQ ID NO: B33;

[0682] a variable light chain region comprising SEQ ID NO: B45 and a variable heavy region comprising SEQ ID NO: B41;

[0683] a variable light chain region comprising SEQ ID NO: B53 and a variable heavy region comprising SEQ ID NO: B49;

[0684] a variable light chain region comprising SEQ ID NO: B59 and a variable heavy region comprising SEQ ID NO: B55;

[0685] a variable light chain region comprising SEQ ID NO: B67 and a variable heavy region comprising SEQ ID NO: B63; or

[0686] a variable light chain region comprising SEQ ID NO: B75 and a variable heavy region comprising SEQ ID NO: B71.

[0687] The light chain variable domain can precede the heavy chain variable domain and they can be joined by a linker that includes 5-20 amino acids, preferably G and S. In some embodiments, the heavy chain variable domain can precede the light chain variable domain and they can be joined by a linker that includes 4-20 amino acids, preferably G and S. In some embodiments, such linkers can compriseAttorney Docket No. 40056-0091WO1 GGGS (SEQ ID NO: D7) or repeats thereof, including the sequence: GGGGSGGGGSGGGGS (SEQ ID NO: D8) or GGGGSGGGGS (SEQ ID NO: D9), and can be located between the VH and VL domains.

[0688] Other examples and GPC3-targeted moieties and GPC3 scFvs are known in the art; for example, CAR (hYP7)-T cells (US Clinical Trial NCT05003895), CT017 CAR T cells (US Clinical Trial NCT03980288), C-CAR031 (NCT05155189), GLYCART cells (NCT02905188), GPC3-CART cells (NCT06641453); 21.15. GPC3 -CART cells (NCT06198296), GPC3 / TGFβ-CAR-T cells

[0689] (NCT03198546), GPC3-CART (NCT03130712), CAR-GPC3 T cells (NCT02876978), GPC3-CAR-T cells (NCT05344664), CBG166 (NCT06461624), and as described throughout.

[0690] (b) Transmembrane Domain

[0691] A CAR disclosed herein can contain a transmembrane domain, which can be a hydrophobic alpha helix that spans the membrane. As used herein, a transmembrane domain refers to any protein structure that is thermodynamically stable in a cell membrane, preferably a eukaryotic cell membrane.

[0692] Transmembrane domains that can be used include those shown below in Table 1.

[0693] Table 1: Examples of Transmembrane Domains

[0694] Name Accession Length Sequence

[0695] CD3z J04132.1 21 aa LC YLLDGILFIYGVILTALFL (SEQ ID NO: 15)

[0696] CD28 NM_006139 27aa FWVLVWGGVLACYSLLVTVAFIIFVW (SEQ ID NO:

[0697] 16)

[0698] CD28(M) NM_006139 28aa MFWVLVWGGVLACYSLLVTVAFIIFWV (SEQ ID NO:

[0699] 17)

[0700] CD4 M35160 22aa MALIVLGGVAGLLLFIGLGIFF (SEQ ID NO: 18)

[0701] CD8tm NM_001768 21aa IYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 19)

[0702] CD8tm2 NM_001768 23aa IYIWAPLAGTCGVLLLSLVITLY (SEQ ID NO: 20)

[0703] CD8tm3 NM_001768 24aa IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 21)

[0704] 4-1BB NM_001561 27aa IISFFLALTSTALLFLLFF LTLRFSVV (SEQ ID NO: 22)

[0705] NKG2D NM_007360 21aa PFFFCCFIAVAMGIRFIIMVA (SEQ ID NO: 23)

[0706]

[0707] Attorney Docket No. 40056-0091WO1 (c) Spacer Domain

[0708] A GPC3 CAR described herein can include a spacer located between the GPC3 targeting domain (i.e., a GPC3 targeted scFv or functional variant thereof) and the transmembrane domain. Without being bound by theory, the spacer can function to provide flexibility to the CAR, or domains thereof, or to prevent steric hindrance of the CAR, or domains thereof. A variety of different spacers can be used. Some of them include at least portion of a human Fc region, for example a hinge portion of a human Fc region or a CH3 domain, or variants thereof. Table 2 below provides various spacers that can be used in the CARs or polypeptides described herein.

[0709] Table 2: Examples of Spacer Domains

[0710] Name Length Sequence

[0711] a3 3 aa AAA

[0712] linker 10 aa GGGSSGGGSG (SEQ ID NO: 24)

[0713] IgG4 hinge (S→P) 12 aa ESKYGPPCPPCP (SEQ ID NO: 25)

[0714] (S228P)

[0715] lgG4 hinge 12 aa ESKYGPPCPSCP (SEQ ID NO: 26)

[0716] lgG4 hinge (S228P) + 22 aa ESKYGPPCPPCPGGGSSGGGSG (SEQ ID NO: 27)

[0717] linker

[0718] Also called HL

[0719] CD28 hinge 39 aa IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP

[0720] (SEQ ID NO: 28)

[0721] CD8 hinge-48aa 48 aa AKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAV HTRGLDFACD (SEQ ID NO: 29)

[0722] CD8 hinge-45aa 45aa TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTR GLDFACD (SEQ ID NO: 30)

[0723] IgG4 (HL-CH3) 129 aa ESKYGPPCPPCPGGGSSGGGSGGQPREPQVYTLPPS QEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY

[0724] Also called: lgG4(HL- KTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVM

[0725] ACH2) HEALHNHYTQKSLSLSLGK (SEQ ID NO: 31)

[0726] (includes S228P in hinge)

[0727]

[0728] Attorney Docket No. 40056-0091WO1 IgG4(L235E, N297Q) 229 aa ESKYGPPCPSCPAPEFEGGPSVFLFPPKPKDTLMISRT PEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPRE EQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPS SIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLS LGK (SEQ ID NO: 32)

[0729] IgG4(S228P, 229 aa ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRT L235E, N297Q) PEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPRE EQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPS SIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLS LGK (SEQ ID NO: 33)

[0730] IgG4(CH3) 107 aa GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKS

[0731] Also called lgG4(ACH2) RWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ

[0732] ID NO: 34)

[0733]

[0734] Some spacer regions include all or part of an immunoglobulin (e.g., IgGl, IgG2, IgG3, IgG4) hinge region, i.e., the sequence that falls between the CHI and CH2 domains of an immunoglobulin, e.g., an IgG4 Fc hinge or a CD8 hinge. Some spacer regions include an immunoglobulin CH3 domain (called CH3 or ACH2) or both a CH3 domain and a CH2 domain. The immunoglobulin derived sequences can include one or more amino acid modifications, for example, 1, 2, 3, 4 or 5 substitutions, e.g., substitutions that reduce off-target binding.

[0735] The hinge / linker region can also comprise an IgG4 hinge region having the sequence ESKYGPPCPSCP (SEQ ID NO: 26) or ESKYGPPCPPCP (SEQ ID NO: 25). The hinge / linker region can also comprise the sequence ESKYGPPCPPCP (SEQ ID NO: 25) followed by the linker sequence GGGSSGGGSG (SEQ ID NO: 24) followed by IgG4 CH3 (HL-CH3) sequence GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 34). Thus, the entire linker / spacer region can comprise the sequence:

[0736] ESKYGPPCPPCPGGGSSGGGSGGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSL GK (SEQ ID NO: 31). In some cases, the spacer has 1, 2, 3, 4, or 5 single amino acid changes (e.g., conservative changes) compared to SEQ ID NO: 31. In some cases, the IgG4 Fc hinge / linker region that is mutated at two positions (L235E; N297Q) in a manner that reduces binding by Fc receptors (FcRs).Attorney Docket No. 40056-0091WO1 (d) Intracellular Signaling Domains

[0737] A CAR construct described herein contains one or more intracellular signaling domains (e.g., CD3ζ, and optionally one or more co-stimulatory domains), which are the functional end of the receptor. Following antigen recognition, receptors cluster and a signal is transmitted to the cell.

[0738] CD3ζ is the cytoplasmic signaling domain of the T cell receptor complex. CD3g contains three immunoreceptor tyrosine-based activation motifs (IT AMs), which transmit an activation signal to the T cell after the T cell is engaged with a cognate antigen. In some cases, CD3 provides a primary T cell activation signal but not a fully competent activation signal, which requires a co-stimulatory signal.

[0739] Accordingly, in some examples, the CAR constructs disclosed herein may further comprise one or more co-stimulatory signaling domains in addition to CD3ζ. For example, the co-stimulatory domain CD28 and / or 4-1 BB can be used to transmit a proliferative / survival signal together with the primary signaling mediated by CD3^.

[0740] The co-stimulatory domain(s) are located between the transmembrane domain and the CD3C signaling domain. Table 3 includes examples of suitable co-stimulatory domains together with the sequence of the CD3ζ signaling domain.

[0741] Table 3: CD3^ Domain and Examples of Co-stimulatory Domains

[0742] Name Accession Length Sequence

[0743] CD3^ J04132.1 113 aa RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRG R DPEMGGKPRRKN PQEGLYNELQKDKMAEAYSEIGMKG ERR RG KG H DG LYQG LSTATKDTYDALH MQALPP R (SEQ ID NO:

[0744] 35)

[0745] ITAMS 1-3 underlined

[0746] CD3^ 113 aa RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGR DPEMGGKPRRKNPQEGLFNELQKDKMAEAFSEIGMKGERR

[0747] variant

[0748] RGKGHDGLFQGLSTATKDTFDALHMQALPPR (SEQ ID

[0749] NO:50)

[0750] CD3^ 113 aa RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGR DPEMGGKPRRKNPQEGLFNELQKDKMAEAFSEIGMKGERR

[0751] variant

[0752] RG KG HDG LYQG LSTATKDTYDALH MQALPPR (SEQ ID

[0753] NO:51)

[0754]

[0755] Attorney Docket No. 40056-0091WO1 CD3^ 113 aa RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGR DPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERR

[0756] variant

[0757] RGKGHDGLFQGLSTATKDTFDALHMQALPPR (SEQ ID

[0758] NO:52)

[0759] CD3t; 113 aa RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGR DPEMGGKPRRKNPQEGLYNELQKDKMAEAFSEIGMKGERR

[0760] variant

[0761] RGKGHDGLFQGLSTATKDTFDALHMQALPPR (SEQ ID

[0762] NO:53)

[0763] CD3^ 113 aa RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGR DPEMGGKPRRKNPQEGLFNELQKDKMAEAYSEIGMKGERR

[0764] variant

[0765] RGKGHDGLFQGLSTATKDTFDALHMQALPPR (SEQ ID

[0766] NO:54)

[0767] CD3^ 113 aa RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGR DPEMGGKPRRKNPQEGLFNELQKDKMAEAFSEIGMKGERR

[0768] variant

[0769] RGKGHDGLYQGLSTATKDTFDALHMQALPPR (SEQ ID

[0770] NO:55)

[0771] CD3^ 113 aa RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGR DPEMGGKPRRKNPQEGLFNELQKDKMAEAFSEIGMKGERR

[0772] variant

[0773] RGKGHDGLFQGLSTATKDTYDALHMQALPPR (SEQ ID

[0774] NO:56)

[0775] CD28 NM_0061 42aa RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

[0776] 39 (SEQ ID NO: 36)

[0777] CD28gg NM_0061 42aa RSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYR

[0778] * 39 S (SEQ ID NO: 37)

[0779] 41BB NM_0015 42 aa KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0780] 61 (SEQ ID NO: 38)

[0781] 0X40 NM_0033 42 aa ALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI

[0782] 27 (SEQ ID NO:39)

[0783] 2B4 NM_0163 120 aa WRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQEQTFPGG

[0784] 82 GSTIYSMIQSQSSAPTSQEPAYTLYSLIQPSRKSGSRKRNHSPS FNSTIYEVIGKSQPKAQNPARLSRKELENFDVYS (SEQ ID NO:

[0785] 40)

[0786]

[0787] Attorney Docket No. 40056-0091 WO1 The CD3 signaling domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 98% identical to SEQ ID NO: 35. In such instances, the CD3£ signaling domain has 1, 2, 3, 4, or 5 single amino acid changes (preferably conservative substitutions) compared to SEQ ID NO: 35, In other examples, the CD3ζ signaling domain is SEQ ID NO: 35.

[0788] The co-stimulatory domain can be: a co-stimulatory domain depicted in Table 3 or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications, a CD28 co-stimulatory domain or a variant thereof having 1-5 e.g., 1 or 2) ammo acid modifications, a 4-1BB co-stimulatory domain or a variant thereof having 1-5 (e.g, 1 or 2) amino acid modifications and an 0X40 co-stimulatory domain or a variant thereof having 1-5 (e.g, 1 or 2) amino acid modifications. In certain embodiments, a 4-1BB co- stimulatory domain or a variant thereof having 1-5 (e.g, 1 or 2) amino acid modifications is present in the CAR polypeptides described herein.

[0789] A CAR can include two co-stimulatory domains, for example, a CD28 co-stimulatory domain or a variant thereof having 1-5 (e.g, 1 or 2) amino acid modifications (e.g, substitutions) and a 4-1BB co-stimulatory domain or a variant thereof having 1-5 (e.g, 1 or 2) amino acid modifications (e.g, substitutions). In various embodiments the 1-5 (e.g., 1 or 2) amino acid modification are substitutions. In various embodiments, the co-stimulatory domain is ammo terminal to the CD3ζ signaling domain and a short linker consisting of 2 - 10, e.g, 3 ammo acids (e.g, GGG) can be positioned between the co- stimulatory domain and the CD3ζ signaling domain.

[0790] The costimulatory domain can be: a CD28 costimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2 acid modifications, a 4-1 BB costimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications and an 0X40 costimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications; a 4-1BB costimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications: CD28 co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions) and a 4-1 BB co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions). In various embodiments the 1-5 (e.g., 1 or 2) amino acid modification are substitutions.

[0791] There can be a short sequence of 1-6 amino acids (e.g. GGG) between the co-stimulatory domains and the CD3ζ signaling domain and / or between the two co- stimulatory domains.

[0792] A useful population of human immune cells (e.g., human T cells or human NK cells) includes immune cells transduced by a nucleic acid molecule encoding a chimeric antigen receptor, wherein chimeric antigen receptor comprises:

[0793] a GPC3 targeting domain (e.g., an scFv targeted to GPC3 or functional variant thereof);Attorney Docket No. 40056-0091 WO1 a transmembrane domain selected from: a CD4 transmembrane domain or variant thereof having 1-5 single amino acid modifications (e.g,, 1 or 2) ammo acid modifications (e.g., substitutions), a CD8 transmembrane domain or variant thereof having 1-5 single amino acid modifications (e.g,, 1 or 2) amino acid modifications (e.g., substitutions), a CD28 transmembrane domain or a variant thereof having 1-5 single amino acid modifications (e.g., 1 or 2) amino acid modifications (e.g., substitutions), and a CD3ζ transmembrane domain or a variant thereof having 1-5 single amino acid modifications (e.g., 1 or 2) amino acid modifications (e.g., substitutions);

[0794] a costimulatory domain (e.g., a CD28 co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions); or a 4-1 BB co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) ammo acid modifications (e.g., substitutions); or both a CD28 co-stimulatory domain or a variant thereof having 1 -5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions) and a 4-1BB co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions); and

[0795] a CD3ξ signaling domain or a variant thereof having 1-5 single amino acid modifications (e.g., 1 or 2) amino acid modifications (e.g., substitutions).

[0796] The CAR can include a spacer region located between the GPC3 targeting domain (e.g., a GPC3 scFv) and the transmembrane domain. A variety of different spacers can be used. Some of them include at least portion of a human Fc region, for example a hinge portion of a human Fc region or a CH3 domain or variants thereof.

[0797] The population of human T cells can include central memory T cells (TCM cells) e.g., at least 20%, 30%, 40%, 50% 60%, 70%, 80% of the cells are TCM cells, or the population of T cells comprises a combination of central memory T cells, naive T cells and stem central memory cells (TCM / SCM / N cells) e.g., at least 20%, 30%, 40%, 50% 60%, 70%, 80% of the cells are TCM / SCM / N cells. In either case, the population of T cells includes both CD4+ cells and CD8+ cells (e.g., at least 20% of the CD3+ T cells are CD4+ and at least 3% of the CD3+ T cells are CD8+ and at least 70, 80 or 90% are either CD4+ or CD8+; at least 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60% of the cells CD3+ cells are CD4+ and at least 4%, 5%, 8%, 10%, 20 of the CD3+ cells are CD8+ cells).

[0798] Combination Therapies

[0799] The therapeutic methods described herein further comprise administering to the subject an effective amount of a population of immune cells expressing a C AR or harboring a nucleic acid encoding a CAR targeted to cancer cells (e.g., solid tumor cells; e.g., HER2-expressing cells). In someAttorney Docket No. 40056-0091 WO1 cases, the CAR is targeted to HER2. In some cases, a HER2 CAR comprises a scFv targeted to HER2. In some cases, the solid tumor is a HER2-expressing cancer. In some embodiment, the population of immune cells expressing a CAR or harboring a nucleic acid encoding a CAR is a population of HER2 CAR T cells. In some cases, the population of immune cells expressing a CAR or harboring a nucleic acid encoding a CAR (e.g., HER2 CAR T cells) are autologous or allogeneic. In some cases, the population of immune cells expressing a CAR or harboring a nucleic acid encoding a CAR (e.g., HER2 CAR T cells) are administered simultaneously or subsequently to administering to the subject a GPC3-targeted therapy described herein (e.g., TCE-GPC3 or GPC3 CAR T cells). In some cases, the HER2 CAR T cells are administered at least or about 0.1, 0.25, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 26, 17, 18, 19, or 20 days after administration of the TCE. In some embodiments, the HER2 CAR T cells are administered at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 26, 17, 18, 19, or 20 weeks after administration of the TCE.

[0800] Useful HER2 CAR constructs are described in WO 2017 / 079694.

[0801] In general, HER2 CAR include a HER2 scFv, a spacer domain, a transmembrane domain, one or more co-stimulatory domains, and a CD3zeta cytoplasmic domain. For example, a HER2 CAR can comprise or consist of the amino acid sequence:

[0802] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKI’GKAPKLLIYSASFLYSGVPS RFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTITPTFGQGIKVEIKGSTSGGGSGGGSGGGGSSE VQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSV KGRFTISADTSKNTAYLQN'INSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSESKYGPP CPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKT KPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPP SQEEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRW QEGNVFSCSVMHEALHNHYTQKSLSLSLGKMFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRS RGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSGGGRVKFSRSADAPAYQQGQNQLYNE LNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGK GHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:ZZ1), or a variant thereof have 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., substitutions), wherein the amino acid modifications are not in the HER2 targeting domain (e.g., the CDRs of the HER2 scFv). A useful HER2 CAR can comprise or consist of the amino acid sequence:

[0803] DIQMTQSPSSLSASVGDRVTITCRASQDWTAVAWYQQKPGKAPKLLIYSASFLYSGVPS RFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKYTIKGSTSGGGSGGGSGGGGSSEAttorney Docket No. 40056-0091 WO1 VQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSV KGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSESKYGPP CPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKT KPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPP SQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRW QEGNVFSCSVMHEALHNHYTQKSLSLSLGKIYIWAPLAGTCGVLLLSLVITKRGRKKLLYIFKQP FMRPVQTTQEEDGCSCRFPEEEEGGCELGGGRVKFSRSADAPAYQQGQNQLYNELNLGRREEY DVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGL STATKDTYDALHMQALPPR (SEQ ID NO:ZZ2), or a variant thereof have 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 single amino acid modifications (e.g., substitutions), wherein the ammo acid modifications are not in the HER2 targeting domain (e.g., the CDRs of the HER2 scFv).

[0804] A useful HER2 scFv can comprise or consist of the amino acid sequence:

[0805] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGS RSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGGGSGGGGSGGGGSEQVLVESG GGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISA DTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO:ZZ3) or is least 90%, 95%. 98%, 99% identical to SEQ ID NO:ZZ3. AHER2 scFv can comprise: a light chain variable domain that is at least 90%, 95%, 98%, 99% or 100% identical to:

[0806] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGS RSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: ZZ4) and comprises light chain CDRs 1-3 (underlined) and a heavy chain variable domain that is at least 90%, 95%, 98%, 99% or 100% identical to:

[0807] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADS VKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO: ZZ5) and comprises heavy chain CDRs 1-3 (underlined). In some embodiments, the light chain variable domain can precede the heavy chain variable domain and they can be joined by a linker that includes 5-20 amino acids, preferably Gand S; as described above.

[0808] A useful scFv can target HER2 A useful HER2 scFv can comprise or consist of the amino acid sequence:

[0809] DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGS GSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKGGGGSGGGGSGGGGSEQVLVESG GGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSAttorney Docket No. 40056-0091 WO1 WRSKNTLYLOMNSLRAEDTAVYYCARNLGPSFYFDYWGOGTLVTVSS (SEQ ID NO: ZZ6) or is least 90%, 95%, 98%, 99% identical to SEQ ID NO: ZZ6. A HER2 scFv can comprise: a light chain variable domain that is at least 90%, 95%, 98%, 99% or 100% identical to:

[0810] DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGS GSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIK (SEQ ID NO: ZZ7) and comprises light chain CDRs 1-3 (underlined) and a heavy chain variable domain that is at least 90%, 95%, 98%, 99% or 100% identical to:

[0811] EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQ RFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSS (SEQ ID NO: ZZ8) and comprises heavy chain CDRs 1-3 (underlined). In some embodiments, the light chain variable domain can precede the heavy chain variable domain and they can be joined by a linker that includes 5-20 ammo acids, preferably G and S, as described above.

[0812] Generally, a useful I IER2 CAR can comprise:

[0813] any HER2 scFv described herein or a variant thereof having 1 -5 (e.g., 1 or 2) single amino acid modifications (e.g., substitutions), wherein the modifications are not in a CDR region;

[0814] a transmembrane domain selected from: a CD4 transmembrane domain or variant thereof having 1-5 (e.g., 1 or 2) single amino acid modifications (e.g., substitutions), a CD8 transmembrane domain or variant thereof having 1-5 (e.g., 1 or 2) single ammo acid modifications (e.g., substitutions), a CD28 transmembrane domain or a variant thereof having 1-5 (e.g., 1 or 2) single ammo acid modifications (e.g., substitutions), and a CD3ξ transmembrane domain or a variant thereof having 1-5 (e.g., 1 or 2) single amino acid modifications (e.g., substitutions);

[0815] a costimulatory domain (e.g., a CD28 co-stimulatory domain or a variant thereof having 1- 5 (e.g., 1 or 2) single amino acid modifications (e.g., substitutions); or a 4-1 BB co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) single amino acid modifications (e.g., substitutions); or both a CD28 co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) single amino acid modifications (e.g., substitutions) and a 4-1 BB co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) single amino acid modifications (e.g., substitutions); and

[0816] a CD3ζ signaling domain or a variant thereof having 1-5 (e.g., 1 or 2) single amino acid modifications.

[0817] Useful populations of human immune cells (e.g., human T cells or human NK cells) include those transduced by a nucleic acid molecule encoding a chimeric antigen receptor, wherein chimeric antigen receptor comprises: an domain targeted to HER2 (e.g., a HER2 scFv); a transmembrane domainAttorney Docket No. 40056-0091 WO1 selected from: a CD4 transmembrane domain or variant thereof having 1-5 single amino acid modifications (e.g,, 1 or 2 amino acid modifications) (e.g., substitutions), a CD8 transmembrane domain or variant thereof having 1-5 single amino acid modifications (e.g., 1 or 2 amino acid modifications) (e.g., substitutions), a CD28 transmembrane domain or a variant thereof having 1-5 single amino acid modifications (e.g., 1 or 2 amino acid modifications) (e.g., substitutions), and a CD3ξ transmembrane domain or a variant thereof having 1-5 single amino acid modifications (e.g., 1 or 2 amino acid modifications) (e.g., substitutions); a costimulatory domain (e.g., a CD28 co-stimulatory domain or a variant thereof having 1 -5 (e.g., 1 or 2) single amino acid modifications (e.g., substitutions); or a 4-1 BB co-stimulatory domain or a variant thereof having 1-5 single (e.g., 1 or 2) ammo acid modifications (e.g., substitutions); or both a CD28 co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) single amino acid modifications (e.g., substitutions) and a 4-1BB co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) single amino acid modifications (e.g., substitutions); and CD3ξ signaling domain of a variant thereof having 1-5 single amino acid modifications (e.g., 1 or 2 amino acid modifications) (e.g., substitutions).

[0818] The CAR can include a spacer region located between the tumor targeting domain (e.g., a scFv; e.g., a HER2 scFv) and the transmembrane domain. A variety of different spacers can be used as described above (e.g., in Table 2). Some of them include at least portion of a human Fc region, for example a hinge portion of a human Fc region or a CH3 domain or variants thereof.

[0819] The population of human T cells include: central memory T cells (TCM cells) e.g., at least 20%, 30%, 40%, 50% 60%, 70%, 80% of the cells are TCM cells, or the population of T cells comprises a combination of central memory T cells, naive T cells and stem central memory cells (TCM / SCM / N cells) e.g., at least 20%, 30%, 40%, 50% 60%, 70%, 80% of the cells are TCM / SCM / N cells. In either case, the population of T cells includes both CD4+ cells and CD8+ cells (e.g., at least 20% of the CD3+ T cells are CD4+ and at least 3% of the CD3+ T cells are CD8+ and at least 70, 80 or 90% are either CD4+ or CD8+; at least 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60% of the cells CD3+ cells are CD4+ and at least 4%, 5%, 8%, 10%, 20 of the CD3+ cells are CD8+ cells).

[0820] Any of the methods described herein can further comprise administering a population of autologous or allogeneic HER2-targeted human immune cells (e.g., macrophages, NK cells, NKT cells, T cells, subpopulations of one or more thereof, and combinations thereof). In some embodiments, useful autologous or allogenic T cells comprise central memory T cells (TCM cells) or a combination of central memory T cells, naive T cells, and stem central memory cells (i.e., the T cells are TCM / SCM cells). In some embodiments, the population of T cells includes both CD4+ cells and CD8+ cells. InAttorney Docket No. 40056-0091 WO1 some embodiments, the immune cells express a CAR or are transduced by a nucleic acid encoding a CAR (e.g., a HER2 CAR).

[0821] Therapies described herein (e.g,, a TCE) can be administered alone or as a component of a pharmaceutical formulation (composition). The compounds may be formulated for administration, in any convenient way for use in human or veterinary medicine. Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, preservatives and antioxidants can also be present in the compositions.

[0822] Pharmaceutical formulations can be prepared according to any method known to the art for the manufacture of pharmaceuticals. Formulations of the compositions of the invention include those suitable for intradermal, subcutaneous, intravenous, transdermal, intraperitoneal, intramuscular, pulmonary, and / or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient (e.g., a GPC3 TCE) which can be combined with a carrier to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration, e.g., subcutaneous or intravenous. More detail on formulating and administering a TCE is known in the art and can be found, for example, in US 2022 / 0041742 Al, WO 2024 / 044545, and WO 2024 / 044548. The amount of active ingredient which can be combined with a carrier to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect, e.g., an antigen specific T cell response. Useful carriers are well known in the art and can include phosphate buffered saline solutions, water, liposomes, various types of wetting agents, sterile solutions, etc. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, or suspensions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, aqueous solutions, or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, or lactated Ringer's. Intravenous vehicles include fluid and nutrient replenishes, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. In addition, the composition might comprise proteinaceous carriers, like, e.g., serum albumin or immunoglobulin, preferably of human origin.

[0823] More information on formulations, dosage, and administration of therapies such as immune cells (e.g., CART cells), TCEs, Abs, and Ab-drug conjugates is known in the art, for example, in US 2021 / 0403587 Al, US 2022 / 0041742 Al, US 2023 / 0235053 Al, US 2023 / 0340137 Al, WOAttorney Docket No. 40056-0091 WO1 2014 / 209855, WO 2016 / 166629, WO 2018 / 204907, WO 2021 / 226321, WO 2022 / 053990, WO 2023 / 199235, WO 2023 / 215738, WO 2023 / 062188, WO 2024 / 044545, WO 2024 / 044548, WO 2024 / 082051, US 12,264,190, and US 11,447,564.

[0824] The present application is directed to, inter alia, the ability of OV to deliver truncated GPC-3 to solid tumors that can be targeted by a GPC -specific therapy to induce potent anti-tumor responses.

[0825] EXAMPLES

[0826] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0827] MATERIALS AND METHODS

[0828] Study design.

[0829] All in vitro assays were performed with at least duplicate samples and were repeated in at least two independent experiments. All in vivo studies were performed using 6- to 8-week-old NSG, using at least three mice per group for all studies, and four to nine mice were included within each group for all therapeutic and survival studies to ensure statistical power and evenly distributed tumor sizes across groups at treatment initiation. The health condition of mice was monitored daily and euthanasia applied according to the American Veterinary Medical / Association Guidelines. Investigators were not blinded when monitoring mouse survival. All studies were performed under approved protocols of the Institutional Animal Care and Use Committee and the institutional review board.

[0830] Cell lines and viruses

[0831] CV-1 (African green monkey kidney), HepG2 and SK-Hepl (liver cancer), SNU-16 (gastric cancer) and As-PC-1 (pancreatic cancer) were obtained from ATCC. HepG2GPC3-KOcells were generated by CRISPR / Cas9 editing. CV-1 cells were grown m DMEM; cancer lines were grown m RPMI-1640 with 10% FBS, 2 mmol / L L-glutamine, and 100 U / mL pemcillin-streptomycin (Corning). All cultures were maintained at 37 °C, 5% CO2.

[0832] Human triple-negative breast cancer cell lines MDA-MB-468 [American Type Culture Collection (ATCC); HTB-132] and MDA-MB-231 (ATCC, HTB-26) were cultured in complete RPMI-1640 medium (Corning) supplemented with 10% fetal bovine serum (FBS; Omgea Scientific) and lx antibiotic / antimycotic (AA; Gibco). Likewise, human pancreatic cancer cell line AsPC-1 (ATCC, CRL-Attorney Docket No. 40056-0091 WO1 1682) was also cultured in complete RPMI-1640 medium (Corning) supplemented with 10% fetal bovine serum (FBS; Omgea Scientific) and lx antibiotic / antimycotic (AA; Gibco).

[0833] Human liver cancer cell lines HEP3B (ATCC, HB-8064), HEPG2 (ATCC, HB-8065), SK-Hepl (ATCC, HTB-52) and HEPG2-GPC3-KO (gift from Eureka Therapeutics Inc.) were cultured Eagle’s minimum essential medium (EMEM, from ATCC) supplemented with 10% fetal bovine serum (FBS; Omgea Scientific) and lx antibiotic / antimycotic (AA; Gibco). African green monkey kidney cell line CV-1 (ATCC, CCL-70) and human aembryomc kidney cell line HEK-293 (ATCC, CRL-1573) were cultured in Dulbecco’s modified eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Omgea Scientific) and l x antibiotic / antimycotic (A / X; Gibco).

[0834] PACBIO Single Molecule, Real-Time (SMRT) Sequencing

[0835] To construct the SMRTbell libraries, a PacBio standard protocol (20-kb Template Preparation Using BluePippin Size-Selection System) was applied. Using Covaris g-TUBE devices (Woburn, MA), the DNA samples were sheared at 4800 rpm for 2 minutes m Eppendorf Mini Spin plus (Hauppauge, NY), and 0.45X volume of AMPure PB magnetic beads of PacBio (Menlo Park, CA) was applied to concentrate the DN A fragments. To remove single-stranded ends from the DNA fragments, 0.2 U / ul of Exo VII in SMRTbell Template Prep Kit 1.0 of PacBio (Menlo Park, CA) was treated at 37°C for 15 minutes. To repair the damages on the DNA templates, IX DN A Damage Repair Mix in SMRTbell Template Prep Kit 1.0 of PacBio (Menlo Park, CA) was treated at 37°C for 20 minutes. To generate the blunt ends of the DNA, IX End Repair Mix in SMRTbell Template Prep Kit 1.0 of PacBio (Menlo Park, CA) was treated at 25°C for 5 minutes. To ligate the SMRTbell adapters onto the blunt ended DNA, 5 uM of Annealed Blunt Adapters were applied with 0.75 U / ul of Ligase in SMRTbell Template Prep Kit 1.0 of PacBio (Menlo Park, CA). The ligation mixture was incubated at 25°C overnight and then incubated 65°C for 10 minutes. To degrade failed ligation products, ExoIII and ExoVH in SMRTbell Template Prep Kit 1.0 of PacBio (Menlo Park, CA) was treated at 37°C for 1 hour. After ExoIII and ExoVII Treatments of Library, the same amount (38.6 ng) of each sample was pooled.

[0836] All reagents for this protocol were provided by PacBio (Menlo Park, CA) and the reagent concentrations in the protocol were calculated by the Sample Setup module in PacBio SMRT Link (vlO.0.0.108728). The Sequencing Primer (1 ul) was diluted by 29 ul of Elution Buffer and the mixture was incubated at 80°C for 2 minutes. And, the conditioned sequencing primers were incubated with the library at 20°C for 1 hour. 1 ul of Sequel Polymerase 3.0 was diluted by 9 ul of Sequel Binding Buffer.Attorney Docket No. 40056-0091 WO1 1.5 ul of the diluted polymerase was applied to the library with the sequencing primers, and 14.9 ul of the mixture was incubated at 30°C for 1 hour.

[0837] The proper volume of all solutions in this step was calculated by SMRT bank’s Setup module. Complex Dilution Buffer was added mto the polymerase complex, first, and Qubit checked the concentration. After adding AMPure PB beads mto the diluted complex, it was incubated for 5 minutes on a rotator. The collected beads on a magnetic rack was resuspended by Complex Dilution buffer, directly, without any ethanol rinse and the polymerase complex was eluted for 15 minutes on a rotator. The concentration of the eluate was measured by Qubit again to calculate the final loading dilution. Each calculated volume of Complex Dilution Buffer, Polymerase-Complex, Diluted Control, DTT and Sequel Additive was mixed in 85 ul as the final loading dilution on the sample plate and it was loaded into Sequel UI v10.0.0.108728. The concentration of the sample on the plate was 8 pM and Movie Time was 20 hours and 2 hours of Pre-Ex tension Time was applied.

[0838] The primary analyses, including real-time signal processing and base calling were processed by a built-in PacBio Blade Center through Sequel ICS, and result stream directly to SMRT Link

[0839] (v10.0.0.108728). The demultiplexing process was carried out by the Demultiplex Barcode module which Infer Barcode Used option was set as false in SMRT Link (v10.0.0.108728). The high-fidelity (HiFi) CCS reads were generated by SMRT Link (vlO.0.0.108728) Circular Consensus Sequences (CCS) module. CCS reads shorter than 5,000bp were removed before aligning to the reference. Mapping to the reference was performed using pbmm2v 1.0.0 with CCS preset parameters and “-N 1 -12500” option. Samtools vO.1.19 was used to pile up the reads and VarScan v2.3.9 was used to call variants and indels using the following option “-mm- coverage 20 — min-reads2 10 — min-var-freq 0.9 — min-avg-qual 1 -strand-filter 0”. The CCS reads were also filtered using samclip (github.com / tseemann / samclip) for soft and hard clipped alignments, with 50 bp as maximum clip length. To perform the de novo assembly, the HiFi CCS reads were produced by the Circular Consensus Sequences (CCS) module in SMRT Link (vlO.0.0.108728) using the default setting. The fasta file was subjected to the de novo assembly using Canu (v2.1) with the genome size (190 kb) and the batMemory=200 options. If a mis-assembly were suspected, another Canu assembly was carried out using the randomly selected reads by a QIIME Script (subsample fasta.py). The structural feature of the contig sequence was investigated using dotted plot via Seqtools (v4.44.0) and the comparison of the sequences between the contig and its reference was performed by Blastn to discover any sequence variants.Attorney Docket No. 40056-0091 WO1 The global sequence alignment was done using Stretcher

[0840] (ebi.ac.uk / jdispatcher / psa / emboss_stretcher) with default parameters. Blastn

[0841] (blast.ncbi.nlm.nih.gov / Blast.cgi) was used for local sequence alignment.

[0842] Generation of recombinant chimeric orthopoxviruses expressing human GPC3 (including variants, truncations, and mutations)

[0843] To generate a shuttle vector containing the human GPC3 expression cassette with the VACV PSE, the GPC3 complementary DNAs (cDNAs) for variant 1 (GPC3-V1) or variant 2 (GPC3-V2) were polymerase chain reaction (PCR) amplified from the plasmids OHu22791D and OHu26910D using Q5 High-Fidelity 2X Master Mix (New England Biolabs Inc., Ipswich, MA) and the following primers: 5'- TAAGTCGACCACCATGGCCGGGACCGTGCGC-3' and

[0844] 5'-AGGTTAATTAATCAGTGCACCAGGAAGAAGAAG-3'. The PCR fragment was digested with Sall and PacI and cloned into the same-cut p33NCTK-SE-hCD19t replacing hCD19t to yield pSC30 (p33NCTK-SE-GPC3-Vl) and pSC31 (p33NCTK-SE-GPC3-V2). Single or double substitution mutations were introduced in the GPC3-V2 cDNA in the plasmid pSC31 to make the GPC3 protein nonfunctional. F41E (Phenylalanine at position 41 replaced by glutamic acid) has been shown to abolish the ability of GPC3 to bind to wnt and hence abolishes its oncogenic property (10). Another substitution mutation W260R (tryptophan replaced by arginine) also blocks wnt binding(10). Therefore, in order to construct CF33 OV encoding GPC3-V2 with one or both of the above substitution mutations, these mutations were introduced in the GPC3-V2 cDNA in the plasmid pSC31. The shuttle plasmid with singles substitution mutation pSC31(J2R-GPC3-V2-F41E) and pSC31(J2R-GPC3-V2-F41E-W260R) were created through using a site-directed mutagenesis kit following the manufacturers instruction (NEB, cat# E0554S). cDNA sequences in all shuttle plasmids were confirmed by Sanger’s sequencing method.

[0845] These shuttle vectors were used to construct the recombinant viruses expressing GPC3-V1, GPC3-V2, GPC3-V2-F41E or GPC3-V2-F41E-W260R. CRISPR / Cas9 technology was used to assist with the selection of recombinant virus, as previously described (11). Briefly, J2R specific oligo was designed and purchased from IDT. The oligo (5’-GTTATAGTAGCCGCACTCG-3’j was used to synthesize single guide RNA (sgRNA) using the EnGen sgRNA Synthesis Kit (Cat#E3322S; NEB) following the manufacturer’s instruction. Next, ~70% confluent HEK293 cells in a 24 well plate were infected with CF33 virus at an MOI of 0.1. Two hours after infection, medium was changed with fresh DMEM medium supplemented with 10% FBS, and the cells were transfected with ribonucleoproteinAttorney Docket No. 40056-0091 WO1 complex of sgRNA and Cas9 together with the template for homologous recombination i.e., the J2R-shuttle plasmids containing GPC3 expression cassettes, Lipofectamine CRISPRMAX transfection reagent (Cat#CMAX00008; ThermoFisher) was used for transfection; 1250 ng of Cas9 and 240 ng sgRNA was used per well. The following day, cell lysate was collected and subjected to 3 rounds of freeze-thaw cycles. The lysate was then used to infect CV1 cells in 6 well plates and methylcellulose overlay was applied. Two days after incubation, plaques were picked and PCR verified. Two more rounds of plaque purification were performed in 6-well plates using methylcellulose overlay. Finally, limiting-dilution procedure was performed in A549 cells in 96-well plate for final clone selection, which was then used to produce a seed stock in a 100 mm dish of A549 cells. The seed stock was then used to further amplify the virus, and the virus was purified first on a sucrose cushion followed by further purification on sucrose gradient using high-speed centrifugation. The transgene in the recombinant virus was sequence verified.

[0846] Generation ofGPC3 ARTEMIS T cells (ECT204), human T cell enrichment, lentivirus production and transduction, and ex vivo expansion

[0847] GPC3 specific CAR-T cells and mock CAR T cells were gift from Eureka Therapeutics Inc. GPC3 antibody sequences were engineered and engrafted into ARTEMIS constructs containing AbTCR and costimulatory receptor recognizing extracellular domains of GPC3. Constructs were cloned into pCDH lentiviral vectors and used to transduce CD3 / CD28-activated primary human T cells.

[0848] Transduction efficiency was assessed by flow cytometry, showing -30% AbTCR-positive cells.

[0849] Generation of GPC3-specific BiTE (also called GPC3xCD3 TCE herein)

[0850] A BiTE (also called TCE herein) was designed by fusing scFvs of anti-GPC3 (sequence obtained from US7919086B2) and anti-CD3 (OKT3) with glycine-serine linkers, cloned into an expression plasmid, and transiently expressed in CHO cells. BiTEs were purified by nickel affinity and validated for binding by flow cytometry.

[0851] Bioinformatic analyses

[0852] Transcriptomics data from the cancer genome atlas (TCGA, portal.gdc.cancer.gov) for hepatocellular carcinoma cohort (374 tumor samples and 50 adjacent tissue samples) were retrieved using the TCGAbiolinks R package (v 2.32.0). Differential expression analysis was conducted using DESeq2 (v 1.44,0). To identify significantly differentially expressed genes, a threshold of log2foldAttorney Docket No. 40056-0091 WO1 change greater than 1 and an adjusted p-value less than 0.05 was applied. Differentially expressed genes visualized on a volcano plot using ggplot2 (v 3.5.2). Normalized counts for GPC3 were extracted from the DESeq2 output and plotted across tumor and normal samples using ggplot2 (v 3.5.2). A t-test was used to assess statistical significance in GPC3 expression between conditions.

[0853] Proteomics data for hepatocellular carcinoma were obtained from the national cancer institute Proteomics Data Commons (proteomic.datacommons.cancer.gov / pdc / , PDC Study Identifier:

[0854] PDC000198). The dataset includes paired tumor and adjacent liver tissue samples from 165 hepatocellular carcinoma patients analyzed using tandem mass tag (TMT) 11-plex labeling followed by nanoLC-MS / MS. Note that all HCC cases in this dataset are HBV-related. Protein abundance for GPC3 was extracted and visualized using ggplot2 (v 3.5.2), and statistical significance between paired tumor and normal samples was assessed using a paired t-test.

[0855] Staining and flow cytometry

[0856] Flow cytometric analysis was performed as previously described (12, 13). Tumor cells and T cells were discriminated using CD45 staining for all in vitro studies. T cell activation was determined by staining for CD69, CD107, CD137 (4-1BB) (CD137) and interferon-y (IFN-y). Cells were first stained for extracellular markers (CD 107, CD69 and CD 137) followed by fixation / permeabilization before staining for the intracellular activation marker, IFN-y. All antibodies were purchased from Biolegend Inc and the fixation / permeabilization kit was purchased from BD Bioscience.

[0857] GPC3 expression following virus infection were determined using an antibody against GPC3 (Novus Biologics) for all in vitro studies. For the detection of TCE on the surface of T cells and GPC3+ cells, the TCE was first labelled with Alexa-fluor-647 dye (Alexafluor labelling kit; ThermoFisher Scientific). T cells isolated from fresh PBMC and HepG2 cells were stained with live / dead fixable Zombie Dye (Biolegend) and then incubated with the labelled TCE for 30 min. Next, the cells were washed twice, fixed with 4% paraformaldehyde and analyzed by flowcytometry (BD Bioscience).

[0858] Binding of the TCE on live cells was determined by analyzing the data on FlowJo software (vlO, TreeStar).

[0859] For virus-induced GPC3 expression, infected cells were stained with Zombie UV fixable dye and Alexa Fluor 488-anti-GPC3 (Cat#NBP-2-44486; Novus Biologicals), then analyzed on a BD Fortessa. To determine activation status of immune cells, cancer cells were plated in a round-bottom 96-well plate. Virus was added to the cells 1 hour later at different MOIs. Immune cells (CAR-Ts or NK cells) were added to the wells 3 hours post-infection and plates were incubated for 24- or 48-hour. To stain forAttorney Docket No. 40056-0091 WO1 CD 107 (de-granulation marker), PE-conjugated CD 107 antibody was added to the co-culture at the time of adding immune cells. At 24- or 48-h post- infection, cells were harvested by trypsinization and stained with Zombie UV fixable dye (Cat#423108) followed by staining with markers for different immune cells and their activation markers. The antibodies used are: anti-CD45-APC (clone HI30), Anti-CD3-PE / Cy7 (Clone SK7), anti-CD8-AF700 (Clone SKI), anti-IFNy-AF488 (clone 4S. B3), anti-Anti-CD69-APC / Cy7 (clone FN50), anti-CD137-Percp / Cy5,5 (clone 4B4-1), All antibodies were purchased from BioLegend Inc.

[0860] OV transduction and CAR T cell killing assays

[0861] To determine cell killing by the combination of CF33-GPC3 and GPC3-CAR T cells, luciferase¬ expressing cancer cells were plated in a 96-well plate with varying multiplicity of infections (MOIs) of the virus and were co-cultured with mock CAR or GPC3-CAR T cells at 1: 1 ratio. Cell survival was measured at different time point by measuring luciferase activity. Briefly, luciferin was directly added to each well and luminescence signal was measured within 10 minutes using a luminometer. The luminescence signal is proportional to the number of live cells in the well. Therefore, cell killing was determined relative to luminescence signal from the untreated wells.

[0862] Cytotoxicity and Virus growth kinetics

[0863] Target cells (20, 000-60, 000 / well) were infected with virus, then co-cultured with mock or ECT204 T cells at 1: 1 ratio. Cell survival was assessed at 24-72 h by LDH release (CyQUANT LDH assay kit; Cat# C20301; ThermoFisher) or luciferase-based assays.

[0864] For virus growth kinetics analysis, cancer cells were infected at MOI 0.01, lysates were collected at 24, 48, and 72 h, and titers quantified by standard plaque assay on CV-1 cells as previously described

[0865] Binding of TCE to its target

[0866] GPC3xCD3 TCE (also called BiTE herein) was purified from HEK cells transfected with a plasmid containing the expression cassette for this TCE. The TCE was labelled with alexa-fluor-647 dye using Alexa Fluor™ Antibody Labeling Kit (ThermoFisher Scientific) following the manufacturer’s instruction. Next, the labelled TCE was incubated with GPC3+ cells (HepG2) or T cells for 30 minutes. The cells were washed and then analyzed on BD Fortessa cytometer (BD Bioscience) to determine binding of TCE to GPC3 and CD3. Alexa-fluor-647-lablled isotype antibody was used as a control.Attorney Docket No. 40056-0091 WO1 OV transduction and TCE cell killing assays

[0867] For TCE-mediated killing of cancer cells, luciferase encoding MDAMB-468 cells were infected with CF33-GPC3 virus at different MOIs. Freshly isolated T cells were added to the wells in presence or absence of 1 jig / ml TCE. Cell survival was determined at different time points through luciferase assay as described above.

[0868] Immunofluorescence

[0869] SK-Hep1 cells were infected with CF33ΔJ2R or CF33-GPC3 (MOI 0.1), fixed, permeabilized, and stained with anti-GPC3 and anti-vaccinia antibodies, followed by Alexa Fluor-488 and PE-conjugated secondary antibodies. DAPI counterstain was used, and images acquired with EVOS FL Auto.

[0870] Immunogenic cell death (ICD)

[0871] ICD was evaluated using three established markers: cell-surface exposure of calreticulin, extracellular release of ATP, and release of high-mobility group box 1 (HMGB1). Calreticulin exposure was assessed by flow cytometry. Briefly, mock- or CF33-GPC3-infected cells were harvested approximately 24 hours post-infection using 10 mmol / L EDTA, washed, and stained with Zombie UV Live / Dead Fixable dye (Cat#423107; BioLegend Inc.), followed by staining with AlexaFluor 488- conjugated anti-calreticulin antibody (abl96158; Abeam) or the corresponding isotype control

[0872] (ab 199091; Abeam). Cells were subsequently fixed with 4% paraformaldehyde and analyzed using a BD LSRFortessa cytometer. Calreticulin levels were analyzed on live cells.

[0873] Extracellular ATP release was quantified in the culture supernatants (24 hours post-infection) using a luminescence-based ATP determination kit (A22066; Molecular Probes), according to the manufacturer’s instructions. HMGB1 release was assessed by Western blot analysis using a fixed volume (30 LIL) of supernatant collected from mock- or virus-infected cells. HMGB1 was detected using a rabbit anti-HMGB1 antibody (ab18256; Abcam), followed by a donkey anti-rabbit secondary antibody (Cat# ab205718; Abcam).

[0874] Immunohistochemistry

[0875] Tumors were FFPE processed and stained on a Ventana automated Stainer. Antibodies included anti-CD3, anti-GPC3, anti-DYKDDDK, and anti-vaccinia virus. Slides were scanned with NanoZoomer S360.Attorney Docket No. 40056-0091 WO1 The biopsy samples stained for GPC3 (Figure ID) were obtained from 9 individuals with confirmed HCC The slide (MTB80) was generated by City of Hope’s pathology core many years ago and has since been routinely used as a positive / negative control for GPC3 staining. As archival, deidentified material utilized for assay control, these samples did not require IRB approval.

[0876] OV transduction and CAR T activation assays

[0877] To determine activation status of CAR T cells, cancer cells were plated in a 96-well plate with varying multiplicity of infections (MOIs) of the virus and were co-cultured with mock CAR or GPC3-CAR T cells at 1:1 ratio. FITC-conjugated anti-CD107 antibody was added to the wells immediately after addition of CAR T cells. The next day cells were washed and stained with live / dead. Fixable Zombie dye followed by staining with antibodies against CD69 and CD137(4-1BB). The cells were then washed and. Fixed with 4% paraformaldehyde and analyzed on BD Fortessa cytometer (BD bioscience).

[0878] In vivo studies using liver cancer model:

[0879] All animal experiments were performed under protocols approved by the City of Hope Institutional Animal Care and Use Committee. For human tumor xenograft studies, GPC3 knocked-out HepG2 cells HepG2-GPC3-KO (5 × 106cells per mouse) were prepared in 1:1 mixture of PBS and matrigel and injected subcutaneously into the flank of female NSG mice. Tumor growth was monitored 2 times per week by caliper measurement. Once tumor volumes reached about 100 to 200 mm3, CF33-OV-GPC3-V2 virus was prepared and diluted in PBS (pH 7.4) and intratumorally administered at 106pfu per tumor-bearing mice. One week post OV-GPC3virus treatment, GPC3-CAR T cells (5E6 cell / mouse) were injected intratumorally (I T.) or intravenously (I.V.). Tumor volume and weight of mice were measured twice per week. At the end of the study, tumors were harvested from mice and immunohistochemistry was performed to stain for virus, virus-encoded GPC3 as well as CAR T cells. Mice were euthanized if body weight loss exceeded 20% or tumors exceeded 1,500 mm3in volume.

[0880] For in vivo GPC3 expression levels at different time points, sub-cutaneous tumors were generated in NSG mice using HepG2-GPC3-KO cells as described above. When tumor volume reached 100-200 mm3, mice were injected with a single dose of CF33-GPC3-V2 (1E6 pfu) either through I. T. or I. V. route. Mice were euthanized on day 3 or day 7 post-treatment and tumors were harvested. The tumors were fixed in formalin, paraffinized and sectioned. Immunohistochemistry was performed to detect virus-encoded GPC3 on the tumor sections using an unconjugated rabbit anti-GPC3 antibody followed by staining with HRP-conjugated anti-rabbit secondary antibody and final color-developmentAttorney Docket No. 40056-0091 WO1 using DAB. Counterstaining was performed using hematoxylin. The stained tumor sections were scanned using a digital slide scanner (Hamamatsu),

[0881] In vivo studies using triple negative breast cancer model:

[0882] The triple negative breast cancer cell line MDA-MB-231 was used to generate bilateral orthotopic tumors (2 tumors per mouse) in NSG mice. When tumors were ~50 mm3 in volume, mice were injected with 1 E6 pfu of the oncolytic virus CF33-GPC3-V2 through tail vein. Mice were injected with mock CAR or GPC3-CAR T cells (5E6 cell / mouse) through tail vein on day 5 post-virus treatment. Tumor volumes were measured twice weekly.

[0883] Statistical analysis

[0884] Data are presented as means ± SEM, unless otherwise stated. Sample sizes were based on established standards in the field and were not pre-determined using formal statistical power calculations. Statistical comparisons between groups were performed using the unpaired two-tailed Student’s t test to calculate P value, unless otherwise stated. Tumor regression curves were compared using 2-Way ANOVA.

[0885] Example 1: Development and characterization of novel OVs expressing human glypican-3 (GPC3) A series of novel oncolytic viruses encoding a human glypican-3 (OV-GPC3; also called CF33-GPC3) were created and sequenced as described above and below.

[0886] Construction of an oncolytic virus encoding GPC3 (CF33-GPC3):

[0887] The recombinant oncolytic virus encoding a human glypican-3 (CF33-GPC3), was constructed using homologous recombination technique and then CRISPR / Cas9 technology was used to assist with the selection of the recombinant virus. Briefly, the GPC3 cDNA was PCR amplified and cloned into a shuttle plasmid (J2R-shuttle plasmid) where the GPC3 -expression cassette (GPC3 cDNA driven by a promoter, such as a viral synthetic early / late promoter pSE) was flanked by -500 bp of CF33 sequence from around the J2R locus (FIG.34).

[0888] For using CRISPR / Cas9 in the selection of desired recombinant virus, J2R specific oligo was designed and purchased from IDT. The oligo (5’-GTTATAGTAGCCGCACTCG-3’) was used to synthesize single guide RNA (sgRNA) using the EnGen sgRNA Synthesis Kit (Cat#E3322S; NEB) following the manufacturer’s instruction. Next, ~70% confluent HEK293 cells in a 24 well plate were infected with CF33 virus at an MOI of 0.1. Two hours after infection, medium was changed with freshAttorney Docket No. 40056-0091 WO1 DMEM medium supplemented with 10% FBS, and the cells were transfected with ribonucleoprotein complex of sgRNA and Cas9 together with the template for homologous recombination i.e., theJ2R-shuttle plasmid containing GPC3 expression cassette. Lipofectamme CRISPRMAX transfection reagent (Cat# CMAX00008; ThermoFisher) was used for transfection; 1250 ng of Cas9 and 240 ng sgRNA was used per well. The following day, cell lysate was collected and subjected to 3 rounds of freeze-thaw cycles. The lysate was then used to infect CVl cells in 6 well plates and methylcellulose overlay was applied. Two days after incubation, plaques were picked and PCR verified. Two more rounds of plaque purification were performed in 6-well plates using methylcellulose overlay. Finally, limiting-dilution procedure was performed in A549 cells in 96-well plate for final clone selection, which was then used to produce a seed stock in a 100 mm dish of A549 cells. The seed stock was then used to further amplify the virus, and the virus was purified first on a sucrose cushion followed by further purification on sucrose gradient using high-speed centrifugation. The transgene in the recombinant virus was sequence verified.

[0889] CF33-GPC3 can efficiently convert GPC3-negative cells into GPC3-positive Experiments were conducted to determine the ability of OV-GPC3 to infect tumor cells. In order to test the ability of the virus to convert GPC3-negative cells into GPC3+, we used cancer cell lines representing different cancer types. We used MDAMB-231 & MDAMB-468 cell lines, which are representative of human triple negative breast cancer (TNBC). Likewise, we used AsPcl as representative of pancreatic cancer and SK-HEP1 as a representative of GPC3-negative liver cancer. For further proof-of concept, we also used another liver cancer cell line, which is highly positive for GPC3 (HEPG2), but we knocked-out the GPC3 gene in this cell line to create a GPC3-negative HEG2 (HEPG2GPC3-KO).

[0890] We infected all these cell lines with the virus (CF33-GPC3) at different multiplicities of infection (MOIs). Next, we harvested these infected cells at different time points (24 h & 48 h post-infection; FIGS. 2A-2B and FIGS.2C-2D, respectively). Flow cytometry was used to analyze GPC3 expression, and the GPC3 levels were compared in the various samples. In all the cell lines tested, the virus was found to efficiently induce GPC3 expression on the cell surface (FIGS.2A-2D).

[0891] For further visual confirmation of transgene expression, we used an immunofluorescence technique. Cancer cells were infected with the oncolytic virus CF33-GPC3 or a control virus lacking the transgene (CF33AJ2R) at an MOI of 0.1. Approximately 18 hours post-infection, cells were fixed using 4% PFA and stained with anti-CF33 virus (rabbit) and anti-GPC3 (mouse) primary antibodies followed by staining with PE-conjugated anti-rabbit secondary’ and Alexafluor-488-conjugated anti-mouseAttorney Docket No. 40056-0091 WO1 secondary. While virus staining (red) was clearly visible in the cells infected with CF33-GPC3 or the control, GPC3 signal (green) was observed only in the cells infected with CF33-GPC3 (FIGS. 3A-3B).

[0892] Virus-mediated GPC3 expression activates GPC3-CAR T cells

[0893] Using 0V-GPC3, we next assessed whether virus-encoded GPC3 is capable of activating GPC3-targeted CAR-T cells or non-targeting T cells.

[0894] HEPG2GPC3-KOcells were either mock infected (MOI 0) or infected with CF33-GPC3 at varying MOIs and co-cultured with mock CAR or GPC3-CAR T cells at effector: target (E: T) ratio of 1:1 for 24 hours. After the 24 h incubation, cells were stained for T-cell activation markers (CD69, CD137, CD107 and IFNy). The CD 107 antibody was added to the cells at the start of co-culture. After harvesting the cells at 24 h, cells were first stained for activation markers present on cell surface (CD69 & CD137), Next, the cells were fixed and permeabilized before staining for intracellular activation marker interferon-y (IFN-y). Activation markers on T cells were found to increase in an MOI-dependent manner (FIGS. 4A-4E).

[0895] Similar activation of GPC3 CAR T cells were observed after co-culture with CF33-GPC3 infected MDAMB-468 cells (Fig. 5A) and CF33-GPC3 infected SK-Hep1 cells (Fig. 5B). Regular HEPG2 cells (high GPC3 level) were used as positive control in the co-culture.

[0896] Virus-encoded GPC3 facilitates killing of cancer cells by GPC3-CAR T cells

[0897] After confirming the activation of GPC3-CAR T cells by virus-encoded GPC3 (above), we assessed whether this activation of CAR-T cells leads to killing of cancer cells. We used the liver cancer cell line HEPG2 with the GPC3 gene knocked out (HEPG2GPC3-KO), the TNBC cell line, MDAMB-468, stably expressing firefly luciferase (MDAMB468-Luc), and the pancreatic cancer cell line SNU16-Luc. Cells were either mock infected (MOI 0) or infected with different MOIs of CF33-GPC3 in a round bottom 96-w'ell plate. Approximately, 6 h after infection with virus, GPC3-CAR T cells or mock CAR cells were added to the w^ells at an E: T of 1: 1. Cells were incubated for 24, 48, or 72 h. Following incubation, cell viability was measured through LDH-release assay for HEPG2GPC3-KOcells and through luciferase-based assay for MDAMB468-Luc. Survival of cancer cells at each time point was calculated relative to the mock infected wells (FIGS. 6A-6C).

[0898] There is a noticeable increase in killing cancers when treating with the OV-GPC3 alone as the MOI increases for all three cancer cell types: HEPG2GPC3’KOcells (FIG 6 A), MDAMB468 cells (FIG.

[0899] 6B), and SNU16 cells (FIG 6C) in each of the time points tested (24, 48, or 72 h).Attorney Docket No. 40056-0091 WO1 Further, compared to killing by virus alone, there was a huge increase in killing of cancer cells by the combination of virus and GPC3-CAR T cells, especially at late time points (48 and 72 hours) (FIGS. 6A-6C).

[0900] For example, at 48 and 72 hours, the lowest MOI (0.025) of virus alone induced low virus-mediated lysis of cancer cells (<20% killing), whereas the combination of MOI 0.025 virus with GPC3-CAR T cells showed potent tumor cell killing (75% at 48 h and greater than 90% at 72 h) in the MD AMB468-Luc cells (FIG. 6A), Similar increases in the killing of cell by the GPC3-CAR T cells in presence of the CF33-GPC3 was observed in the HEPG2GPC3-KOcells (FIG. 6B). In the pancreatic cancer cells (SNU16-Luc), the combination resulted in even more drastic increase in cancer cell killing with greater than 90% killing at 48 h and 100% killing at 72 h at the lowest MOI (0.025) of virus tested (FIG.

[0901] 6C). Together, these data suggest that the oncolytic virus encoding GPC3 is capable of converting GPC3-negative cells into GPC3-positive and inducing potent antigen-specific CAR T cell-mediated killing of cancer cells.

[0902] Example 2. Anti-tumor efficacy with OV-GPC3 and in combination with GPC3-CAR T cells in human solid tumor xenograft model.

[0903] After confirming the ability for the virus to deliver GPC3 antigen to GPC3-negative cancer cells and the ability of the virally-encoded GPC3 to activate GPC3-CAR T cells, we proceeded to test OV-GPC3 activity in in vivo experiments using human solid tumor models.

[0904] CF33-GPC3 can robustly convert GPC3-negative tumors into GPC3+ tumors in vivo in mice in a human solid tumor xenograft model

[0905] We used the GPC3-negative liver cancer cell line HEPG2GPC3’KOto generate subcutaneous bilateral tumors in NSG mice. Next, the mice were given a single injection of either PBS or the virus (CF33-GPC3) either through intra-tumoral (I. T.) or intravenous (I. V.) route. In the LT. group, each tumor was injected with 1E6 PFU virus and in the I. V. group each mouse was injected with 2E6 PFU virus. Mice were then euthanized either on day 3 or day 7 post-treatment and tumors were harvested. The tumors were formalin fixed, paraffin embedded and sectioned in 5 μm thin sections. The tumor sections were then stained for GPC3 using a mouse anti-GPC3 (Cat#GTX34764, clone#rGPC3 / 863; GenTex) primary antibody followed by staining with an HRP-conjugated anti-mouse secondary antibody. Finally, the HRP substrate DAB was used to develop the brown color.

[0906] The PBS treated tumors were negative for GPC3 (FIGS. 7A-7B). Tumors from mice treated with I. T. or I. V. injection of virus had clearly visible GPC3 signal spread throughout the tumors on DayAttorney Docket No. 40056-0091 WO1 3 (FIGS. 7A-7B). On day 7, the GPC3 signal was found to further increase in all the mice covering most part of the tumors (FIGS. 7A-7B).

[0907] For comparison purpose, we also used an array of tumor sections from 9 liver cancer patients for GPC3 staining (FIG. 7C), Staining on these human samples was done in parallel with the mouse samples. Out of the 9 patients’ tumors, only 3 were found to be positive for GPC3 (FIG, 7C). Out of those 3, one showed very low level of GPC3 and one showed high GPC3 expression (FIG 7C). This tumor array from liver cancer patients, shows wide variations in GPC3 levels in liver cancer. Compared to the levels of GPC3 observed in the tumors of human patients, the virus-treated tumors (from mice) show higher levels of GPC3 expression, which is disseminated throughout the tumors.

[0908] CF33-GPC3 treatment and CF33-GPC3 combination treatment with GPC3-CAR T cells results in strong therapeutic efficacy and reduction in tumor volume in a human tumor xenograft model.

[0909] To test the antitumor efficacy of the OV and the combination with GPC3 CAR T cells, we used the GPC3-negative liver cancer cell line HEPG2GPC3-KOto generate subcutaneous tumors in NSG mice. When the tumors reached ~150 mm3in volume, mice were divided into 5 treatment groups (n=8 mice / group) such that each group had similar average tumor volume. The mice were then treated with PBS or virus (CF33-GPC3) on day 0. Mock CAR T or GPC3-CAR T cells were administered to respective groups on day 7 (1 -week post-virus injection).

[0910] Virus was administered I. T., and CAR T cells were administered either through LT. or I. V. route. A single injection of virus (1E6 PFU / mouse) or PBS was administered, and a single injection of CAR (5E6 / mouse) was given. Tumor volume was measured twice a week using a digital caliper.

[0911] Tumors in the mice treated with only GPC3-CAR T cells (filled triangle indicator) grew rapidly and the tumor growth was very similar to the PBS treated group (open diamond indicator). Mice treated with virus alone (open triangle indicator) showed signs of tumor regression starting day 7 post-treatment (FIG 8). This OV-GPC3 -mediated tumor regression was sustained throughout the experiment with continued tumor regression seen up to the last day tested (20 days post treatment).

[0912] Notably, the combination of virus and GPC3-CAR T cells (administered I.T. (open star) or I.V. (closed star)) resulted in significantly improved regression of tumors, even compared to the virus alone group (FIG. 8). Taken together, the in vivo studies suggest that the virus could robustly convert GPC3- negtaive tumors into GPC3 -positive tumors, leads to potent tumor cell killing, and allows for GPC3-CAR T cells to exert their significant anti-tumor activity.Attorney Docket No. 40056-0091 WO1 Example 3: OV expression of GPC3 Variant 1 (VI) and truncated GPC3-V1 (GPC3t-Vl):

[0913] Comparison and Characterization of CF33-hNIS-GPC3-Vl and CF33-hNIS-GPC3t-Vl

[0914] A senes of novel oncolytic viruses encoding a human glypican-3 (OV-GPC3; also called CF33-GPC3) were created and sequenced as described above and below. There are 4 isoforms (variant # VI-V4), and this example features OV-GPC3s encoding two variants and a truncation of one variant, inserted at the F14.5L locus of a CF33 with an hNIS already inserted to generate CF33-hNIS-GPC3-Vl, CF33-hNIS-GPC3-Vl, and CF33-hNIS-GPC3-V2. In these constructs, the JR2 locus is partially deleted and the FI 5.5L locus is deleted.

[0915] Construction of oncolytic viruses encoding GPC3 (CF33-GPC3):

[0916] Glypican 3 (GPC3) is a membrane-bound protein that is overexpressed in many types of tumors, including hepatocellular carcinoma (HCC) There are 4 isoforms (variant# VI -V4), which are alternatively spliced variants of the gene. The variant VI is the longest (1812 bp) among all. For our first virus construct, we inserted the full-length cDNA for GPC3-V1 (GPC3 Variant 1) at the Fl 4.5L locus of CF33-hNIS virus to generate CF33-hNIS-GPC3-Vl (Fig. 9). We also generated another construct, CF33-hNIS-GPC3t, through insertion of truncated VI of GPC3 (GPC3t), which only included the C-terminal domain (CTD) of GPC3 (669 bp) (Fig. 9, bottom panel).

[0917] OV mediated conversion of GPC3-negative cancer cells into GPC3-positive

[0918] These two viruses were compared for their ability to express GPC3 from infected cancer cells. The triple negative breast cancer cell line MDAMB468 was infected with CF33-hNIS-GPC3-Vl or CF33-hNIS-GPC3t at MOI of 0 (uninfected) and 0.3. Twenty-four hours later, cells were harvested, stained with live / dead zombie dye followed by staining with PE-conjugated anti-GPC3 antibody.

[0919] Finally, GPC3 expression was determined by flow cytometry (Fig. 10). Similar expression levels were detected from both CF33-hNIS-GPC3-VI (Fig. 10, top panel) and CF33-hNIS-GPC3t-Vl (Fig. 10, bottom panel).

[0920] The virus constructs were also used to infect a glioma cell line, U251, to compare GPC3 expression. Expression from both CF33-11NIS-GPC3-V1 (FIG. 11A) and CF33-hNIS-GPC3t-Vl (FIG.

[0921] 1 IB) were comparable in this cell line. The mean fluorescence intensities (MFI) for the GPC3 (PE) in virus-infected cells were significantly higher than those in uninfected cells (MOI =0) (FIGS. 11 A-l IB).

[0922] Virus-encoded GPC3 facilitates killing of cancer cells by GPC3-CAR T cellsAttorney Docket No. 40056-0091 WO1 We used the U251 cell line to test tumor cell killing by CF33-hNIS-GPC3-Vl and CF33-hNIS-GPC3t-Vl and also test the tumor cell killing of the combination of the viruses with GPC3-CAR T cells. Cells were either mock infected (MOI 0) or infected with CF33-hNIS-GPC3-Vl -full or CF33-hNIS-GPC3t at an MOI of 1, Approximately 6 h after infection with virus, GPC3-CAR T or mock CAR cells were added to the wells at an E: T (effector: tumor ratio) of 1:1. Cells were incubated for 24 hours.

[0923] Following incubation, cell viability was measured using flow cytometry. Survival of cancer cells was calculated relative to the mock infected wells. In the presence of the virus encoding full-length GPC3, CF33-hNIS-GPC3-Vl, killing by GPC3-CAR was significantly higher compared to killing by mock- C / XR (FIG. 12). However, in presence of virus encoding the truncated GPC3, CF33-hNIS-GPC3t, no statistical difference was observed in tumor cell killing between cells co-cultured with GPC3-CAR T cells and cells co-cultured with mock-CAR T cells (FIG. 12).

[0924] Virus-mediated GPC3 expression activates GPC3-CAR T cells

[0925] Furthermore, activation of GPC3-CAR T cells and mock CAR T cells were determined by measuring interferon-? (IFNy) levels in those cells. U251 cells were infected with CF33-hNIS-GPC3-Vl (FIG. 13A) or CF33-hNIS-GPC3t (FIG. 13B) at an MOI 1 and co-cultured with mock CAR (third panel) or GPC3-CAR (fourth panel) at 1: 1 for 24 hours. Cells were harvested 24 h later and stained with live / dead Zombie dye, APC-conjugated anti-CD45, PE-conjugated anti-IFNy and APC-Cy7-conjugated anti-CD69 antibodies. IFNy as well as CD69 expression was measured on T cells (CD45+ cells). While >20% CAR-T cells were found positive for IFNy in presence of full-length GPC3-encoding virus, there were <5% CAR cells positive for IFNy in presence of truncated-GPC3 -encoding virus (FIGS. 13A-13B).

[0926] These experiments showed, inter alia, that there was potent killing of cancer cells by GPC3-targeted CAR-T cells in presence GPC3 -encoding virus.

[0927] CF33-GPC3 can efficiently convert GPC3-negative cells into GPC3-positive Experiments were conducted to determine the ability of OV-GPC3 to infect tumor cells. In order to test the ability of the virus to convert GPC3 -negative cells into GPC3+, we used cancer cell lines representing different cancer types. We used MDAMB-231 & MDAMB-468 cell lines, which are representative of human triple negative breast cancer (TNBC). Likewise, we used AsPcl as representative of pancreatic cancer, and SK-HEP1 as a representative of GPC3 -negative liver cancer. For further proof-of concept, we also used another liver cancer cell line, which is highly positive for GPC3 (HEPG2), but we knocked-out the GPC3 gene in this cell line to create a GPC3-negative HEG2Attorney Docket No. 40056-0091 WO1 (HEPG2GPCJ'KO). We infected all these cell lines with the virus (CF33- hNIS-GPC3-V2) at different multiplicities of infection (MOIs). Next, we harvested these infected cells at different time points (24 h, 48 h, and 72 h post-infection; FIGS. 26-27). Flow cytometry was used to analyze GPC3 expression, and the GPC3 levels were compared m the various samples. In all the cell lines tested, the virus was found to efficiently induce GPC3 expression on the cell surface of each of Sk-Hepl (human liver cancer cells), AsPCl-Luc (human pancreatic cancer cell line), MDA-MB231 and MDAMB468 (human triple negative breast cancer cell lines) (FIGS. 26-27).

[0928] For further visual confirmation of transgene expression, we used an immunofluorescence technique. Cancer cells were infected with the oncolytic virus CF33-GPC3-V2 or a control virus lacking the transgene (CF33AJ2R) at an MOI of 0.1. Human liver cancer cells (SK-Hep-1) were infected with CF33ΔTK or CF33-GPC3-V2 virus at an MOI of 0.1 in a 8-well chamber slide. 24 h post-infection, cells were fixed using 4%PFA and stained with anti-CF33 virus (rabbit) and anti-GPC3 (mouse) primary antibodies followed by staining with PE-conjugated anti-rabbit secondary and Alexafluor-488 (FITC)-conjugated anti-mouse secondary. Images were acquired at 10X using fluorescent microscope. While virus staining (red) was clearly visible in the cells infected with CF33-GPC3 or the control, GPC3 signal (green) was observed only m the cells infected with CF33-GPC3 (FIGS. 3A-28).

[0929] Human TNBC cells (MDA-MB231) were infected with CF33ΔTK or CF33-GPC3-V2 virus at an MOI of 0.1 in an 8-well chamber slide. 24 h post-infection, cells were fixed using 4% PF A and stained with anti-CF33 virus (rabbit) and anti-GPC3 (mouse) primary antibodies followed by staining with PE-conjugated anti-rabbit secondary and Alexafluor-488 (FITC)-conjugated anti-mouse secondary. Images were acquired at 10X using fluorescent microscope. Similarly, the virus staining (red) was clearly visible in the cells infected with CF33-GPC3 or the control (CF33ΔTK). The GPC3 signal (green) was observed only in the cells infected with CF33-GPC3-V2 (FIG.29).

[0930] The GPC3 copy number in un-infected and CF33-GPC3 infected cells 24 h post-infection wTas measured and quantified (FIG. 30). Cells were plated in a 96-well plate and infected with mock (PBS) or CF33-GPC3-V2 (encoding variant 2 of GPC3). The data is based on two independent experiments: one with single well per sample and one with three wells per samples (samples were in triplicates). Plates were incubated overnight and the next day, cells were harvested using 10 mM EDTA, and stained for GPC3 using AF488-conjugated and anti- GPC3 antibody (NBP2-47762AF488 (clone: gpc3 / 863); Novus). For quantification of GPC3 copy number, Quantum beads pre-labelled with Al exa-fluor-488 (Bangs Laboratories, Cat#488) were used according to manufacturer’s instruction and calculations wereAttorney Docket No. 40056-0091 WO1 performed using manufacturer-provided template called QuickCal. BD Fortessa was used for running samples.

[0931] Virus-mediated GPC3 expression activates GPC3-CAR T cells

[0932] We next assessed whether virus-encoded GPC3 is capable of activating GPC3-targeted CAR-T cells or non-targeting T cells. SK-Hep-1 cancer cells were either mock infected (MOI 0) or infected with the virus (CF33-GPC3-V2) at MOI 0.1 in a 96-well round bottom plate (FIG. 31). 6h post-infection Mock CAR or GPC3-CAR T cells were added to the wells at effector:target (E: T) of 1: 1. After the 24 h incubation, cells were stained for T-cell activation markers (CD69, CD 107 and IFNy). The CD 107 antibody was added to the cell at the start of co-culture. After harvesting the cells at 24 h, cells were first stained for activation markers present on cell surface (CD69). Next, the cells were fixed and permeabilized before staining for intracellular activation marker interferon-y (IFN-y). Cells were analyzed and activation markers on T cells were determined (FIGS. 31-32).

[0933] Similar activation of GPC3 CAR T cells were observed after co-culture with CF33-GPC3 infected MDAMB-468 cells (Fig. 32, middle panel) and CF33-GPC3 infected AspC-1 cells (Fig. 32, left panel).

[0934] Virus-encoded GPC3 facilitates killing of cancer cells by GPC3-CAR T cells

[0935] After confirming the activation of GPC3-CAR T cells by virus-encoded GPC3 (above), we assessed whether this activation of CAR-T cells leads to killing of cancer cells. We used the liver cancer cell line HEPG2 with the GPC3 gene knocked out (HEPG2GPC3-KO), the TNBC cell line, MDAMB-468, stably expressing firefly luciferase (MDAMB468-Luc), and the pancreatic cancer cell line SNU16-Luc. Cells were either mock infected (MOI 0) or infected with the virus (CF33-hNIS-GPC3-V2) at indicated MOI in a 96-well round bottom plate. 6 h post-infection, mock CAR or GPC3-CAR T cells were added to the wells at E: T of 1:1. The plates were incubated and cell survival was determined at 24, 48 and 72 h using a luciferase-based assay for MDAMB-468-Luc cells (FIG. 25A) and SNU16-Luc cells (FIG. 25B) or LDH-release assay for SK-Hep-1 cells (FIG. 25C) and AsPCl cells (FIG. 25D). Survival of cancer cells at each time point was calculated relative to the mock infected wells (FIGS. 25A-25D).

[0936] There is a noticeable increase in killing cancer cells when treating with the O V-GPC3 alone as the MOI increases for all four cancer cell types: MDAMB-468-Luc cells (FIG. 25A), SNU16-Luc cells (FIG. 25B), SK-Hep-1 cells (FIG. 25C), and AsPCl cells (FIG. 25D) in each of the time points tested (24, 48, or 72 h).Attorney Docket No. 40056-0091 WO1 Further, compared to killing by virus alone, there was a marked increase in killing of cancer cells by the combination of virus and GPC3-CAR T cells, especially at late time points (48 and 72 hours) (FIGS. 25A-25B).

[0937] Together, these data suggest, that the oncolytic virus encoding GPC3 is capable of converting GPC3 -negative cells into GPC3 -positive and inducing potent antigen-specific CAR T cell-mediated killing of cancer cells.

[0938] Example 4: O V expression of GPC3 Variant 1 (VI ) and Variant 2: Comparison and Characterization of CF33-GPC3-V1 and CF33-GPC3-V2

[0939] A series of novel oncolytic viruses encoding a human glypican-3 were created and sequenced as described above and below. There are 4 isoforms (variant # VI -V4), and this example features OV-GPC3s encoding two variants inserted at the J2R locus of CF33 to generate CF33-GPC3-V1 and CF33-GPC3-V2.

[0940] Construction of oncolytic viruses encoding GPC3 (CF33-GPC3);

[0941] Glypican 3 (GPC3) is a membrane- bound protein that is overexpressed in many types of tumors, including hepatocellular carcinoma (HCC). There are 4 isoforms (variant# VI -V4), which are alternatively spliced variants of the gene. The variant V I is the longest (1812 bp) among all.

[0942] The constructs m Example 3 had GPC3 inserted at the F14.5L locus, and there were 2 transgenes (e.g., CF33-hNIS-GPC3-Vl as depicted in Fig. 9, top panel).

[0943] Here, we inserted the GPC3-full length Variant 1 (GPC3 VI) or GPC3-full length Variant 2 (GPC V2) to generate CF33-GPC3-V1 (Fig. 14, top panel) or CF33-GPC3-V2 (Fig. 14, bottom panel).

[0944] OV mediated conversion of GPC3-negative cancer cells into GPC3-positive

[0945] These two viruses were compared for their ability to express GPC3 from infected cancer cells. The triple negative breast cancer cell line MDAMB468 was infected with CF33-GPC3-V1 or CF33- GPC3-V2 at an MOI of 0.5 and incubated the cells for 24 h. After incubation, cells were harvested using 10 uM EDTA and stained with live / dead Zombie dye, followed by staining with Alexafluor-488-conjugated GPC3 antibody (Cat# NBP2-47762AF488 (clone: gpc3 / 863); Novus). GPC3 expression was analyzed on Live cells only. Virus encoding VI of GPC3, CF33-GPC3(V1), demonstrated minimal expression of GPC3 in the tumor cells as compared to CF33-GPC3(V2) (Fig, 15).Attorney Docket No. 40056-0091 WO1 Comparison of virus-encoded VI and V2 of GPC3 in facilitating CAR-T mediated killing of cancer cells:

[0946] We used the MDAlVlB-468-Luc cel 1 line to compare cell killing by the OV-GPC3s and the combination of viruses and CAR-T cells. Cells were either mock infected (MOI 0) or infected with different MOIs of CF33-GPC3-V1 or CF33-GPC3-V2.

[0947] Approximately 6 h after infection with viruses, GPC3-CAR T or mock CAR T cells were added to the well at an E: T of 1:1, Cells were incubated for 48 h. Following incubation, cell viability was measured using luciferase-based assay. Survival of cancer cells was calculated relative to the mock infected wells.

[0948] Both CF33-GPC3-V1 and CF33-GPC3-V2 facilitated killing cancer cells as the MOI increases (FIG. 16 “OV alone”).

[0949] In presence of CF33-GPC3-V2, tumor cell killing by GPC3-CAR T cells was significantly higher compared to killing by virus alone or virus plus mock-CAR (FIG. 16). In case of the VI- encoding virus (CF33-GPC3-V1), the combination of virus and GPC3-CAR T cells resulted in increased killing of cancer cells compared to killing by virus alone or virus plus mock-CAR, but not as potent as the CF33-GPC3-V2 (FIG. 16).

[0950] Example 5: O V expression of truncated GPC3 Varian t 2: Comparison and Characterization o f CF33-GPC3- N500 and CF33-GPC3- C500

[0951] A series of novel oncolytic viruses encoding a human glypican-3 were created and sequenced as described above and below. There are 4 isoforms (variant # VI -V4), and this example features OV-GPC3s encoding two truncations of variant 2 inserted at the J2R locus of CF33 to generate CF33-GPC3-AN500 and CF33-GPC3-AC5OO.

[0952] We constructed two viruses using V2 of GPC3: one encoding GPC3-V2 with a 500 bp truncation from N-terminal end, CF33-GPC3-AN5OO (depicted in Fig. 17, top panel), and another encoding GPC3-V2 with 500 bp truncation from C-terminal end, CF33-GPC3-AC500 (depicted in Fig. 17, bottom panel).

[0953] We selected 6 clones for each of the two viruses and compared their ability to express GPC3 m infected cells. The CF33-GPC3-AC500 virus clones all expressed low levels of GPC3 (<8%; FIG. 18A). For CF33-GPC3-AN500, clone#4 was found to be the best (>40% positive cells; FIG. 18B), so this clone was selected for further analysis.Attorney Docket No. 40056-0091 WO1 We assessed CF33-GPC3-AN500 virus for its ability to activate GPC3-targeted CAR-T cells. For this, we infected MDAMB468 cells and incubated the infected cells with mock CAR or GPC3-CAR T cells. Twenty-four hours later, we analyzed activation markers (IFNy and CD69; FIGS. 19A and 19B, respectively) on the T cells. The truncated GPC3 did not appear to be able to activate the CAR T cells (FIGS. 19A-19B).

[0954] Example 6: OV expression of mutated GPC3 Variant 2: Comparison and Characterization of CF33-GPC3, CF33-GPC3-F41E, & CF33-GPC3-G41E-W260R

[0955] A series of novel oncolytic viruses encoding a human glypican-3 were created and sequenced as described above and below. There are 4 isoforms (variant # VI -V4), and this example features OV-GPC3s mutants of the full length GPC3-V2 inserted into the J2R locus as described above.

[0956] Construction of oncolytic viruses encoding GPC3 (CF33-GPC3);

[0957] GPC3 has oncogenic property, which is caused through its interaction with wnt protein, and previous studies have shown that substitution mutations within the GPC3 gene can abolish the oncogenicity of this protem(IO). A single substitution of phenylalanine to glutamine (F41E) at ammo acid #41 can completely inhibit the ability of GPC3 to interact with wnt and greatly reduce oncogenicity of GPC3. Likewise, another amino acid substitution at position 260, tryptophan to arginine (W260R) has also been shown to greatly reduce downstream activity of wnt, with potential to reduce the oncogenicity of GPC3.

[0958] While GPC3 is an oncogenic protein, CF33-GPC3 infected cells are expected to undergo cell death, making it highly unlikely that the virus-encoded GPC3 could induce oncogenic transformation. However, as an added precaution, we have developed two additional CF33 constructsT3, one that encodes GPC3-V2 with a single substitution of phenylalanine to glutamine (F41E) at amino acid #41 (CF33-GPC3-F41E; FIG.20 top panel) and another that encodes GPC3-V2 with two substitution mutations i.e. F41E and W260R (CF33-GPC3-F41E-W260R; FIG.20 bottom panel). These oncolytic virus constructs with substitution mutations within the wild-type GPC3 cDNA were constructed to encode non-functional (i.e. non-oncogenic) GPC3.

[0959] OV mediated conversion of GPC3-negative cancer cells into GPC3-positive

[0960] These two viruses that encode GPC3 with one (CF33-GPC3-F41E) or two point mutations (CF33-GPC3-F41 E-W260R) were compared for their ability to express GPC3 from infected cancer cells. The triple negative breast cancer cell line MDAMB468 was infected with CF33-GPC3 or CF33-Attorney Docket No. 40056-0091 WO1 GPC3-F41E or CF33-GPC3-F41E-W260R at MOIs of 0.025 or 0.1. After either 24 h incubation (FIG.

[0961] 21 A) or 48 h incubation (FIG. 21 B), cells were harvested using 10 uMEDTA and stained with live / dead Zombie dye, followed by staining with Alexafluor-488-conjugated GPC3 antibody (Cat# NBP2-47762AF488 (clone: gpc3 / 863); Novus), GPC3 expression was analyzed on Live cells only. Similar levels of GPC3 expression was achieved by all 3 viruses (FIGS. 21 A-21C).

[0962] Virus-encoded mutant forms of GPC3 can activate GPC3-CAR T cells and facilitate CAR-T mediated killing of cancer cells;

[0963] We assessed whether virus-encoded GPC3 with one point mutation (CF33-GPC3-F41E) or two point mutations (CF33-GPC3-F41E-W260R) are capable of activating GP(^3 -targeted CAR-T cells. To do this, we infected MDAMB-468 tumor cells with CF33-GPC3 or CF33-GPC3-F41E or CF33-GPC3- G41 E-W260R at MOIs 0.025 or 0.1. The infected MDAMB-468 cells were co-cultured with mock C / XR or GPC3-CAR T cells at effectortarget (E: T) ratio of 1: 1 for 24 hours. After the 24 incubation, cells were stained for T-cell activation markers (CD69, CD137, CD107 and IFNy). The CD107 antibody was added to the cells at the start of co-culture. After harvesting the cells at 24 h, cells were first stained for activation markers present on cell surface (CD69 & CD137). Next, the cells were fixed and permeabilized before staining for intracellular activation marker, interferon-y (IFN-y). Activation markers on GPC3 -CAR-T cells, but not on mock-CAR-T, were found to increase in presence of virus encoding mutated GPC3, similar to the wild-type GPC3, suggesting that the mutated GPC3 can be recognized by CAR-T cells (Figs. 22-23D).

[0964] Virus-encoded mutant forms of GPC3 facilitate killing of cancer cells by GPC3-CAR T cells:

[0965] After confirming the activation of GPC3-CAR T cells by virus-encoding mutant forms of GPC3-v2, we assessed whether this activation of CAR-T cells leads to antigen-specific killing of cancer cells. We used the TNBC cell line MDAMB-468, stably expressing firefly luciferase (MDAMB468-Luc), and cells were either mock infected (MOI 0) or infected with different MOIs of viruses in a round bottom 96- well plate. Approximately 6 h after infection with virus, GPC3-CAR T or mock CAR cells were added to the wells at an E: T of 1:1. Cells were incubated for 48-hour. Following incubation, cell viability was measured through luciferase-based assay. Survival of cancer cells was calculated relative to the mock infected wells.Attorney Docket No. 40056-0091 WO1 Compared to killing by virus alone, there was a huge increase in killing of cancer ceils by the combination of virus and GPC3-CAR T (Fig. 24), Efficacy of the mutant forms of GPC3 was comparable to the efficacy of wildtype GPC3-V2 m terms of facilitating killing of cancer cells by GPC3-targeted cancer cells. Together, these data suggest that the oncolytic virus encoding full-length GPC3 (variant 2) either in its wild-type form or mutated forms, is capable of converting GPC3-negative cells into GPC3-positive and inducing antigen-specific CAR T cell-mediated killing of cancer cells.

[0966] Example 7: A GPC3 Targeted T cell Engager facilitates T cell-mediated killing of GPC3+ Cells A GPC3xCD3 T cell engager having the amino sequence shown in FIG. 33 was constructed. The TCE was produced by transfecting HEK cells with a plasmid containing the expression cassette for the TCE To determine the binding of this TCE to GPC3 and T cells, we first labelled the TCE with alexa-fluor-647 dye using Alexa Fluor™ Antibody Labeling Kit (ThermoFisher Scientific) following the manufacturer’s instruction. Next, the labelled TCE was incubated with GPC3+ cells (HEPG2) or T cells for 30 minutes. The cells were washed and then analyzed on BD Fortessa cytometer (BD Bioscience) to determine binding of TCE to GPC3 and CD3.?\lexa-fluor-647-lablled isotype antibody was used as a control. As shown in FIG. 39A, the TCE was found to bind to GPC3+ cells (HEPG2) and CD3 on I' cells.

[0967] We tested the ability of the TCE to facilitate T cell-mediated killing of GPC3+ cells. To do this, we infected luciferase-encoding MDA-MB-468 cells with CF33-GPC3-V2 at different MOIs (0, 0.025 and 0.1) in a 96- well plate and added freshly isolated T cells in presence or absence of 5 pg / ml TCE. Cell survival was determined at different time points through luciferase-based assay. Killing of virus- infected cells (GPC3+) by T cells in presence of TCE was significantly higher compared to killing in the absence of TCE as shown m FIG. 39B.

[0968] Example 8: GPC3-encoding oncolytic vims enhance CAR-T spread within the tumors

[0969] GPC3 knocked-out HepG2 cells (HepG2-GPC3-KO) were used to generate tumors m NSG mice. Once tumor volumes reached about 100 to 200 mm3, CF33-GPC3-V2 virus was prepared and diluted in PBS (pH 7.4) and intratumorally administered at 106pfu per tumor-bearing mice. One week post virus treatment, GPC3-CAR T cells (5E6 cell / mouse) were injected intratumorally (I. T.). Two weeks after CAR-T injection, mice were euthanized, and tumors were harvested. The tumors were stained to detect virus, virus-encoded GPC3 and CAR T cells (CD3), As shown in FIG. 40, the results show that CAR-T cells are beter spread in the tumor in presence of GPC3 -encoding oncolytic virus.Attorney Docket No. 40056-0091 WO1

[0970] Example 9: GPC3-encoding oncolytic virus improves anti-tumor efficacy of GPC3-CAR T cells in a triple negative breast cancer model:

[0971] The triple negative breast cancer cell line MDA-MB-231 was used to generate bilateral orthotopic tumors (2 tumors per mouse) in NSG mice. When tumors were ~50 mm3in volume, mice were injected with 1E6 pfu of the oncolytic virus CF33-GPC3-V2 through tail vein. Mice were injected with mock CAR or GPC3-CAR T cells (5E6 cell / mouse) through tail vein on day 5 post- virus treatment. Tumor volume of the mice were measured twice weekly. As shown in FIG 41, combination treatment resulted in significantly improved anti-tumor efficacy.

[0972] Example 10: Translational Potential of an Oncolytic Vims— Mediated Antigen Delivery to Expand GPC3-Targeted Therapies in Hepatocellular Carcinoma

[0973] Expression of GPC3 is highly heterogeneous in HCC

[0974] GPC3 is an oncofetal protein expressed during fetal development but largely absent in adult tissues. In HCC and certain other malignancies, however, GPC3 is re-expressed and ranks among the most highly upregulated genes m HCC15. To further validate GPC3 expression, we analyzed transcriptomic data from The Cancer Genome Atlas (TCGA, portal.gdc.cancer.gov). Among the top 20 differentially expressed genes between normal tissue and HCC tumors, GPC3 was prominently represented (FIG. 42A). While HCC tumors exhibited elevated GPC3 expression, a substantial subset displayed levels comparable to that in normal tissues (FIG. 42B). We further examined GPC3 protein abundance using a dataset from the National Cancer Institute Proteomics Data Commons (proteomic.datacommons.cancer.gov / pdc / PDC Study Identifier: PDC000198), which included paired tumor and adjacent liver samples from 165 HCC patients. Consistent with the transcriptomic findings, GPC3 protein levels showed considerable variability, with nearly half of the tumor samples exhibiting expression comparable to normal tissue (FIG. 42C). To further validate these observations, we analyzed immunohistochemical (IHC) staining of biopsy samples from 9 HCC patients. Again, marked heterogeneity was evident: 2 samples were strongly positive, 3 were weakly positive, and 4 were negative for GPC3 expression (FIG. 42D). Collectively, these data show that while GPC3 is a tumor- associated marker in HCC, its expression is highly heterogeneous in HCC patients.Attorney Docket No. 40056-0091 WO1 CF33-GPC3 effectively converts GPC3-negative cancer cells into GPC3-positive in vitro and in vivo

[0975] The oncolytic virus CF33-GPC3 was engineered by inserting a GPC3 expression cassette into the J2R locus of the chimeric poxvirus CF33. We first validated transgene expression by RT-PCR and Western blotting, confirming GPC3 expression within 24 hours at levels comparable to GAPDH To assess surface expression of GPC3, we utilized the GPC3-knockout HepG2 line (HepG2GPC3-KO) and the innately GPC3-negative HCC line SK-Hepl, Flow cytometry showed efficient induction of GPC3 in both cell lines: SK-Hepl cells were uniformly GPC3-positive within 24 hours at MOI 1, whereas HepG2GPC3’KOcells exhibited complete expression by 48 hours (FIG. 43A--43B). Similar induction was observed in multiple tumor models, including cholangiocarcinoma, pancreatic, an...

Claims

Attorney Docket No. 40056-0091 WO1 WHAT IS CLAIMED:

1. An oncolytic virus (OV) expressing a glypican-3 (GPC3), wherein the OV expressing a GPC3 (OV-GPC3) comprises a nucleotide sequence comprising:(i) nucleotides 6,302 - 80,395 of SEQ ID NO: Al having no more than 300 single nucleotide modifications (e.g., modifications that do not change the amino acid sequence of the encoded proteins);(ii) a nucleotide sequence encoding a human GPC3; and(lii) nucleotides 82,356 - 190,102 of SEQ ID NO: Al having no more than 300 single nucleotide modifications (e.g,, modifications that do not change the amino acid sequence of the encoded proteins).

2. The OV-GPC3 of claim 1, wherein the nucleotide sequence comprises:(i) nucleotides 6,302 - 80,395 of SEQ ID NO: Al having no more than 200, 100, 75, 50, or 30 single nucleotide modifications (e.g., nucleotide substitutions), wherein the modifications do not change the amino acid sequence of the encoded proteins; and(ii) nucleotides 82,356 - 190,102 of SEQ ID NO: Al having no more than 200, 100, 75, 50, or 30 single nucleotide modifications (e.g., nucleotide substitutions), wherein the modifications do not change the amino acid sequence of the encoded proteins.

3. The OV-GPC3 of claim 1 or claim 2, wherein the GPC3 comprises an amino acid sequence selected from SEQ ID NOs: 1-6 and 1B-6B.

4. The OV-GPC3 of any one of claims 1-3, wherein the GPC3 comprises at least one or at least two amino acid substitutions selected from F41E, L66A, W260R, Y264K, L268E, M269S, Y277A, Y408D, L421E, W423A, L428R, Y432A using SEQ ID NO: 1 as a reference sequence (e.g., F41E corresponds to F17E in SEQ ID NO:1B, F41E in SEQ ID NO:4, and F17E in SEQ ID NO:5B).

5. The OV-GPC3 of any one of claims 1-4, wherein the GPC3 comprises at least one amino acid substitution selected from F41E, L66A, W260R, Y264K, L268E, M269S, Y277A, Y408D, L421E, W423A, L428R, Y432A using SEQ ID NO:1 as a reference sequence (e.g., F41E corresponds to F17E in SEQ ID NO: 1B, F41E m SEQ ID NO:4, and F17E in SEQ ID NO:5B).

6. The OV-GPC3 of any one of claims 1-5, wherein the GPC3 comprises two amino acid substitutions selected from F41E, L66A, W260R, Y264K, L268E, M269S, Y277A, Y408D, L421E,Attorney Docket No. 40056-0091 WO1 W423A, L428R, Y432A using SEQ ID NO: 1 as a reference sequence (e.g., F41E corresponds to F17E in SEQ ID NO: 1B, F41E in SEQ ID NO:4, and Fl 7E in SEQ ID N0 5B)7. The 0V-GPC3 of claim 6, wherein the two amino acid substitutions are F41E and W260R,8 The 0V-GPC3 of any one of claims 1-7, wherein the GPC3 comprises the amino acid sequence of SEQ ID NO: 3 or 3B.9 The 0V-GPC3 of any one of claims 1-8, further comprising a promoter that is operably linked to the GPC3.10 The OV-GPC3 of any one of claims 1-9, wherein the nucleotide sequence comprises:(i) nucleotides 6,302 - 80,395 of SEQ ID NO: Al; and(ii) nucleotides 82,356 - 190,102 of SEQ ID NO: Al.11 A method of treating a subject having a solid tumor comprising:administering to the subject an effective amount of an oncolytic virus expressing a human glypican-3 (GPC3);wherein the nucleotide sequence of the oncolytic virus expressing GPC3 (OV-GPC3) comprises: (a) an oncolytic virus nucleotide sequence encoding at least 25, 50, 60, 70, 80, 90, 100, 110, 120, or 121 of SEQ ID NOs: Z1, Z17, Z26, Z27, Z28, Z32, Z34, Z36, Z41, Z52, Z53, Z55, Z63, Z65, Z66, Z76, Z77, Z79, Z81, Z82, Z87, Z90, Z91, Z92, Z93, Z104, Z108, Z110, Z111, Z116, Z118, Z120, Z123, Z128, Z129, Z130, Z138, Z140, Z142, Z143, Z145, Z146, Z147, Z149, Z150, Z151, Z152, Z153, Z154, Z155, Z156, Z157, Z158, Z160, Z161, Z165, Z168, Z169, Z170, Z172, Z173, Z174, Z175, Z176, Z177, Z178, Z181, Z182, Z186, Z192, Z193, Z194, Z195, Z197, Z198, Z199, Z203, Z206, Z211, Z212, Z216, Z219, Z220, Z222, Z223, Z224, Z225, Z226, Z227, Z228, Z229, Z231, Z232, Z233, Z234, Z236, Z238, Z240, Z241, Z243, Z244, Z252, Z253, Z254, Z257, Z258, Z259, Z260, Z261, Z263, Z264, Z265, Z266, Z267, Z268, Z269, Z270, Z271, Z272, Z274, and Z275; and(b) a transgene comprising a nucleotide sequence encoding the GPC3.Attorney Docket No. 40056-0091 WO1 12. The method of claim 11, wherein the method further comprises administering to the subject an effective amount of a GPC3 -targeting therapy,13. The method of claim 11 or claim 12, wherein the OV-GPC3 does not encode a functional thymidine kinase and / or wherein the human GPC3 comprises a GPC variant 1 (GPC3-V1) or comprises a GPC variant 2 (GPC3-V2).

14. The method of any one of claims 11-13, wherein the OV-GPC3 does not encode any of an? XFP, a BCMA, a BCMAt, a CA125, a GDI 9, a CD19t, a CD20, a CD33, a CD22, a GDI 23, a CD30, a CD38, a CEA, a IIER2, a GD2, a PSMA, a Claudin 18.2, a EpCAM, a GD2, a MSLN, an EGFR, an EGFRVIII, a Trop-2, a c-MET, a Nectin-4, a CD79b, a CCK4, a GPA33, a HL / X-A2, a CLEC12A, a p-cadherin, a TD02, a MART-I, a MUCI, a Pmel 17, a MAGE-I, a TRP-1, a TRP-2, a NY-ESQ, a PSA, a CDK4, a BCA225, a GA 125, a MG7-Ag, a NY-CO-I, a ROAS 1, a SDCCAG16, a TAAL6, and a TAG72, and optionally including functional variants thereof.

15. The method of any one of claims 11-14, wherein the transgene comprises a promoter that drives expression the nucleotide sequence encoding the GPC3.

16. The method of any one of claims 11-15, wherein GPC3 has one or two point mutations that decreases GPC3 binding to Wnt (e.g., one or two of F41E, L66A, W260R, Y264K, L268E, M269S, Y277A, Y408D, L421E, W423A, L428R, and Y432A in GPC3-V2).

17. The method of any one of claims 11-16, wherein GPC3 comprises an amino acid sequence that comprises or consists of any one of SEQ ID NOs: 1-6, 1B-6B, or a variant thereof having 1, 2, 3, 4, or 5 single amino acid modifications (e.g., conservative amino acid substitutions).

18. The method of any one of claims 11-17, wherein the 0V-GPC3 comprises a nucleotide sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100% identical to or has no more than 100 single nucleotide changes compared to nucleotides (nt) 6,302 - 190,102 of SEQ ID NO: Al over the entire length of nt 6,302 - 190,102 of SEQ ID NO: Al;wherein the OV-GPC3 comprises a nucleotide sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100% identical to or has no more than 100 single nucleotide changesAttorney Docket No. 40056-0091 WO1 compared to nucleotides (nt) 6,302 - 190,102 of SEQ ID NO: A3 over the entire length of nt 6,302 - 190,102 of SEQ ID NO: A3;wherein the OV-GPC3 comprises a nucleotide sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100% identical to or has no more than 100 single nucleotide changes compared to nucleotides (nt) 6,302 - 190,102 of SEQ ID NO: A4 over the entire length of nt 6,302 -190,102 of SEQ ID NO: A4; orwherein the OV-GPC3 comprises a nucleotide sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100% identical to or has no more than 100 single nucleotide changes compared to nucleotides (nt) 6,302 - 191,594 of SEQ ID NO: A5 over the entire length of nt 6,302 - 191,594 of SEQ ID NO: A5.The method of any one of claims 11-17, wherein oncolytic virus nucleotide sequence comprises a nucleotide sequence that is at 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to or has no more than 100 single nucleotide changes compared to SEQ ID NO: A2 over the entire length of SEQ ID NO: A2 excluding the 5’ ITR sequence and the 3’ ITR sequence of SEQ ID NO: A2, but comprises a deletion of all or a portion of the JR2 gene sequence of SEQ ID NO: A2, wherein the 5’ ITR consists of nt 1- 4,054 SEQ ID NO: A2 and the 3’ ITR consists of nt 185,351-189,404 of SEQ ID NO: A2; or wherein the OV-GPC3 comprises a nucleotide sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100% identical to or has no more than 100 single nucleotide changes compared to nucleotides (nt) 10,001-179,403 of SEQ ID NO: A2 over the entire length of nt 10,001-179,403 of SEQ ID NO: A2.The method of claim 19, wherein the deletion comprises at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 250, 275, 400, 425, 450, 475, or 500 contiguous nucleotides of nt 77,603-78,136 of SEQ ID NO: A2.The method of any one of claims 11-16, wherein the oncolytic virus nucleotide sequence comprises:a nucleotide sequence that is at 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to nt 4,055-77,602 of SEQ ID NO: A2 and a nucleotide sequence that is at. 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to nt 78,137-158,350 of SEQ ID NO: A2;Attorney Docket No. 40056-0091 WO1 a nucleotide sequence that is at 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to nt 4,055-77,681 of SEQ ID NO: A2 and a nucleotide sequence that is at 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to nt 78,085-158,350 of SEQ ID NO: A2;a nucleotide sequence comprising nt 4,055-77,602 of SEQ ID NO: A2 having no more than 100 single nucleotide changes and a nucleotide sequence comprising nt 78,137-158,350 of SEQ ID NO: A2 having no more than 100 single nucleotide changes; ora nucleotide sequence comprising nt 4,055-77681 of SEQ ID NO: A2 having no more than 100 single nucleotide changes and a nucleotide sequence comprising nt 78085-158,350 of SEQ ID NO: A2 having no more than 100 single nucleotide changes.

22. The method of any one of claims 12-21, wherein the GPC3 -targeting therapy comprises a bispecific T cell engager (TOE) comprising a scFv that binds GPC3 and an scFv that binds CD3.

23. The method of any one of claims 12-22, wherein the GPC3 -targeting therapy is selected from: a TCE comprising or consisting of SEQ ID NO: 11, ERY-974, ABP-110, Codrituzumab (GC33), hYP7, HN3, Anti-Glypican 3 antibody [SP86](ab95363), ECT-204, Anti-CD3 / 'MUCl-armed-cytokme induced killer cells, GPC-3298306, MDX-1414, B010-A, HLX-63, LQ-102, GSK2857916, BMS- 986182, and BMS-986183.

24. The method of any one of claims 12-22, wherein the GPC3 -targeting therapy comprises:(a) a GPC3 target domain comprising:a variable heavy (VH) chain as set forth in table A2, or a variant thereof having 1, 2, 3, 4, or 5 single amino acid modifications (e.g., conservative substitutions), wherein the modifications are not in a CDR region, anda variable light (VL) chain as set forth in table A2, or a variant thereof having 1, 2, 3, 4, or 5 single amino acid modifications (e.g., conservative substitutions), wherein the modifications are not in a CDR region; and(b) a CD3 target domain comprising:a variable heavy (VH) chain as set forth in table A3, or a variant thereof having 1, 2, 3, 4, or 5 single amino acid modifications (e.g,, conservative substitutions), wherein the modifications are not in a CDR region, andAttorney Docket No. 40056-0091 WO1 a variable light (VL) chain as set forth in table A3, or a variant thereof having 1, 2, 3, 4, or 5 single amino acid modifications (eg., conservative substitutions), wherein the modifications are not in a CDR region.

25. The method of any one of claims 12-21, wherein the GPC3 -target! ng therapy comprises a population of autologous or allogeneic human immune cells expressing a chimeric antigen receptor (CAR) or harboring a nucleic acid encoding a CAR, wherein the CAR comprises a GPC -targeting domain, a transmembrane domain, a costimulatory domain, and a CD3ζ signaling domain26. The method of claim 25, wherein the GPC 3 -targeting domain comprises a GPC 3 -targeting domain as set forth in table Al or A2 (e.g., the GPC3-targeting domain of ERY-974, the GPC3 -targeting domain of ABP-110, the GPC3 -targeting domain of Codrituzumab (GC33), the GPC3 -targeting domain of hYP7, an scFv comprising SEQ ID NO: B28).

27. The method of claim 25 or 26, wherein the C AR comprises: a GPC3 -targeting domain comprising a GPC3 scFv or a variant thereof having 1-5 (e.g,, 1 or 2) single amino acid modifications (e.g., substitutions), wherein the modifications are not in a CDR region;a transmembrane domain is selected from: a CD4 transmembrane domain or variant thereof having 1-5 (e.g., 1 or 2) single ammo acid modifications (e.g., substitutions), a CD8 transmembrane domain or variant thereof having 1-5 (e.g,, 1 or 2) single ammo acid modifications (e.g., substitutions), a CD28 transmembrane domain or a variant thereof having 1-5 (e.g., 1 or 2) single amino acid modifications (e.g., substitutions), and a CD3£ transmembrane domain or a variant thereof having 1-5 (e.g., 1 or 2) single amino acid modifications (e.g., substitutions);a costimulatory domain is selected from: a CD28 co-stimulatory domain or a variant thereof having 1- 5 (e.g., 1 or 2) single amino acid modifications (e.g., substitutions); or a 4-1 BB costimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) single amino acid modifications (e.g., substitutions); or both a CD28 co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) single ammo acid modifications (e.g., substitutions) and a 4-1 BB co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) single ammo acid modifications (e.g., substitutions); and a CD32, signaling domain or a variant thereof having 1-5 (e.g., 1 or 2) single ammo acid modifications.Attorney Docket No. 40056-0091 WO1 28. The method of any one of claims 25-27, wherein the CAR further comprises a spacer domain located between the GPC3 targeting domain and the transmembrane domain and comprising any one of SEQ ID NOs: 24-34, or a variant of one or more thereof having 1-5 (e.g,, 1 or 2) single amino acid modifications,29. The method of any one of claims 25-28, wherein the CAR comprises SEQ ID NO: DI or D2, or a variant thereof having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 single amino acid modifications (e.g,, conservative substitutions), wherein the modifications are not in a CDR region or the GPC3 scFv domain.

30. The method of any one of claims 26-29, wherein the immune cells are selected from: macrophages, NK cells, NKT cells, T cells, subpopulations of one or more thereof, and combinations thereof.

31. The method of any one of claims 11 -30, wherein the solid tumor is HER2 positive and the method further comprises administering an effective amount of a population of HER2 CAR T cells.

32. The method of claim 31, wherein the population of HER2 CAR T cells expresses a CAR comprising SEQ ID NO: ZZ1 orZZ2.

33. A method of treating a subject having a solid tumor comprising:administering to the subject an effective amount of an oncolytic virus expressing a human glypican-3 (GPC3), wherein the nucleotide sequence of oncolytic virus (OV-GPC3) comprises a nucleotide sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to or has no more than 300 single nucleotide changes compared to:a) SEQ ID NO: Al over the entire length of SEQ ID NO: Al;b) nucleotides (nt) 6,302 - 190,102 of SEQ ID NO: Al over the entire length of nt 6,302 - 190, 102 of SEQ ID NO: Al;c) nt 8,000-187,000 of SEQ ID NO: A1 over the entire length of nt 8,000-187,000 SEQ ID NO: Al;d) SEQ ID NO: A3 over the entire length of SEQ ID NO: A3;e) nucleotides (nt) 6,302 - 190,102 of SEQ ID NO: A3 over the entire length of nt 6,302 - 190, 102 of SEQ ID NO: A3;Attorney Docket No. 40056-0091 WO1 f) nt 8,000-187,000 of SEQ ID NO: A3 over the entire length of nt 8,000-187,000 SEQ ID NO: A3;g) SEQ ID NO: A4 over the entire length of SEQ ID NO: A4;h) nucleotides (nt) 6,302 - 190,102 of SEQ ID NO: A4 over the entire length of nt 6,302 - 190, 102 of SEQ ID NO: A4;i) nt 8,000-187,000 of SEQ ID NO: A4 over the entire length of nt 8,000-187,000 SEQ ID NO: A4;j) SEQ ID NO: A5 over the entire length of SEQ ID NO: A5;k) nucleotides (nt) 6,302 - 191,594 of SEQ ID NO: A5 over the entire length of nt 6,302 - 191,594 of SEQ ID NO: A5; orl) nt 8,000-190,000 of SEQ ID NO: A5 over the entire length of nt 8,000-190,000 SEQ ID NO: A5.

34. A method of treating a subject having a solid tumor comprising administering to the subject an effective amount of an oncolytic virus (OV) expressing a human glypican-3 (GPC3), wherein the nucleotide sequence of oncolytic virus (OV-GPC3) comprises a nucleotide sequence comprising:(i) nucleotides 6,302 - 80,395 of SEQ ID NO: Al having no more than 300 single nucleotide modifications (e.g., modifications that do not change the amino acid sequence of the encoded proteins);(ii) a nucleotide sequence encoding a human GPC3; and(iii) nucleotides 82,356 - 190,102 of SEQ ID NO: Al having no more than 300 single nucleotide modifications (e.g., modifications that do not change the amino acid sequence of the encoded proteins).

35. A method of treating a subject having a solid tumor comprising:administering to the subject an effective amount of an oncolytic virus expressing a human glypican-3 (GPC3), wherein the nucleotide sequence of oncolytic virus (OV-GPC3) comprises:(a) an oncolytic virus nucleotide sequence; and(b) a nucleotide sequence encoding a GPC3;wherein the oncolytic virus nucleotide sequence comprises a nucleotide sequence that is at 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to or has no more than 300 single nucleotide changes compared to:Attorney Docket No. 40056-0091 WO1 a) SEQ ID NO: A2 over the entire length of SEQ ID NO: A2, but lacks a functional JR2 gene sequence of SEQ ID NO: A2;b) SEQ ID NO: A2 over the entire length of SEQ ID NO: A2, but lacks a functioning JR2 gene sequence, the 5’ ITR sequence and the 3’ ITR sequence of SEQ ID NO: A2 (e g., a nucleotide sequence that is at 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to nt 4,055-77,602 of SEQ ID NO: A2 and a nucleotide sequence that is at 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to nt 78,137-158,350 of SEQ ID NO: A2); orc) nucleotides 4,055-185,350 of SEQ ID NO: A2 over the entire length of nucleotides 4,055-185,350 SEQ ID NO: A2, but all or a portion of the J2R sequence.

36. The method of any one of claims 33-35, wherein the method further comprises administering to he subject an effective amount of a therapy that binds to the GPC3.

37. The method of any of claims 33-36, wherein the oncolytic virus nucleotide sequence has no modifications in the coding regions comprising SEQ ID NOs: X1-X275, or wherein any modifications within the coding regions (SEQ ID NOs: X1-X275) do not change the ammo acid sequence of the encoded protein; and / orwherein the OV-GPC3 does not encode functional thymidine kinase; and / or wherein the OV-GPC3 does not encode an APP, a BCMA, a BCMAt, a CA125, a CD19, a CD19t, a CD20, a CD33, a CD22, a CD123, a CD30, a CD38, a CEA, a HER2, a GD2, a PSMA, a Claudin 18.2, a EpCAM, a GD2, a SLN, an EGFR, an EGFRVffl, a Trop-2, a c-MET, a Nectm-4, a CD79b, a CCK4, a GPA33, a HLA-A2, a CLEC12A, a p-cadhenn, a TDO2, a MART-I, a MUCI, a Pmel 17, a MAGE-I, a TRP-1, a TRP-2, a NY-ESQ, a PSA, a CDK4, a BCA225, a CA 125, a MG7-Ag, a NY-CO-I, a RCAS 1, a SDCCAG16, a TAAL6, and a TAG72.

38. The method of any of claims 35-37, wherein the oncolytic virus nucleotide sequence comprises a nucleotide sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or 100% identical to or has no more than 100 single nucleotide changes compared to: SEQ ID NO: A2 over the entire length of SEQ ID NO: A2, but lacks all or a portion of the JR2 gene sequence and comprises nucleotide sequences that encode the proteins having SEQ ID NOs: Z1-Z275, or a variant thereof having 1 2, 3, 4, or 5 single amino acid substitutions.Attorney Docket No. 40056-0091 WO139. The method of claim 33 or 34, wherein the 0V-GPC3 comprises a nucleotide sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99% or 100% identical to SEQ ID NO: Al, A3, A4, or A5 over the entire length of SEQ ID NO: Al, A3, A4, or A5;and encodes proteins having the amino acid sequences of SEQ ID NOs: SEQ ID NO: Z1, Z17, Z26, Z27, Z28, Z32, Z34, Z36, Z41, Z52, Z53, Z55, Z63, Z65, Z66, Z76, Z77, Z79, Z81, Z82, Z87, Z90, Z91, Z92, Z93, Z104, Z108, Z110, Z111, Z116, Z118, Z120, Z123, Z128, Z129, Z130, Z138, Z140, Z142, Z143, Z145, Z146, Z147, Z149, Z150, Z151, Z152, Z153, Z154, Z155, Z156, Z157, Z158, Z160, Z161, Z165, Z168, Z169, Z170, Z172, Z173, Z174, Z175, Z176, Z177, Z178, Z181, Z182, Z186, Z192, Z193, Z194, Z195, Z197, Z198, Z199, Z203, Z206, Z211, Z212, Z216, Z219, Z220, Z222, Z223, Z224, Z225, Z226, Z227, Z228, Z229, Z231, Z232, Z233, Z234, Z236, Z238, Z240, Z241, Z243, Z244, Z252, Z253, Z254, Z257, Z258, Z259, Z260, Z261, Z263, Z264, Z265, Z266, Z267, Z268, Z269, Z270, Z271, Z272, Z274, and Z275, or a variant thereof having 1, 2, 3, 4, or 5 single amino acid substitutions.

40. The method of claim 33 or 34, wherein the OV-GPC3 comprises a nucleotide sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to or has no more than 100 single nucleotide changes compared to:(i) nt 6,302 - 190,102 of SEQ ID NO: Al over the entire length of nt 6,302 -190,102 SEQ ID NO: Al;(ii) nt 8,000-187,000 of SEQ ID NO: Al over the entire length of nt 8,000-187,000 SEQ ID NO: Al; (iii) nt 6,302 - 190,102 of SEQ ID NO: A3 over the entire length of nt 6,302 - 190,102 of SEQ ID NO: A3;(iv) nt 8,000-187,000 of SEQ ID NO A3 over the entire length of nt 8,000-187,000 SEQ ID NO: A3; (v) nt 6,302 - 190,102 of SEQ ID NO: A4 over the entire length of nt 6,302 - 190,102 of SEQ ID NO: A4;(vi) nt 8,000-187,000 of SEQ ID NO: A4 over the entire length of nt 8,000-187,000 SEQ ID NO: A4; (vii) nt 6,302 - 191,594 of SEQ ID NO: A5 over the entire length of nt 6,302 - 191,594 of SEQ ID NO: A5; or(viii) nt 8,000-190,000 of SEQ ID NO: A5 over the entire length of nt 8,000-190,000 SEQ ID NO: A5;and encodes proteins having the amino acid sequences of SEQ ID NOs: Zl, Zl 7, Z26, Z27, Z28, Z32, Z34, Z36, Z41, Z52, Z53, Z55, Z63, Z65, Z66, Z76, Z77, Z79, Z81, Z82, Z87, Z90, Z91, Z92, Z93,Attorney Docket No. 40056-0091 WO1 Z104, Z108, Z110, Z111, Z116, Z118, Z120, Z123, Z128, Z129, Z130, Z138, Z140, Z142, Z143, Z145, Z146, Z147, Z149, Z150, Z151, Z152, Z153, Z154, Z155, Z156, Z157, Z158, Z160, Z161, Z165, Z168, Z169, Z170, Z172, Z173, Z174, Z175, Z176, Z177, Z178, Z181, Z182, Z186, Z192, Z193, Z194, Z195, Z197, Z198, Z199, Z203, Z206, Z211, Z212, Z216, Z219, Z220, Z222, Z223, Z224, Z225, Z226, Z227, Z228, Z229, Z231, Z232, Z233, Z234, Z236, Z238, Z240, Z241, Z243, Z244, Z252, Z253, Z254, Z257, Z258, Z259, Z260, Z261, Z263, Z264, Z265, Z266, Z267, Z268, Z269, Z270, Z271, Z272, Z274, and Z275, or a variant thereof having 1, 2, 3, 4, or 5 single amino acid substitutions,.

41. The method of claim 35 or claim 36, wherein: the oncolytic virus sequence is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100% identical to or has no more than 300 single nucleotide changes compared to nt 4,055-77,602 of SEQ ID NO: A2 and nt 78,137-158,350 SEQ ID NO: A2 over the entire length of nt 4,055-77,602 of SEQ ID NO: A2 and nt 78,137-158,350 SEQ ID NO: A2.

42. The method of claim 35 or claim 36, wherein OV-GPC3 is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100% identical to or has no more than 300 single nucleotide changes compared to: SEQ ID NO: A2 over the entire length of SEQ ID NO: A2 except that the nucleotide sequence encoding GPC3 and a promoter sequence for expressing GPC3 replaces at least 10 contiguous nucleotides of the JR2 gene sequence.

43. The method of any one of claims 35, 36, and 42, wherein at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 250, 275, 400, 425, 450, 475, or 500 contiguous nucleotides of the J2R gene sequence (nt 77,603-78,136 of SEQ ID NO: A2) have been deleted.

44. The method of claim 43, wherein the deletion comprises nucleotides 77,682-78,084 of SEQ ID NO: A2.

45. The method of any one of claims 35-36 and 42-44, wherein the nucleotide sequence encoding GPC3 is inserted into a noncoding region of SEQ ID NO: A2.Attorney Docket No. 40056-0091 WO1 46. The method of any of claims 34-36, and 42-45, wherein the nucleotide sequence encoding the GPC3 encodes one or two single amino acid substitutions (e.g., one or two of F41E, L66A, W260R, Y264K, L268E, M269S, Y277A, Y408D, L421E, W423A, L428R, and Y432A in GPC3-V2).

47. The method of any of claims 34-36 and 42-46, wherein the GPC3 comprises the amino acid sequence of any one of SEQ ID NOs: 1, IB, 2, 2B, 3, 3B, 4, 4B, 5, 5B, 6, and 6B, or a variant thereof having 1, 2, 3, 4, or 5 single amino acid substitutions.

48. The method of any of claims 34-36 and 42-47, wherein the GPC3 comprises an amino acid sequence that comprises or consists of SEQ ID NO: 1, 2, 3, 4, 5, 6, IB, 2B, 3B, 4B, 5B, or 6B.

49. The method of any of claims 34-36 and 42-48, wherein the nucleotide sequence encoding GPC3 is operably linked to a promoter.

50. The method of any one of claims 36-49 and 89, wherein the therapy that binds to the GPC3 is any one or more of a population of immune cells expressing a receptor comprising a domain that binds GPC3, an antibody or antibody fragment targeted to GPC3, and a TCE comprising a domain that binds GPC3 and a domain that binds CD3.

51. The method of claim 50, wherein the domain that binds GPC3 comprises:a VH comprising an amino acid sequence selected from SEQ ID NOs: B8, B16, B26, B33, B41, B49, B55, B63, and B71, or a variant thereof having no more than 5 single amino acid substitutions, wherein the substitutions are not in the CDRs, anda VL comprising an amino acid sequence selected from SEQ ID NOs: B4, B12, B20, B27, B37, B45, B53, B59, B67, and B75, or a variant thereof having no more than 5 single amino acid substitutions, wherein the substitutions are not in the CDRs.

52. The method of claim 50, wherein the domain that binds GPC3 (e.g., a GPC3-targeted scFv) comprises any one of the following:a variable light chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B1-B3 and a variable heavy chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B5-B7;Attorney Docket No. 40056-0091 WO1 a variable light chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B9-B11 and a variable heavy chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B13-B15;a region having CDRl, CDR2, and CDR3 comprising SEQ ID NOs: B17-B19 or a region having CDRl, CDR2, and CDR3 comprising SEQ ID NOs: B21-B23;a variable light chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B32-B34 and a variable heavy chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B29-B3I;a variable light chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B38-B40 and a variable heavy chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B34-B36;a variable light chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B46-B48 and a variable heavy chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B42-B44;a variable light chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B46, B54, and B48 and a variable heavy chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B50-B52;a variable light chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B60-B62 and a variable heavy chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B56-B58;a variable light chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B68-B70 and a variable heavy chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B64-B66; anda variable light chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B76-B78 and a variable heavy chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B72-B74.

53. The method of any one of claims 50-52, wherein the domain that binds CD3 (e.g., a CD3-targeted scFv) comprises:a variable light chain region having CDR1, CDR2, and CDR3 regions comprising SEQ ID NOs: C1-C3 and a variable light chain region having CDR1, CDR2, and CDR3 regions comprising SEQ ID NOs: C5-C7;Attorney Docket No. 40056-0091 WO1 a variable light chain region having CDR1, CDR2, and CDR3 regions comprising SEQ ID NOs: C9-C11 and a variable light chain region having CDR1, CDR2, and CDR3 regions comprising SEQ ID NOs: C13-C15;a variable light chain region having CDR1, CDR2, and CDR3 regions comprising SEQ ID NOs: C17-C19 and a variable light chain region having CDR1, CDR2, and CDR3 regions comprising SEQ ID NOs: C21-C23;a variable light chain region comprising SEQ ID NO: C4 and a variable heavy region comprising SEQ ID NO: C8;a variable light chain region comprising SEQ ID NO: C12 and a variable heavy region comprising SEQ ID NO: Cl 6;a variable light chain region comprising SEQ ID NO: C20 and a variable heavy region comprising SEQ ID NO: C24; ora VII comprising an ammo acid sequence selected from SEQ ID NOs: C8, C24, C33, C41, C49, C59, C69, and C70, or a variant thereof having no more than 5 single amino acid substitutions, wherein the substitutions are not in the CDRs, anda VL comprising an amino acid sequence selected from SEQ ID NOs: C4, C20, C29, C37, C45, C55, C62, and C74, or a variant thereof having no more than 5 single ammo acid substitutions, wherein the substitutions are not in the CDRs.

54. The method of any one of claims 50-53, wherein the therapy that binds GPC3 comprises any one of the constructs in Table Al.

55. The method of any one of claims 11-54 and 89, wherein the solid tumor is HER2 positive and the method further comprises administering an effective amount of a population of HER2 CAR T cells.

56. An oncolytic virus expressing a GPC3 (OV-GPC3), the nucleotide sequence of OV-GPC3 comprising:(a) an oncolytic virus nucleotide sequence; and(b) a nucleotide sequence encoding the GPC3;wherein the oncolytic virus nucleotide sequence encodes at least 25, 50, 60, 70, 80, 90, 100, 110, 120, or 121 of SEQ ID NO: Z1, Z17, Z26, Z27, Z28, Z32, Z34, Z36, Z41, Z52, Z53, Z55, Z63, Z65, Z66, Z76, Z77, Z79, Z81, Z82, Z87, Z90, Z91, Z92, Z93, Z104, Zl 08, Zl 10,Attorney Docket No. 40056-0091 WO1 Z111, Z116, Z118, Z120, Z123, Z128, Z129, Z130, Z138, Z140, Z142, Z143, Z145, Z146, Z147, Z149, Zl 50, Zl 51, Z152, Z153, Z154, Z155, Z156, Zl 57, Zl 58, Z160, Z161, Z165, Z168, Z169, Z170, Z172, Z173, Z174, Z175, Z176, Z177, Z178, Z181, Z182, Z186, Z192, Z193, Z194, Z195, Z197, Z198, Z199, Z203, Z206, Z211, Z212, Z216, Z219, Z220, Z222, Z223, Z224, Z225, Z226, Z227, Z228, Z229, Z231, Z232, Z233, Z234, Z236, Z238, Z240, Z241, Z243, Z244, Z252, Z253, Z254, Z257, Z258, Z259, Z260, Z261, Z263, Z264, Z265, Z266, Z267, Z268, Z269, Z270, Z271, Z272, Z274, and Z275; and / orwherein the OV-GPC3 nucleotide sequence is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identical to or has no more than 300 single nucleotide changes compared to:(i) nt 6,302 - 190,102 of SEQ ID NO: Al over the entire length of nt 6,302 -190,102 SEQ ID NO: Al;(ii) nt 8,000-187,000 of SEQ ID NO: Al over the entire length of nt 8,000-187,000 SEQ ID NO: Al;(in) nucleotides 10,000-185,000 of SEQ ID NO: Al over the entire length of nucleotides 10,000-185,000 SEQ ID NO: Al;(iv) nt 6,302 - 190,102 of SEQ ID NO: A3 over the entire length of nt 6,302 - 190,102 of SEQ ID NO: A3;(v) nt 8,000-187,000 of SEQ ID NO: A3 over the entire length of nt 8,000-187,000 SEQ ID NO: A3;(vi) nucleotides 10,000-185,000 of SEQ ID NO: A3 over the entire length of nucleotides 10,000-185,000 SEQ ID NO: A3;(vii) nt 6,302 - 190,102 of SEQ ID NO: A4 over the entire length of nt 6,302 - 190,102 of SEQ ID NO: A4;(vi) nt 8,000-187,000 of SEQ ID NO: A4 over the entire length of nt 8,000-187,000 SEQ ID NO: A4;(vii) nucleotides 10,000-185,000 of SEQ ID NO: A4 over the entire length of nucleotides 10,000- 185,000 SEQ ID NO: A4;(viii) nt 6,302 - 191,594 of SEQ ID NO: A5 over the entire length of nt 6,302 - 191,594 of SEQ ID NO: A5;(ix) nt 8,000-190,000 of SEQ ID NO: A5 over the entire length of nt 8,000-190,000 SEQ ID NO: A5; orAttorney Docket No. 40056-0091 WO1 (x) nucleotides 10,000-185,000 of SEQ ID NO: A5 over the entire length of nucleotides 10,000-185,000 SEQ ID NO: A5.

57. The OV-GPC3 of claim 56, wherein the OV-GPC3 nucleotide sequence is at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to or has no more than 100 single nucleotide changes compared to:(i) nt6,302 - 190,102 of SEQ ID NO: Al over the entire length of nt 6,302 -190,102 SEQ ID NO: Al;(li) nt 8,000-187,000 of SEQ ID NO: Al over the entire length of nt 8,000-187,000 SEQ ID NO: Al;(lii) nucleotides 10,000-185,000 of SEQ ID NO: Al over the entire length of nucleotides 10,000-185,000 SEQ ID NO: Al;(iv) nt 6,302 - 190,102 of SEQ ID NO: A3 over the entire length of nt 6,302 - 190,102 of SEQ ID NO: A3;(v) nt 8,000-187,000 of SEQ ID NO: A3 over the entire length of nt 8,000-187,000 SEQ ID NO: A3;(vi) nucleotides 10,000-185,000 of SEQ ID NO: A3 over the entire length of nucleotides 10,000-185,000 SEQ ID NO: A3;(vii) nt 6,302 - 190,102 of SEQ ID NO: A4 over the entire length of nt 6,302 - 190,102 of SEQ ID NO: A4;(vi) nt 8,000-187,000 of SEQ ID NO: A4 over the entire length of nt 8,000-187,000 SEQ ID NO: A4;(vii) nucleotides 10,000-185,000 of SEQ ID NO: A4 over the entire length of nucleotides 10,000- 185,000 SEQ ID NO: A4;(viii) nt 6,302 - 191,594 of SEQ ID NO: A5 over the entire length of nt 6,302 - 191,594 of SEQ ID NO: A5;(ix) nt 8,000-190,000 of SEQ ID NO A5 over the entire length of nt 8,000-190,000 SEQ ID NO: A5;(x) nucleotides 10,000-185,000 of SEQ ID NO: A5 over the entire length of nucleotides 10,000-185,000 SEQ ID NO: A5;(xi) SEQ ID NO: Al over the entire length of SEQ ID NO: Al;(xii) SEQ ID NO: A3 over the entire length of SEQ ID NO: A3;Attorney Docket No. 40056-0091 WO1 (xiii) SEQ ID NO: A4 over the entire length of SEQ ID NO: A4; or(xiv) SEQ ID NO: A5 over the entire length of SEQ ID NO: A5.

58. The OV-GPC3 of claim 56 or claim 57, wherein any nucleotide modifications in the oncolytic virus nucleotide sequence do not change the ammo acid sequence of encoded proteins having an ammo acid sequences SEQ ID NO: Z1, Z17, Z26, Z27, Z28, Z32, Z34, Z36, Z41, Z52, Z53, Z55, Z63, Z65, Z66, Z76, Z77, Z79, Z81, Z82, Z87, Z90, Z91, Z92, Z93, Z104, Z108, Z110, Zi ll, Z116, Z118, Z120, Z123, ZI28, Z129, Z130, Z138, Z140, Z142, Z143, ZI45, Z146, Z147, Z149, Z150, Z151, Z152, Z153, Z154, Z155, Z156, Z157, Z158, ZI60, Z161, Z165, Z168, Z169, Z170, Z172, ZI73, Z174, Z175, Z176, Z177, ZI78, Z18I, Z182, Z186, Z192, Z193, Z194, ZI95, Z197, Z198, Z199, Z203, Z206, Z21I, Z212, Z216, Z219, Z220, Z222, Z223, Z224, Z225, Z226, Z227, Z228, Z229, Z231, Z232, Z233, Z234, Z236, Z238, Z240, Z24I, Z243, Z244, Z252, Z253, Z254, Z257, Z258, Z259, Z260, Z261, Z263, Z264, Z265, Z266, Z267, Z268, Z269, Z270, Z27I, Z272, Z274, and Z275.

59. The OV-GPC3 of any one of claims 56-58, wherein any nucleotide modifications in the oncolytic virus nucleotide sequence do not change the amino acid sequence of encoded proteins having an amino acid sequences SEQ ID NO: Z1-Z275.

60. The OV-GPC3 of any one of claims 56-59, wherein the OV-GPC3 does not encode any of an AFP, a CA125, a CD19, a CD19t, a CD20, a CD33, a CD22, a CD123, a CD30, a CD38, a GPC-3, a CEA, a HER2, a GD2, a PSMA, a Claudin 18.2, a EpCAM, a GD2, a MSLN, an EGFR, an EGFRVIII, a Trop-2, a c-MET, a Nectin-4, a CD79b, a CCK4, a GPA33, a HLA-A2, a CLEC12A, a p-cadherin, a TDO2, a MART-I, a MUCI, a Pmel 17, a MAGE-I, a TRP-1, a TRP-2, a NY-ESQ, a PSA, a CDK4, a BCA225, a CA 125, a MG7-Ag, a NY-CO-I, a RCAS 1, a SDCCAG16, a TAAL6, and a TAG72, and optionally functional variants of one or more thereof.

61. The OV-GPC3 of any one of claims 56-60, wherein the GPC3 comprises one or two single amino acid substitutions, wherein the substitutions are selected from: F41E, L66A, W260R, Y264K, L268E, M269S, Y277A, Y408D, L421E, W423A, L428R, and Y432A m GPC3-V2.Attorney Docket No. 40056-0091 WO1 62. The 0V-GPC3 of any one of claims 56-61, wherein the GPC3 comprises the amino acid sequence of any one of SEQ ID NOs: 1, IB, 2, 2B, 3, 3B, 4, 4B, 5, 5B, 6, and 6B, or a variant thereof having 1, 2, 3, 4, or 5 single amino acid substitutions.

63. The 0V-GPC3 of any one of claims 56-62, wherein the GPC3 comprises an amino acid sequence hat comprises or consists of SEQ ID NO: 1, 2, 3, 4, 5, 6, IB, 2B, 3B, 4B, 5B, or 6B.

64. The OV-GPC3 of any one of claims 56-63, wherein the nucleotide sequence encoding GPC3 is operably linked to a promoter.

65. An oncolytic virus expressing a GPC3 (0V-GPC3) comprising a nucleotide sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to or has no more than 100 single nucleotide changes compared to nucleotides 6,302 - 190,102 of SEQ ID NO: Al over the entire length of nucleotides 6,302 - 190,102 of SEQ ID NO: Al.

66. An oncolytic virus expressing a GPC3 (OV-GPC3), the nucleotide sequence of OV-GPC3 comprising:(a) an oncolytic virus nucleotide sequence; and(b) a nucleotide sequence encoding a human GPC3;wherein the oncolytic virus nucleotide sequence encodes at least 25, 50, 60, 70, 80, 90, 100, 110, 120, or 121 of SEQ ID NO: Z1, Z17, Z26, Z27, Z28, Z32, Z34, Z36, Z41, Z52, Z53, Z55, Z63, Z65, Z66, Z76, Z77, Z79, Z81, Z82, Z87, Z90, Z91, Z92, Z93, Z104, Z108, Z110, Z111, Z116, Z118, Z120, Z123, Z128, Z129, Zl 30, Z138, Z140, Z142, Z143, Z145, Z146, Z147, Z149, Z150, Z151, Z152, Z153, Z154, Z155, Z156, Z157, Z158, Z160, Z161, Z165, Z168, Z169, Z170, Z172, Z173, Z174, Z175, Z176, Z177, Z178, Z181, Z182, Z186, Z192, Z193, Z194, Z195, Z197, Z198, Z199, Z203, Z206, Z211, Z212, Z216, Z219, Z220, Z222, Z223, Z224, Z225, Z226, Z227, Z228, Z229, Z231, Z232, Z233, Z234, Z236, Z238, Z240, Z241, Z243, Z244, Z252, Z253, Z254, Z257, Z258, Z259, Z260, Z261, Z263, Z264, Z265, Z266, Z267, Z268, Z269, Z270, Z271, Z272, Z274, and Z275; and / orwherein the OV-GPC3 nucleotide sequence comprises nucleotides 6,302 - 190,102 of SEQ ID NO: Al or a variant thereof with up to 100, 200, or 300 single nucleotide substitutions.Attorney Docket No. 40056-0091 WO1 67. The 0V-GPC3 of claim 66, wherein the 0V-GPC3 nucleotide sequence comprises nucleotides 6,302 - 190,102 of SEQ ID NO: Al or a variant thereof with up to 100 single nucleotide substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotide substitutions).

68. An oncolytic virus expressing a GPC3 (OV-GPC3) comprising a nucleotide sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to or has no more than 100 single nucleotide changes compared to nucleotides 6,302 - 190,102 of SEQ ID NO: A3 over the entire length of nucleotides 6,302 - 190,102 of SEQ ID NO: A3.

69. An oncolytic virus expressing a GPC3 (OV-GPC3), the nucleotide sequence of OV-GPC3 comprising:(a) an oncolytic virus nucleotide sequence; and(b) a nucleotide sequence encoding a human GPC3;wherein the oncolytic virus nucleotide sequence encodes at least 25, 50, 60, 70, 80, 90, 100, 110, 120, or 121 of SEQ ID NO: Z1, Z17, Z26, Z27, Z28, Z32, Z34, Z36, Z41, Z52, Z53, Z55, Z63, Z65, Z66, Z76, Z77, Z79, Z81, Z82, Z87, Z90, Z91, Z92, Z93, Z104, Z108, Z110, Z111, Z116, Z118, Z120, Z123, Z128, Z129, Z130, Z138, Z140, Z142, Z143, Z145, Z146, Z147, Z149, Z150, Z151, Z152, Z153, Z154, Z155, Z156, Z157, Z158, Z160, Z161, Z165, Z168, Z169, Z170, Z172, Z173, Z174, Z175, Z176, Z177, Z178, Z181, Z182, Z186, Z192, Z193, Z194, Z195, Z197, Z198, Z199, Z203, Z206, Z211, Z212, Z216, Z219, Z220, Z222, Z223, Z224, Z225, Z226, Z227, Z228, Z229, Z231, Z232, Z233, Z234, Z236, Z238, Z240, Z241, Z243, Z244, Z252, Z253, Z254, Z257, Z258, Z259, Z260, Z261, Z263, Z264, Z265, Z266, Z267, Z268, Z269, Z270, Z271, Z272, Z274, and Z275; and / orw’herein the OV-GPC3 nucleotide sequence comprises nucleotides 6,302 - 190,102 of SEQ ID NO: A3 or a variant thereof with up to 100, 200, or 300 single nucleotide substitutions.

70. The OV-GPC3 of claim 69, wherein the OV-GPC3 nucleotide sequence comprises nucleotides 6302 - 190,102 of SEQ ID NO: A3 or a variant thereof with up to 100 single nucleotide substitutions (eg 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,Attorney Docket No. 40056-0091 WO1 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotide substitutions),71. An oncolytic virus expressing a GPC3 (OV-GPC3) comprising a nucleotide sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to or has no more than 100 single nucleotide changes compared to nucleotides 6,302 - 190,102 of SEQ ID NO: A4 over the entire length of nucleotides 6,302 - 190,102 of SEQ ID NO: A4.

72. An oncolytic virus expressing a GPC3 (OV-GPC3), the nucleotide sequence of OV-GPC3 comprising:(a) an oncolytic virus nucleotide sequence; and(b) a nucleotide sequence encoding a human GPC3;wherein the oncolytic virus nucleotide sequence encodes at least 25, 50, 60, 70, 80, 90, 100, 110, 120, or 121 of SEQ ID NO: Z1, Z17, Z26, Z27, Z28, Z32, Z34, Z36, Z41, Z52, Z53, Z55, Z63, Z65, Z66, Z76, Z77, Z79, Z81, Z82, Z87, Z90, Z91, Z92, Z93, Z104, Z108, Zl 10, Z111, Z116, Z118, Z120, Z123, Z128, Z129, Z130, Z138, Z140, Z142, Z143, Z145, Z146, Z147, Z149, Z150, Z151, Z152, Z153, Z154, Z155, Z156, Z157, Z158, Z160, Z161, Z165, Z168, Z169, Z170, Z172, Z173, Z174, Z175, Z176, Z177, Z178, Z181, Z182, Z186, Z192, Z193, Z194, Z195, Z197, Z198, Z199, Z203, Z206, Z211, Z212, Z216, Z219, Z220, Z222, Z223, Z224, Z225, Z226, Z227, Z228, Z229, Z231, Z232, Z233, Z234, Z236, Z238, Z240, Z241, Z243, Z244, Z252, Z253, Z254, Z257, Z258, Z259, Z260, Z261, Z263, Z264, Z265, Z266, Z267, Z268, Z269, Z270, Z271, Z272, Z274, and Z275; and / orwherein the OV-GPC3 nucleotide sequence comprises nucleotides 6,302 - 190,102 of SEQ ID NO: A4 or a variant thereof with up to 100, 200, or 300 single nucleotide substitutions.

73. The OV-GPC3 of claim 72, wherein the OV-GPC3 nucleotide sequence comprises nucleotides 6302 - 190,102 of SEQ ID NO: A4 or a variant thereof with up to 100 single nucleotide substitutions (eg 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 31 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58 59 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86 87 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotide substitutions).Attorney Docket No. 40056-0091 WO174. An oncolytic virus expressing a GPC3 (0V-GPC3) comprising a nucleotide sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to or has no more than 100 single nucleotide changes compared to nucleotides 6,302 - 191,594 of SEQ ID NO: A5 over the entire length of nucleotides 6,302 - 191,594 of SEQ ID NO: A5.

75. An oncolytic virus expressing a GPC3 (OV-GPC3), the nucleotide sequence of OV-GPC3 comprising:(a) an oncolytic virus nucleotide sequence; and(b) a nucleotide sequence encoding a human GPC3;wherein the oncolytic virus nucleotide sequence encodes at least 25, 50, 60, 70, 80, 90, 100, 110, 120, or 121 of SEQ ID NO: Z1, Z17, Z26, Z27, Z28, Z32, Z34, Z36, Z41, Z52, Z53, Z55, Z63, Z65, Z66, Z76, Z77, Z79, Z81, Z82, Z87, Z90, Z91, Z92, Z93, Z104, Z108, Z110, Z111, Z116, Z118, Z120, Z123, Z128, Z129, Z130, Z138, Z140, Z142, Z143, Z145, Z146, Z147, Z149, Z150, Z151, Z152, Z153, Z154, Z155, Z156, Z157, Z158, Z160, Z161, Z165, Z168, Z169, Z170, Z172, Z173, Z174, Z175, Z176, Z177, Z178, Z181, Z182, Z186, Z192, Z193, Z194, Z195, Z197, Z198, Z199, Z203, Z206, Z211, Z212, Z216, Z219, Z220, Z222, Z223, Z224, Z225, Z226, Z227, Z228, Z229, Z231, Z232, Z233, Z234, Z236, Z238, Z240, Z241, Z243, Z244, Z252, Z253, Z254, Z257, Z258, Z259, Z260, Z261, Z263, Z264, Z265, Z266, Z267, Z268, Z269, Z270, Z271, Z272, Z274, and Z275; and / orwherein the OV-GPC3 nucleotide sequence comprises nucleotides 6,302 - 191,594 of SEQ ID NO: A5 or a variant thereof with up to 100, 200, or 300 single nucleotide substitutions.

76. The OV-GPC3 of claim 65, wherein the OV-GPC3 nucleotide sequence comprises nucleotides 6302 - 191,594 of SEQ ID NO: A5 or a variant thereof with up to 100 single nucleotide substitutions (eg 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 31 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58 59 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86 87 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotide substitutions).77 The OV-GPC3 of any one of claims 65-76, wherein the oncolytic virus comprises at least 25, 50, 75 100 125, 150, 175, 200, 225, or 250 of SEQ ID NO: X1-X275.Attorney Docket No. 40056-0091 WO178. The 0V-GPC3 of any one of claims 65-77, wherein the OV-GPC3 does not encode any of an AFP, a CAI 25, a CD19, a CD19t, a CD20, a CD33, a CD22, a CD123, a CD30, a CD38, a GPC-3, a CEA, a HER2, a GD2, a PSMA, a Claudin 18.2, a EpC AM, a GD2, a MSLN, an EGFR, an EGFRVIII, a Trop-2, a c-MET, a Nectin-4, a CD79b, a CCK4, a GPA33, a HLA-A2, a CLEC12A, a p-cadherin, a TD02, a MART-I, a MUCI, a Pmel 17, a MAGE-I, a TRP-1, a TRP-2, a NY-ESQ, a PSA, a CDK4, a BCA225, a CA 125, a MG7-Ag, a NY-CO-I, a RCAS 1, a SDCCAG16, a TAAL6, and a TAG72, and optionally functional variants of one or more thereof.

79. The OV-GPC3 of any one of claims 65-78, wherein the GPC3 comprises one or two single animo acid substitutions selected from: F41E, L66A, W260R, Y264K, L268E, M269S, Y277A, Y408D, L421E, W423 A, L428R, and Y432A m GPC3-V2.

80. The OV-GPC3 of any one of claims 65-69, wherein the GPC3 comprises SEQ ID NO: 1, IB, 2, 2B, 3, 3B, 4, 4B, 5, 5B, 6, and 6B, or a variant thereof having 1, 2, 3, 4, or 5 single ammo acid substitutions.

81. The OV-GPC3 of any one of claims 65-70, wherein the GPC3 comprises an amino acid sequence that comprises or consists of SEQ ID NO: 1, 2, 3, 4, 5, 6, IB, 2B, 3B, 4B, 5B, or 6B.

82. The OV-GPC3 of any one of claims 65-81, wherein the nucleotide sequence encoding GPC3 is operably linked to a promoter.

83. An oncolytic virus expressing GPC3 (OV-GPC3) comprising nucleotides 6,302 - 190,102 of SEQ ID NO: Al or a variant thereof with up to 100 single nucleotide modifications (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotide substitutions).

84. An oncolytic virus expressing GPC3 (OV-GPC3) comprising nucleotides 6,302 - 190,102 of SEQ ID NO: A3 or a variant thereof with up to 100 single nucleotide modifications (e.g., 1, 2, 3, 4, 5, 6,Attorney Docket No. 40056-0091 WO1 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotide substitutions).

85. An oncolytic virus expressing GPC3 (OV-GPC3) comprising nucleotides 6,302 - 190,102 of SEQ ID NO: A4 or a variant thereof with up to 100 single nucleotide modifications (e.g., 1, 2, 3, 4, 5, 6, 7 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92 93, 94, 95, 96, 97, 98, 99, or 100 nucleotide substitutions).86 An oncolytic virus expressing GPC3 (OV-GPC3) comprising nucleotides 6,302 - 191,594 of SEQ ID NO: A5 or a variant thereof with up to 100 single nucleotide modifications (e.g., 1, 2, 3, 4, 5, 6, 7 8 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92 93, 94, 95, 96, 97, 98, 99, or 100 nucleotide substitutions).

87. A T cell engager (TCE) comprising a GPC3-targeting domain and a CD3 -targeting domain, wherein the GPC3 -targeting domain comprises a VH comprising a CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: B29, B30, and B31, respectively, and a VL comprising a CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: B32, 33 and B34, respectively, andwherein the CD3 -targeting domain comprises a VH comprising a CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: C71, C72, and C73, respectively, and a VL comprising a CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: C75, C76 and C77, respectively.88 AT cell engager (TCE) comprising a GPC3 -targeting domain and a CD3 -targeting domain, wherein the GPC3 -targeting domain comprises:mAttorney Docket No. 40056-0091 WO1 a VH comprising an amino acid sequence selected from SEQ ID NOs: B8, B16, B26, B33, B41, B49, B55, B63, and B71 or a variant thereof having no more than 5 single amino acid substitutions, wherein the substitutions are not in the CDRs, anda VL comprising an amino acid sequence selected from SEQ ID NOs: B4, B12, B20, B27, B37, B45, B53, B59, B67, and B75, or a variant thereof having no more than 5 single amino acid substitutions, wherein the substitutions are not in the CDRs; andwherein the CD3-targeting domain comprises:a VI I comprising an ammo acid sequence selected from SEQ ID NOs: C8, C24, C33, C41, C49, C59, C69, and C70, or a variant thereof having no more than 5 single ammo acid substitutions, wherein the substitutions are not in the CDRs, anda VL comprising an ammo acid sequence selected from SEQ ID NOs: C4, C20, C29, C37, C45, C55, C62, and C74, or a variant thereof having no more than 5 single amino acid substitutions, wherein the substitutions are not in the CDRs.

89. A method of treating a subject having a solid tumor comprising administering to the subject an effective amount of the OV-GPC3 of any one of claims 1-10 and 56-88.

90. The method of claim 89, wherein the method further comprises administering to the subject an effective amount of a therapy that binds to the GPC3.

91. The method of claim 90, wherein the therapy that binds to the GPC3 comprises an amino acid sequence selected from:a variable light chain region having CDR1, CDR2, and CDRS regions comprising SEQ ID NOs: B1-B3 and a variable heavy chain region having CDR1, CDR2, and CDRS regions comprising SEQ ID NOs: B5-B7;a variable light chain region having CDR1, CDR2, and CDR3 regions comprising SEQ ID NOs: B9-B11 and a variable heavy chain region having CDR1, CDR2, and CDRS regions comprising SEQ ID NOs: B13-B15;a region having CDR1, CDR2, and CDR3 comprising SEQ ID NOs: B17-B19;a region having CDRl, CDR2, and CDR3 comprising SEQ ID NOs: B21-B23;a variable light chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B32-B34 and a variable heavy chain region having CDRl, CDR2, and CDRS regions comprising SEQ ID NOs: B29-B31;Attorney Docket No. 40056-0091 WO1 a variable light chain region having CDR1, CDR2, and CDR3 regions comprising SEQ ID NOs: B38-B40 and a variable heavy chain region having CDR1, CDR2, and CDR3 regions comprising SEQ ID NOs: B34-B36;a variable light chain region having CDR1, CDR2, and CDR3 regions comprising SEQ ID NOs: B46-B48 and a variable heavy chain region having CDR1, CDR2, and CDR3 regions comprising SEQ ID NOs: B42-B44;a variable light chain region having CDR1, CDR2, and CDR3 regions comprising SEQ ID NOs: B46, B54, and B48 and a variable heavy chain region having CORI, CDR2, and CDR3 regions comprising SEQ ID NOs: B50-B52;a variable light chain region having CDR1, CDR2, and CDR3 regions comprising SEQ ID NOs: B60-B62 and a variable heavy chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B56-B58;a variable light chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B68-B70 and a variable heavy chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B64-B66; anda variable light chain region having CDR1, CDR2, and CDR3 regions comprising SEQ ID NOs: B76-B78 and a variable heavy chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B72-B7492. The method of claim 90 or claim 91, wherein the therapy that binds to the GPC3 comprises any¬ one of:a variable light chain region comprising SEQ ID NO: B4 and a variable heavy region comprising SEQ ID NO: B8;a variable light chain region comprising SEQ ID NO: B12 and a variable heavy region comprising SEQ ID NO: Bl 6;a single domain comprising SEQ ID NO: B20;an scFv comprising SEQ ID NO: B24;a variable light chain region comprising SEQ ID NO: B27 and a variable heavy- region comprising SEQ ID NO: B26;an scFv comprising SEQ ID NO:28;a variable light chain region comprising SEQ ID NO: B37 and a variable heavy region comprising SEQ ID NO: B33;Attorney Docket No. 40056-0091 WO1 a variable light chain region comprising SEQ ID NO: B45 and a variable heavy region comprising SEQ ID NO: B41;a variable light chain region comprising SEQ ID NO: B53 and a variable heavy region comprising SEQ ID NO: B49;a variable light chain region comprising SEQ ID NO: B59 and a variable heavy region comprising SEQ ID NO: B55;a variable light chain region comprising SEQ ID NO: B67 and a variable heavy region comprising SEQ ID NO: B63; anda variable light chain region comprising SEQ ID NO: B75 and a variable heavy region comprising SEQ ID NO: B71.

93. The method of claim 90, wherein the therapy that binds to the GPC3 comprises the TOE of claim 87 or claim 88.

94. The method of claim 90, wherein the therapy that binds to the GPC3 is any one or more of a population of immune cells expressing a receptor comprising a domain that binds GPC3, an antibody or antibody fragment comprising a domain that binds GPC3, and a TCE comprising a domain that binds GPC3 and a domain that binds CD3.

95. The method of claim 94, wherein the domain that binds GPC3 comprises:a VH comprising an ammo acid sequence selected from SEQ ID NOs: B8, B16, B26, B33, B41, B49, B55, B63, and B71, or a variant thereof having no more than 5 single amino acid substitutions, wherein the substitutions are not in the CDRs, anda VL comprising an amino acid sequence selected from SEQ ID NOs: B4, B12, B20, B27, B37, B45, B53, B59, B67, and B75, or a variant thereof having no more than 5 single amino acid substitutions, wherein the substitutions are not in the CDRs.

96. The method of claim 94, wherein the domain that binds GPC3 (e.g., a GPC3-targeted scFv) comprises any one of the following:a variable light chain region having CDR1, CDR2, and CDR3 regions comprising SEQ ID NOs: B1-B3 and a variable heavy chain region having CDR1, CDR2, and CDR3 regions comprising SEQ ID NOs: B5-B7;Attorney Docket No. 40056-0091 WO1 a variable light chain region having CDR1, CDR2, and CDR3 regions comprising SEQ ID NOs: B9-B11 and a variable heavy chain region having CDR1, CDR2, and CDR3 regions comprising SEQ ID NOs: B13-B15;a region having CDRl, CDR2, and CDR3 comprising SEQ ID NOs: B17-B19;a region having CDR1, CDR2, and CDR3 comprising SEQ ID NOs: B21 -B23;a variable light chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B32-B34 and a variable heavy chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B29-B3I;a variable light chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B38-B40 and a variable heavy chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B34-B36;a variable light chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B46-B48 and a variable heavy chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B42-B44;a variable light chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B46, B54, and B48 and a variable heavy chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B50-B52;a variable light chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B60-B62 and a variable heavy chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B56-B58;a variable light chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B68-B70 and a variable heavy chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B64-B66; anda variable light chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B76-B78 and a variable heavy chain region having CDRl, CDR2, and CDR3 regions comprising SEQ ID NOs: B72-B74.

97. The method of any one of claims 94-96, wherein the domain that binds CD3 (e.g., a CD3-targeted scFv) comprises:a variable light chain region having CDR1, CDR2, and CDR3 regions comprising SEQ ID NOs: C1-C3 and a variable light chain region having CDR1, CDR2, and CDR3 regions comprising SEQ ID NOs: C5-C7;Attorney Docket No. 40056-0091 WO1 a variable light chain region having CDR1, CDR2, and CDR3 regions comprising SEQ ID NOs: C9-C11 and a variable light chain region having CDR1, CDR2, and CDR3 regions comprising SEQ ID NOs: C13-C15;a variable light chain region having CDR1, CDR2, and CDR3 regions comprising SEQ ID NOs: C17-C19 and a variable light chain region having CDR1, CDR2, and CDR3 regions comprising SEQ ID Os: C21-C23;a variable light chain region comprising SEQ ID NO: C4 and a variable heavy region comprising SEQ ID NO: C8;a variable light chain region comprising SEQ ID NO: C12 and a variable heavy region comprising SEQ ID NO: Cl 6;a variable light chain region comprising SEQ ID NO: C20 and a variable heavy region comprising SEQ ID NO: C24; ora VI I comprising an amino acid sequence selected from SEQ ID NOs: C8, C24, C33, C41, C49, C59, C69, and C70, or a variant thereof having no more than 5 single amino acid substitutions, wherein the substitutions are not in the CDRs, anda VL comprising an amino acid sequence selected from SEQ ID NOs: C4, C20, C29, C37, C45, C55, C62, and C74, or a variant thereof having no more than 5 single ammo acid substitutions, wherein the substitutions are not in the CDRs.

98. The method of any one of claims 50-53, wherein the therapy that binds GPC3 comprises any one of the constructs in Table Al.

99. The method of any one of claims 36-55 and 90-98, w’herein the therapy that binds GPC3 comprises any one of the sequences or domains listed in any one of Tables Al, A2, and A3.

100. The method of any one of claims 89-99, wherein the solid tumor is HER2 positive and the method further comprises administering an effective amount of a population of HER2 CAR T cells.

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