Compositions and methods regarding engineered and non-engineered γδ -T cells for treatment of solid tumors

Abstract

Aspects of the invention include compositions and methods for treatment of solid tumors with engineered or non-engineered γδ-Τ cells. In some embodiments, the γδ-Τ cells comprise a chimeric antigen receptor (CAR) construct. The CAR construct can contain an anti-TryD binding domain, a CD8α hinge and transmembrane domain, a costimulatory domain, a 003ζ signalling domain, a combination thereof, or all thereof. The CAR construct can contain an anti- GPC3 binding domain, a CD8α hinge and transmembrane domain, a costimulatory domain, a CD3ζ signalling domain, a combination thereof, or all thereof. The CAR construct can contain a domain encoding for a secreted common gamma chain cytokine such as a sIL 15 domain.

Description

COMPOSITIONS AND METHODS REGARDING ENGINEERED AND NON- ENGINEERED ¥3-T CELLS FOR TREATMENT OF SOLID TUMORS CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 62 / 739,826. filed October 1, 2018. the contents of which arc hereby incorporated for any and all purposes. SEQUENCE LISTING [0001.1] The instant application contains a Sequence Listing which has been submitted electronically in ASCI] format and is hereby incorporated by reference in its entirety. Said ASCH copy, created on January 8, 2020, is named ADC-0006-PCT_SL.txt and is 48,406 bytes in size. BACKGROUND OF THE INVENTION

[0002] Adoptive cellular therapy has undergone near constant iteration for more than thirty (30) years, from early days focusing on basic lymphokine activation and / or tumor infiltration to more recent strategies engineering these immune cells to express genetically engineered antigen receptors, such as chimeric antigen receptors (CARs)s. While there have been some hints and indications of the curative potential of these approaches along the way, much still remains to be done. In particular, successful tumor eradication by CAR-T lymphocytes depends on CAR-T cell persistence and effector function, but an excess of either can trigger graft-versus-host effects in the patient. Moreover, solid tissues in particular present a problem due to the lack of available positive stimulation and the presence of an inhibitory environment. As such the art is testing myriad co-stimulation strategies in both T and NK cells, and in «ff T cells in particular, with a view to balancing efficacy with safety. Notably, the practical translation of any of these various approaches to va T cells is at best uncertain, given the current lack of understanding around the co-stimulation requirements of v8 I cells as compared to af I" cells. See, e.g. Ribot ef al, “Searching for “signal 2”: costimulation requirements of v8 T cells”, Cell. Mol. Life Sci. (2011) 68:2345-2355.

[0003] Accordingly, improved strategies are still needed to improve the specificity or selectivity of the cells, to improve safety of the cells, for example by reducing or avoiding graft versus host (GVH) effects, to improve efficacy of the cells against solid tumor cells, for example, by avoiding suppression of effector functions, and to improve the activity and / or survival of the cells upon administration to subjects. Provided are methods, cells, compositions, kits, and systems that meet such needs. SUMMARY OF INVENTION

[6004] Aspects of the invention include an isolated nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises a binding domain that specifically binds to a protein-peptide complex comprising a tumor associated antigen (TAA) peptide and an MHC protein, wherein the complex is expressed on a surface of a solid tumor cell, optionally wherein the binding domain binds the complex in an HLA restricted manner, a CD8a hinge domain; a CD8a transmembrane domain; a costimulatory signaling region selected trom a 4- IBB costimulatory signaling region and ga CD27 costimulatory signaling region: and a CD3{ signaling domain. Aspects of the invention further include a non-engineered y8 T cell describad herein and an engineered y8 T cell comprising a nucleic acid encoding a CAR construct described herein, wherein the v8 T cell functionally expresses the nucleic acid encoded CAR ont the surface of the v3 T cell. 10005] Aspects of the invention further include a plurality of v8 T cells as described herein. Aspects of the invention further include a method of making the v8 T cell or plurality of y8 T cells described herein, Aspects of the invention further include a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a v8 T cell or plurality of 8 T cells described herein. Aspects of the invention further include contacting a solid tumor cell with a tumor cell killing effective amount of a ¥8 T cell as described herein or plurality of ¥8 T cells described herein.

[6006] In one aspect, the present invention provides an isolated nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises (a) a binding domain that specifically binds to a protein-peptide complex comprising a tumor associated antigen (TAA) peptide and an MHC protein. wherein the complex is expressed on a surface of a solid tumor cell, optionally wherein the binding domain binds the complex in an HLA restricted manner; {b) 4 hinge domain, such as a CD8u hinge domain; (¢) a transmembrane domain, such as a CD8a transmembrane domain: {d) a costimulatory signaling region or combination of costimulatory signaling regions, optionally wherein the costimulatory signaling region(s) are selected from a 4-1BB (CD137) costimulatory signaling region and a CD27 costimulatory signaling region; and (e) a signaling domuin, such as a CD3{ signuling domain. In some embodiments, the foregoing elements (a)-(¢) are encoded on the sense strand of the isolated nucleic acid in 5" to 3° order.

[0007] In some embodiments, thc TAA comprises a contiguous region of TyrD. In some embodiments, the contiguous region of TvrD comprises at least, or at least about, 4 and no more than, or no more than about, 12 contiguous amino acids of TyrD, preferably. or preferably about, 7. 8, or 9 contiguous amino acids of TyrD. In some embodiments, the contiguous region of TyrD is TyrDsso.377. In some embodiments, the binding domain that specifically binds the TAA peptide MHC complex specifically binds HLA-A2 / TyrDiso.377. 10008] In some embodiments. the binding domain specifically binds to the epitope bound by, or competes with, an antibody comprising: a CDRH comprising TSGMGVS (SEQ 1D NO: 33); a CDRH2 comprising HIYWDDDKRYNPSLKS (SEQ TD NO: 34); a CDRH3 comprising KDYGSSFYAMHY (SEQ ID NO: 35); a CDRL1comprising KASQDIHNYIA (SEQ ID NO: 36); a CDRL1 comprising YTSTLQP (SEQ ID NO: 37). and a CDRL2 comprising LQYDNLWT (SEQ ID NO: 38).

[0009] [n another aspect, the binding domain specifically binds to a tumor associated antigen (TAA) expressed on a surface of a solid tumor cell, optionally wherein the antigen is a protein- peptide complex, wherein the protein is an MHC protein, wherein the binding domain binds the protein-peptide complex in an HLA restricted manner, and the encoded CAR of the isolated nucleic acid sequence comprises (b) a hinge domain such as a CD8a hinge domain; (¢) a transmembrane domain, such as a CDB8a transmembrane domain: (d) a costimulatory signaling region or combination of costimulatory signaling regions, optionally wherein the costimulatory signaling region(s) are selected from a 4-1BB (CD137) costimulatory signaling region and a CD27 costimulatory signaling region: and (e) a signaling domain, such as a CD3{ signaling domain. In some embodiments. the foregoing elements (a)-(e) are encoded on the sense strand of the isolated nucleic acid in 5° to 3° order.

[0010] In some embodiments the binding domain specifically binds an epitope within GPC3 expressed on the surface of a solid tumor cell, In some embodiments, the binding domain comprises the following complementarity determining regions (CDRs) binds the same GPC3 epitope as an antibody comprising the following CDRs, and / or competes for binding to an epitope of GPC3 with an antibody comprising the following CDRs: a CDRH! comprising a sequence of DYEMH (SEQ ID NO: 39) (or GYTFTDYEMH (SEQ ID NO: 40)); a CDRH2 comprising a sequence of ALDPKTGDTAYSQKFKG (SEQ ID NO: 41); a CDRH3 comprising a sequence of FYSYTY (SEQ ID NO: 42) a CDRLIcomprising a sequence of RSSQSLVHSNRNTYLH (SEQ ID NO: 43); a CDRL.2 comprising a sequence of KVSNRFS (SEQ ID NO: 44); and / or a CDRL3 comprising a sequence of SQNTHVPPT (SEQ ID NO: 45). 16011) In some embodiments of any one of the foregoing aspects or embodiments or any of the CAR encoding nucleic acids described herein, the encoded CAR comprises: a CD8a hinge domain comprising SEQ in NO: (PTPAPTIASQPLSLLRPEACRPAAGGAVHTRGLDFACDIY) or SEQ 1D NO:2 (TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY), or a CD8a transmembrane domain comprising SEQ TD NQ:3 (IWAPLAGTCGVLLLSLVITLYC); and / or a CD3¢ signaling domain. Tn some cascs, the CD3¢ signaling domain compriscs the sequence of SEQ ID NO:4 (RVKFSRSADAPAYQQGONQLYNELNLGRREEYDVLDKRRGRDPEMG GKPQRRKNPQEGLYNELQKDKMAFAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYD ALHMQALPPR); or SEQ ID NO:5 (RVKFSRSADAPAYQOQGONQLYNELNLGRREEYD VLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGI. YQGLSTATKDTYDALHMQALPPR).

[0012] In some embodiments, the CAR comprises a 4-1BB costimulatory signaling region comprising SEQ ID NO:6 (KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL); or a CD27 costimulatory signaling region comprising SEQ ID NO:7. {QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP), or the isolated nucleic acid encodes the 4-1BB costimulatory signaling region comprising SEQ ID NO:6 and the (CD27 costimulatory signaling region comprising SEQ 1D NO:7.

[0013] In some embodiments of any one of the foregoing, or as described herein, the isolated nucleic acid further encodes a secreted cytokine; or a secreted common gamma chain interleukin; or a secreted common gamma chain interleukin such as 1L-15. preferably wherein the secreted common gamma chain interleukin such as 11-15 comprises an interleukin polypeptide sequence operably linked to a secretion signal sequence (e.g., a secretion signal of SEQ 1D NO: 12 or 26). In some embodiments, the isolated nucleic acid encodes a secreted 1L-15, preferably wherein the I-15 comprises the sequence of SEQ [DD NO:14, more preferably wherein the 1-15 comprises the sequence of 14 operably linked 10 a secretion signal sequence of SEQ ID NO:12, or wherein the 11-15 comprises the sequence of SEQ ID NO: 14 operably linked to a secretion signal sequence of SEQ ID NO: 26. In some cases, the secreted cytokine, common gamma chain interleukin, and / or 1L-15 are encoded carboxy terminal to the binding region, hinge and transmembrane domains, signaling domain, and / or costimulation endodomain. In some cases, the secreted cytokine, common gamma chain interleukin, and / or 11.-15 are encoded on the sense strand 3’ of the region encoding the binding region, hinge and transmembrane domains, signaling domain. and / or costimulation endodomain. 10014] [n some embodiments, the nucleic acid encodes a multi<cistronic linker region configured to facilitate translation of the CAR and the secreted cytokine, common gamma chain cytokine, or 11-15 as separate polypeptides. In some embodiments, the mulii-cistronic linker region encodes a self cleavage and / or a cleavage polypeptide sequence. In some cases, the self- cleavage sequence is a P2A, F2A, T2A, or E2A self cleavage sequence. In some cases, the cleavage sequence is a furin cleavage sequence. In some cases, the cleavage sequence (e.g., furin cleavage sequence) is amino terminal to a self cleavage sequence. In some embodiments, the multi-cistronic linker region encodes an internal ribosome entry site. In some embodiments, the nucleic acid encodes a multi-cistronic linker region amino terminal to the interleukin or cytokine or interleukin or cytokine secretion signal, preferably wherein the multicistronic linker region comprises a sequence of any one of SEQ ID NOs: 15-17, 25, or 27-30, or a combination thereof, or encodes an internal ribosome entry site, e.g.. SEQ 1D NO: 31 or 32.

[0015] In some embodiments, the secretion signal comprises a sequence of SEQ ID NO: 12 or SEQ ID NO: 26, preferably SEQ ID NO: 12; and / or the si1.15 domain comprises a sequence of SEQ ID NO: 14; and / or the P2A cleavage sequence comprises a sequence of SEQ ID NO: 15 or SEQ ID NO:25; and / or the furin cleavage sequence comprises a sequence of SEQ ID NO: 16; and / or the CAR comprises, in amino to carboxy order, a sequence of SEQ ID NO: 17, SEQ ID NO: 12, and SEQ ID NO: 14.

[0016] In some embodiments, the binding domain specifically binds to HLA-A2 / TyrDig9.377 and the nucleic acid encodes SEQ [D NO: 8, or SEQ ID NO: 18. In some embodiments, the binding domain specifically binds to GPC3 and the nucleic acid encodes SEQ ID NO: 20, or 22. In some embodiments, the nucleic acid comprises the sequence of SEQ ID NO: 9, SEQ ID NO: 19, SEQ ID NO: 21, 23, or 24.

[0017] In another aspect. the present invention provides a polypeptide comprising a CAR binding domain, such as one of the polypeptides encoded by any one of the foregoing nucleic acids, or a polypeptide described herein.

[0018] In another aspect. the present invention provides an, e.g. v8. T cell comprising the foregoing polypeptide, or comprising a nucleic acid encoding a CAR described herein, wherein the cell functionally expresses the a binding domain of the polypeptide or nucleic acid encoded CAR on the surface of the cell. In some embodiments, the cell exhibits in vifro and / or in vivo cell killing activity against a solid tumor cell that exhibits cell surface expression of the tumor associated antigen (TAA). In some embodiments. the solid tumor cell killing activity of said cell is greater than an innate level of ir vitro and / or in vive solid tumor cell killing activity in a control cell that does not comprise a CAR construct. in some embodiments, the cell exhibits the increased solid tumor cell killing activity against HLA class I* solid tumor cells. In some embodiments, the solid tumor cell killing activity or increased solid tumor cell killing activity persists for, for about, for at least, or for at least about, 6 days to 180 days after first contact with the solid tumor cell.

[0019] In some embodiments, the cell proliferates in response to contact with a solid tumor cell that exhibits cell surface expression of the tumor associated antigen (TAA). In some embodiments, the ceil exhibits increased proliferation in response to contact with a solid tumor cell that exhibits cell surface expression of the tumor associated antigen (TAA) as compared to a control cell that does not functionally express the nucleic acid encoded CAR on the surface of the cell. In some embodiments, the cell proliferates in a host organism that comprises the solid tumor cell that exhibits cell surface expression of the tumor associated antigen (TAA). In some embodiments, the cell proliferation or increased cell proliferation persists for, for about, for at least, or for at least about, 6 days to 180 days after first contact with the solid tumor cell. In some embodiments, the cell expresses one or more pro-inflammatory cytokines, optionally wherein the one or mote pro-inflammatory cytokines comprises tumor necrosis factor alpha or interferon gamma, afier contact with the solid tumor cell, preferably in an amount greater than a control cell that does not functionally express the nucleic acid encoded CAR on the surface of the cell. 10020] In some embodiments, the cell exhibits reduced, substantially reduced, essentially none, or no grafl versus host response when introduced into an allogeneic host in comparison to a graft versus host response exhibited by an ap 1 cell administered to an allogeneic host. In some embodiments. the, e.g, v8, T cell exhibits reduced, substantially reduced. essentially no, or no graft versus host response when introduced into an allogeneic host in comparison to a graft versus host response exhibited by an af T cell administered to an allogeneic host. [n some embodiments, the T cell is av T cell. In some embodiments, the T cell is a 3 'I' cell. In some embodiments, the T cell is a v8 T cell. In some embodiments, the T cell is a 81, a 82. 4 83, or a 84 T cell, preferably a 82° 8 T cell, more preferably a 81 8 T cell. In some embodiments, the T' cell isa dl, ad2, ads, orad4 vd T cell, preferably a 82° v8 T cell, more preferably a 81 v3 T cell.

[0021] In another aspect, the present invention provides a plurality of of any one of the foregoing cells such as , e.g, yd. T cells, or a plurality of cells such as, e.g, v3, T cells as described herein. In some embodiments. the plurality comprises at least about 10% cells such as 10%, e g.. v8, T cells, preferably from about 108 cells, e g., v5. T cells to about 101 cells, e. g., v8. T cells. In some embodiments, the plurality comprises a composition that is at least 60%, 80%, or from about 60% or 80% to about 90% or 95% 81, 82, 83, or 84 cells, such as, e.g, v8 T cells, preferably 81 or 82 v8 1 cells, more preferably $27 v8 1 cells, most preferably 81 v8 T cells. 10022] In some embodiments, the present invention provides a method of making a cell, such as an, e.g., vd, T cell as described herein, or a plurality of cells, such as, e.g, v3, T cells as described herein, wherein the method comprises transfecting the cell(s) with a construct comprising an isolated nucleic acid sequence as described herein. In some cases, the method comprises, e.g., gamma, retroviral transduction. In some cases, the method comprises ex vivo expansion of the celi(s), wherein the ex vivo expansion is performed before transfection and / or after transfection of the isolated nucleic acid sequence. [n some cases, the method comprises ex vivo expansion of the cell(s), wherein the ex vivo expansion is performed before transfection and after transfection of the isolated nucleic acid sequence. In some cases, the method comprises ex vivo expansion of the cell(s). wherein the ex vivo expansion is performed after transfection of the isolated nucleic acid sequence. In some embodiments, the method comprises producing the from about 10% cells, such as. e.g, v5. T cells to about 10!" cells, such as, e.g.. ¥5, T cells that functionally express a CAR described herein within about 30 days of transfection.

[0023] In another aspect, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a cell or plurality of cells as described herein, such as, e.g., v8, T cell(s) described herein.

[0024] In another aspect, the present invention provides a method of killing a solid tumor cell, the method comprising contacting the solid tumor cell with a tumor cell killing effective amount of any one of the foregoing cells or plurality. of cells or pharmaceutical compositions, or a cell or plurality of cells or pharmaceutical composition as described herein. In some cases, the cell or plurality of cells are, ¢.g., v8, T cell(s).

[0025] In some embodiments, the method comprises introducing a therapeutically effective amount of the cells such as, e.g.. vd, T cell(s) or the pharmaceutical composition into a host organism comprising the solid tumor cell. In some embodiments, the method comprises introducing into a host organism comprising the solid tumor cell a therapeutically effective amount of the cells, such as, e.g, v8, T cell(s), or a pharmaceutical composition thereol and simultaneously or sequentially administering one or more methods to elevate common gamma chain cytokine(s).

[0026] [n some embodiments, the administering one or more methods to elevate common gamma chain cytokine(s) comprises administering simultaneously with introducing the cell(s) or sequentially an amount of common gamma chain cytokine(s) effective to increase proliferation. cytotoxic activity, persistence, or the combination thereof of the introduced cell(s), preferably wherein the method comprises administering IL-2, more preferably wherein the method comprises administering 11-15. In some embodiments, the one or more methods to elevate common gamma chain cytokine(s) comprise administering an amount of common gamma chain cytokine(s) effective to increase proliferation, cytotoxic activity, persistence, or the combination thereof of the introduced cell(s) before and / or after introducing the cells).

[0027] In some embodiments, the one or more methods to elevate common gamma chain cytokine(s) comprises lymphodeplction before introducing the v8 ‘1 cell(s). In some embodiments, the one or more methods to elevate common gamma chain cytokine(s) comprises secretion of one or more common gamma chain cytokine(s) from the introduced cell(s). In some embodiments, the method reduces the in vivo tumor burden in the host organism, and / or increases the mean survival time of the host organism as compared to a control organism, wherein the control organism is not treated with the cell(s) or the pharmaceutical composition. In some embodiments, the method is a method of treating cancer in a subject in need thereof.

[0028] 1n another aspect, the present invention provides a use of a tumor cell killing effective amount of any one of the foregoing cells or a cell as described herein {such as an, e.g.. v8 T cell); a plurality of such cells or a pharmaceutical composition containing such cells in the manufacture of a medicament for the treatment of a solid tumor cell cancer in a subject in need thereol. In another aspect, the present invention provides a method of treating cancer in a subject in need thereof, the method comprising administering a therapeutically effective amount of the cells, wherein the cancer comprises solid tumor cells that exhibit cell surface expression of TyrD or GPC3.

[0029] [n some embodiments, the method comprises simultaneously with the administering of cells or sequentially, administering one or more methods to elevate common gamma chain cytokine(s). In some embodiments, the method comprises performing a plurality of administrations of the cells, wherein the interval between the plurality of administrations is at least about a week, preferably at least about 2, 3, 4, 5, 6, 7, 8, or 12 weeks, and / or no more than once every 6 or 12 months.

[6030] In yet another aspect, the present invention provides a pharmaceutical composition for use in any one of the foregoing methods or a method described herein. INCORPORATION BY REFERENCE

[0031] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Fig. 1 is a schematic illustration of an embodiment of a chimeric antigen receptor (CAR) containing one costimulatory signaling endodomain (left) or two costimulatory signaling endodomains (right). As used herein costimulatory signaling endodomains are also referred to as costimulation endodomains or costimulatory endodomains. Exemplary costimulatory signaling endodomains useful in exemplary CARs include, without limitation, CD28; CD137 (4-1BB); CD278 (ICOS); CN27; CDI134 (0X40); TLR2, and combinations thereof.

[6033] Fig. 2 illustrates in vitro cytotoxicity of engineered and non-engincered v8 T cells described herein against 526 and WM266.1-Luc melanoma cell lines.

[0034] Fig. 3 illustrates in vivo therapeutic cfticacy of v8 T cells described herein in a

[0035] Fig. 4 illustrates a manufacturing process for production of engineered v8 CAR-T cells and non-engineered v8 CAR-T cells, e.g., for {reatrent of solid tumors.

[0036] Fig. 5 illustrates cytotoxic activity of V8! T cells transduced with control CAR constructs or constructs targeting the tyrosinase polypeptide.

[0037] Fig. 6 illustrates ransduction efficiency of V81 cells with an anu-glypican 3 (GPC3) CAR construct, including soluble 1L-15 (s[L.13) (SEQ ID NO:14) and codon optimized (WO 2007 / 037780A2) s1L.15.

[0038] Tig. 7 illustrates cytotoxic activity of V81 T cells either untransduced or transduced with anti-GPC3 CAR constructs against a panel of liver cancer cell line with different levels of GPC3 expression, DETAILED DESCRIPTION Definitions:

[0039] For purposes of interpreting this specification, the following definitions will apply, and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth conflicts with any document incorporated herein by reference, the definition set forth below shall controi. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains.

[0040] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of 20% or 10%, more preferably £5%, even more preferably +1%, and still more preferably +0.1% from the specified value, as such variations are appropriate to perform the disclosed methods,

[0041] The term “yd T-cells (gamma delta T-cells)” as used herein refers to a subset of T= cells that express a distinct T-cell receptor (TCR), namely v8 TCR, on their surface, composed ol one y-chain and one 8-chain. The term “v8 T-cells” specifically includes all subsets of v8 T- cells, including, without limitation. V81 and V52, V83 v8 T cells. as well as naive, effector memory, central memory, and terminally differentiated v8 T-cells. As a further example. the term “v8 T-cells” includes V84, V85, V47, and V8 v8 T cells, as well as Vy2, Vy3, Vy3, Vy8, Vv9, Vv10, and Vvl1 v8 T cells. In some embodiments, the v8 T-cells are V81-, V&2-, or V&l- and V582". Compositions and methods tor making and using engineered and non-engineered v8 T cells and / or sub-types thereof include, without limitation, those described in US 2016 / 0175358: WO 2017 / 197347; US 9499788; US 2018 / 0169147: US 9907820; US 2018 / 0125889 and US 2017 / 0196910, the contents of each of which are incorporated by reference for all purposes, including the said compositions and methods for making and using engineered and non- engineered v8 T cells and / or sub-types thereof. The present application further contemplates T cells, or other engineered leukocytes or lymphocyies, that express one y-chain or one 8-chain, optionally in combination with a second polypeptide to form a functional TCR. Such engineered leukocytes or lymphocytes, that express one y-chain or one $-chain may be uscd in the methods or present in the compositions described herein.

[0042] As used herein, the term “T lymphocyte” or “T cell” refers to an immune cell that expresses or has expressed CD3 (CD3+) and a T Cell Receptor (TCR+). T cells play a central role in cell-mediated immunity. A T cell that “has expressed CD3 and a TCR” has been engineered to eliminate CD3 and / or TCR cell surface expression.

[0043] As used herein, the term “TCR” or “T cell receptor” refers to a dimeric heterologous cell surface signaling protein forming an alpha-beta or gamma-delta receptor or combinations thereof. aTCRs recognize an antigen presented by an MITC molecule, whereas ySTCR can recognize an antigen independently of MHC presentation.

[0044] The term "MHC" (major histocompatibility complex) refers to a subset of genes that encodes cell-surface antigen-presenting proteins. In humans, these genes are referred to as human leukocyte antigen (HLA) genes. Herein, the abbreviations MHC or HLA are used interchangeably.

[0045] “Activation”, as used herein, refers 10 the state of a T cell that has been sulTiciently stimulated to induce detectable cellular proliferation. Activation can also be associated with induced cytokine production, and detectable effector functions. The term “activated T cells” refers to, among other things, T cells that are undergoing cell division. 10046] The term “antibody,” as used herein, refers to an immunoglobulin molecule which specifically binds with an antigen. Antibodies can be intact immunoglobulins derived from natural sources or [rom recombinant sources and can be immunoreactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. The antibodies in the present invention may exist in a variety of forms including, for example, polyclonal antibodies. monoclonal antibodies, Fv, Fab and F(ab), as well as single chain antibodies and humanized antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al, 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird ct al., 1988, Science 242:423-426).

[0047] The term “antibody fragment” refers to a portion of an intact antibody and refers to the antigenic determining variable regions of an intact antibody. Examples of antibody fragments include. but are not limited to, Tab, Fab’, F{ab')2, and Fv fragments. linear antibodies, scFv antibodies, and multispecific antibodiés formed ftom antibody fragments. 10048] An “antibody heavy chain,” as used herein, refers to the larger of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations. 10049] An “antibody light chain,” as used herein, refers to the smaller of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations. K and A light chains refer to the two major antibody light chain isotypes.

[0050] By the term “synthetic antibody” as used herein. is meant an antibody which is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage as described herein. The term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence technology which is available and well known in the.art.

[6051] The term “antigen” or “Ag” us used herein iy defied as a mohsule that provokes an immune response, This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both. The skilled artisan will understand that any macromolecule, including proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA that comprises a nucleotide sequences or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an “antigen” as that term is used herein. Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full-length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Moreover, a skilled artisan wilf understand that an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be generated, synthesized, or can be derived from a biological sample. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a biological fluid.

[0052] The term “epitope” includes any protein determinant, lipid or carbohydrate determinant capable of specific binding to an immunoglobulin or T-cell receptor. Epitopic determinants usually consist of active surface groupings of molecules such as amino acids, lipids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. An antibody is said to specifically bind an antigen when the equilibrium dissociation constant (Kp) is in a range of 106 — 107M.

[0053] The term "chimeric antigen receptors (CARs)," as used herein, may refer to artificial T-cell receptors, T-bodies, single-chain immunoreceptors, chimeric T-cell receptors, or chimeric immunoreceptors, for example, and encompass engineered receptors that graft an anificial specificity onto a particular immune elfector cell. CARs may be employed to impart the specificity of a monoclonal antibody onto a T cell, thereby allowing a large number of specific T cells to be generated, for example, for use in adoptive cell therapy. In specific embodiments, CARs direct specificity of the cell to a fumor associated antigen, for example. In some embodiments, CARs comprise an intracellular activation domain (allowing the T cell to activate upon engagement of targeting moiety with target cell, such as a target tumor cell), a transmembrane domain, and an extracellular domain that may vary in length and comprises a disease- or disorder-associated, ¢.g., a tumor-antigen binding region. In particular aspects, CARs comprise fusions of single-chain variable fragments (scFv) derived from monoclonal antibodies, fused to CD3-zeta a transmembrane domain and endodomain. The specificity of other CAR designs may be derived from ligands of receptors (e.g., peptides) or from pattern-recognition receptors, such as Dectins. In certain cases, the spacing of the antigen-recognition domain can be modified to reduce activation-induced ceil death. In certain cases, CARs comprise domains for additional co-stimulatory signaling, such as CD3(, FcR, CD27, CD28, CD137, DAP 10 / 12, and / or 0X40, ICOS, TLRs {e.g., TLR2), etc. In some cases, molecules can be co-expressed with the CAR. including co-stimulatory molecules. reporter genes for imaging (e.g. for positron emission tomography), gene products that conditionally ablate the T cells upon addition of a pro- drug, homing receptors, chemokines, chemokine receptors, cytokines, and cytokine receptors. Furthermore, one skilled in the art wifl understand that a costimulatory domain need not be encoded solely by a full-length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response.

[0054] The tertn “anti-tumor effect” as used herein, refers to a biological effect which can be manifested by a decrease in tumor volume. a decrease in the number of tumor ceils, a decrease in the number of metastases, an increase in life expectancy, or amelioration of various physiological symptoms associated with the cancerous condition. An “anti-tumor effect” can also be manifested by the ability of the peptides, polynucleotides, cells and antibodies of the invention in prevention of the occurrence of tumor in the first place.

[0055] The term “auto-antigen” means, in accordance with the present invention, any self- antigen which is mistakenly recognized by the immune system as being foreign. Auto-antigens comprise, but are not limited to, cellular proteins, phosphoproteins, cellular surface proteins, cellular lipids, nucleic acids, glycoproteins, including cell surface receptors.

[0056] As used herein, the term “autologous” is meant to refer to any material derived from an individual which is later to be re-introduced into the same individual.

[0057] As used herein, the term “allogeneic” refers to material derived from an animal which is later introduced into a ditferent animal of the same species. 0058 The term “therapeutically effective amount” refers to the amount of a composition pi y that will elicit a biological or medical response of a tissue, system, or subject that is being sought by the researcher, veterinarian, medical doctor or other clinician. The term “therapeutically effective amount™ includes that amount of a composition that, when administered, is sufficient to prevent development of, or alleviate ta some extent, one or more of the signs or symptoms of the disorder or disease (e.g.. solid tumor} being treated, The therapeutically effective amount will vary depending on the composition, the disease and its severity and the age, weight, etc., of the subject to be treated.

[0059] To “treat” a disease as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject. 16060] Administration “in combination with” one or more further therapeutic agents includes simultaneous (concurrent) and sequential administration in any order.

[0061] The term “pharmaceutically acceptable”, as used herein, refers to a material, including but not limited, to a salt, carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively nontoxic, ie.. the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

[0062] “Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides {i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus. a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA. 10063] “Isolated” means altered or removed from the natural state, For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0064] Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns.

[0065] The terms “patient.” “subject,” “individual,” and the like are used interchangeably herein, and refer to any animal, amenable to the methods described herein. In certain non- limiting embodiments, the patient, subject or individual is a human.

[0066] By the term “specifically binds,” as used herein with respect to an antibody, is meant an antibody which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds fo an antigen from one species may also bind to that antigen from one or more species. But. such cross-species reactivity does not itself alter the classification of an antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antibody as specific. In some instances, the terms “specific binding™ or “specifically binding,” can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope “A”. the presence of a molecule containing epitope A (or free. unlabeled A). in a reaction containing labeled “A” and the antibody, will reduce the amount of labeled A bound to the antibody.

[0067] In some embodiments, specific binding can be characterized by an equilibrium dissociation constant of at least about 1x10" M or less (e.g.. a smaller Ki denotes a tighter binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. Moreover, multi-specific antibodies that bind to a first antigen and one or more additional antigens or a bispecific antibody that binds to two different regions of an antigen are nonetheless considered antibodies that “specifically bind,” as used herein.

[0068] Solid tumors are tumors that comprise a tumor mass of at least about 10 or at least about 100 twmor cells. The solid tumor can be a soft tissue lumor, a primary solid tumor, or a metastatic lesion.

[0069] Examples of solid tumars include, e.g., sarcomas, adenocarcinomas, and carcinomas, of the various organ systems, such as those affecting liver, lung, breast, lymphoid, gastrointestinal (e.g.. colon), genitourinary tract {e.g.. renal, urothelial cells), prostate and pharynx. Adenocarcinomas include malignancies such as most colon cancers, rectal cancer, renal-cell carcinoma, liver cancer, non-small celf carcinoma of the lung, cancer of the small intestine and cancer of the esophagus. In one embodiment, the cancer is a melanoma, e.g., an advanced stage melanoma. Metastatic lesions of the aforementioned cancers can also be treated or prevented using the methods and compositions of the invention. Examples of other cancers that can be treated include bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma. epidermoid cancer, squamous cell cancer, environmentally induced cancers including those induced by asbestos, and combinations of said cancers. In a preferred embodiment, the solid tumor cell expresses, or over-expresses, TyrD, ora fragment thereof. In some embodiments, the solid tumor cell expresses, or over-expresses an HLA:peptide complex containing a TyrD fragment. In some embodiments, the TyrD fragment is TyrDseo-377. In some embodiments, the [HLA is a class [ HLA, such as HLA-A2. In some embodiments, the solid tumor cell expresses, or over-expresses HLA-A2 / TyrDiso.377. 16070] In some embodiments, the solid tumor cel! expresses, or over-expresses, glypican3 (GPC3). In some embodiments, the solid tumor cell expresses. or over-expresses an epitope of GPC3 that is specifically bound by an anti- GPC3 antibody, T cell Receptor, or chimeric antigen receptor described in U.S. 7.919,086; WO 2014 / 180306; WO 2018 / 019772; WO 2016 / 049459, WO 2003 / 000883; WO 2006 / 046751: WO 2007 / 047291;: WO 2016 / 086813; WO 2016 / 047722; WO 2016 / 036973; Cancer Res, 2008:68:9832-9838; Proc Natl Acad Sci U S A. 2013 Mar 19:110(12):E1083-91, the contents of each of which are incorporated by reference in the entirety and for all purposes and in particular for the binding domains, antibodies, antibody fragments, complementarity determining regions, polypeptides containing said complementarity determining regions, nucleic acids encoding for said complementarity determining regions, and epitope specificities and assays for determining epitope specificity described therein. In some embodiments, the solid tumor cell expresses, or over-expresses an epitope of glypican3 that is specifically bound by the anti-GPC3 antibody GC33. In some embodiments, the solid tumor expresses, or over-expresses, an HLA:peptide complex containing a GPC3 fragment. In some embodiments, the HLA is a class | HLA, such as HI.LA-A2. In some embodiments. the solid tumor expresses, or overexpresses, an HLA peptide complex containing a GPC3141-152 peptide. In some embodiments, the solid tumor expresses, or overexpresses, an HLA:peptide complex containing a GPC3298.306 peptide. See, Oncoimmunalogy. 2012 Nov 1; 1(8): 1448-1450.

[0071] “Expression cassette” refers to a nucleic acid comprising expression control sequences operatively linked to a nucleic acid encoding a transcript or polypeptide to be expressed. An expression cassette comprises sufficient cis-acting elements for expression: other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression cassettes can be a component of a vector such as a cosmid, a plasmid (e.g., naked or contained in a liposome), or a virus (e.g.. lentivirus, retrovirus, adenovirus, and adeno-associated virus). An expression casselic can be in a host cell, such as a v8 T cell.

[0072] Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from | to 3. from 1 to 4, from 1 to 5. from 2 to 4, from 2 to 6. from 3 to 6 etc., as well as individual numbers within that range, for example, 1. 2, 2.7, 3. 4, 5, 5.3. and 6. This applies regardless of the breadth of the range. Chimeric Antigen Receptor Constructs: 10073] Aspects of the invention include nucleic acids encoding CARs, and constructs and vectors containing such nucleic acids. In some cases. the nucleic acid is a, e.g., heterologous, component of an expression cassette. In some embodiments, the nucleic acid is a, e.g. heterologous component of a retroviral vector. In some embodiments, the nucleic acid is a, e.r., heterologous, component of an aff or v8 T cell, and preferably a v6 T cell. In some embodiments, the nucleic acid is a, e.g, heterologous, component of an ¥* T cell and / or a 87 T cell. In sone embodiments, the nucleic acid is a, e.g., heterologous, component of an a” T cell and / ora fT cell.

[0074] Described herein are nucleic acids encoding a CAR binding domain that specifically binds to a tumor associated antigen (TAA) expressed on a surface of a solid twmor cell. An exemplary TAA is tyrosinase (TyrD) ar a peptide fragment thereof. In some cases, the TAA is glypican3 or a peptide fragment thereof. In some cases, the TAA is a peptide bound to an HLA molecule, such as a class 1 HLA molecule. Tyrosinase peptides that bind to class I HLA molecules (also referred to herein interchangeably as HLL A-restricted tyrosinase epitopes, HLA- restricted tyrosinase epitopes and MHC-restricted tyrosinase antigens) are derived from the tyrosinase enzyme (Genebank Accession No: NP_000363.1) and are typically 8-10 amino acids long, bind to the heavy chain al-a2 groove via two or three anchor residues that interact with corresponding binding pockets in the HLA molecule.

[0075] Tyrosinase is a membrane-associated N-linked glycoprotein and it is the key enzyme in melanin synthesis, It is expressed in all healthy melanocytes and in nearly all melanoma tumor samples (H. Takeuchi. et al., 2003; S. Reinke, et al., 2005). Peptides derived from this enzyme are presented on MHC class | molecules and are recognized by autologous cytolytic T lymphocytes in melanoma patients [T, Wolfel, et al., 1994; Brichard, et al., 1993; Renkvist et al, Cancer immunology immunotherapy 2001 50:3-15: Novellino L. et al., March 2004 update. Cancer Immunol Immunotherapy. 54:187-207. 2005]. Additional tumor tyrosinase HLA- restricted peptides derived from tumor associated antigens (TAA) can be found at the website of the Istituto Nazionale per lo Studio ¢ la Cura dei Tumori at www. istitutotumori.mi.it .

[0076] Non-limiting examples of MHC class 1 restricted tyrosinase antigenic peptides are provided in WO2008 / 120202, which is fully incorporated herein by reference in its entirety, e.g.. in Table 139 of WO2008 / 120202. According to some embodiments of the invention, the tyrosinase antigenic peptide is the TyrDig.177 peptide. Binding domains that specifically bind TyrD, an epitope within TyrD, including but not limited to those that bind in an HLA (e.g., class I HLA) restricted manner, include without limitation those described in WO 2016 / 199140; WO 2016 / 199141; US 9688739; and in co-pending application PCT / IB2017 / 053539, the contents of each of which are incorporated by reference in the entirety and for all purposes including but not limited to compositions and methods for identifying, making, and using binding domains that specifically bind TyrD, or an epitope within TyrD, e.g. in an HLA-restricted or HLA- independent manner.

[6077] GPC3 peptides that bind to class | HLA molecules (also referred to herein interchangeably as HLA-restricted GPC3 epitopes, HLA-restricted GPC3 epitopes and MHC- restricted GPC3 antigens) are derived from the glypican3 protein (Genebank Accession No: NM_001164617.2) and arc typically 8-10 amino acids Jong, bind to the heavy chain al-a2 groove via twa or three anchor residues that inferact with corresponding binding pockets in’ the HLA molecule. 10078] As used herein, a binding domain, CAR, or CAR T cell, that specifically binds TyrD and / or specifically binds an epitope within Tyr) includes, without limitation, binding domains, CARs, or CAR T cells that specifically bind a TyrD peptide fragment. The binding domains, CARs, or CAR T cells that specifically bind a TyrD peptide fragment can specifically bind the referenced TyrD peptide fragment in an HLA -restricted manner. Similarly, as used herein. a cell that expresses TvrD on the surface of the cell includes cells that express, or over-express, a TyrD peptide fragment on the surface of the cell, such as in a peptide:HLA complex.

[6079] As used herein, a binding domain, CAR. or CAR T cell, that specifically binds GPC3 and / or specifically binds an epitope within GPC3 includes, without limitation, binding domains, CARs, or CAR T cells that specifically bind a GPC3 peptide [ragment. The binding domains, CARs, or CAR T cells that specifically bind a GPC3 peptide fragment can specifically bind the referenced GPC3 peptide fraginent in an HLA-restricted manner. Similarly, as used herein, a cell that expresses TyrD on the surface of the cell includes cells that express. or over-express, a GPC3 peptide fragment on the surface of the cell, such as in a peptide:HLA complex. 16080] In some embodiments, the binding domain binds the antigen as expressed in a full- length functional polypeptide on the surface of a cell. In some embodiments, the binding domain binds the antigen as presented in an MHC:antigen complex. In some embodiments, the binding domain binds the antigen in an HLA-restricted manner. Binding domains exhibiting specificity for MHC:antigen complexes are described, e.g., in WO / 2016 / 199140 and WO / 2016 / 199141.

[0081] In some embodiments, the isolated nucleic acid encodes an anti-TyrD binding domain having a CDRHI comprising TSGMGVS (SEQ ID NO: 33), a CDRH2 comprising HIYWDDDKRYNPSLKS (SEQ ID NO: 34), a CORH3 comprising KDYGSSFYAMHY (SEQ ID NO: 35). CDRLIcomprising KASQDHHINYIA {SEQ ID NO: 36), a CDRLI comprising YTSTLQP (SEQ ID NO: 37), and / or a CDRL2 comprising LQYDNLWT (SEQ ID NO: 38).

[0082] In some embodiments, the isolated nucleic acid encodes an anti-GPC3 binding domain having a CDRH! comprising DY EMH (SEQ ID NO: 39) (or GYTFTDYEMH (SEQ ID NO: 40)). a CDRH2 comprising ALDPKTGDTAYSQKFKG (SEQ ID NO: 41), a CDRH3 comprising FYSYTY (SEQ [D NO: 42), CDRL1comprising RSSQSLVHSNRNTYLH (SEQ ID NO: 43), a CDRL2 comprising KVSNRFS (SEQ ID NO: 44), and / or a CDRL3 comprising SOQNTHVPPT (SEQ ID NO: 43).

[0083] The present disclosure also contemplates anti-TyrD or anti-GPC3 binding domains that compete for binding with a sequence provided herein. One can determine whether an anti- TyrD binding domain binds to the same epitope as, or competes for binding with, a reference antibody or binding domain by using known methods. For example, to determine if a test antibody binds to the same epitope as a reterence binding domain, the reterence binding domain can be allowed to bind to TyrD under saturating conditions, Next, the ability of a test binding domain to bind to TyrD molecule can be assessed. If the test binding domain is able to bind to TyrD following saturation binding with the reference binding domain, it can be concluded that the test binding domain binds to a different epitope than the reference binding domain. On the other hand, if the test binding domain is not able to bind to Tyr} following saturation binding with the reference binding domain. then the test binding domain may bind to the same epitope as the epitope bound by the reference binding domain.

[0084] To determine if a binding domain competes for binding with a reference binding domain, the above-described binding methodology is performed in two orientations: In a first orientation, the reference binding domain is allowed to bind to TyrD under saturating conditions followed by assessment of binding of the test binding domain to the TyrD molecule. In a second orientation, the test binding domain is allowed to bind to a TyrD molecule under saturating conditions followed by assessment of binding of the reference binding domain to the TyrD molecule. If. in both orientations, only the first (saturating) binding domain is capable of binding to the TyrD molecule, then it is concluded that the test binding domain and the reference binding domain compete for binding to TyrD. As will be appreciated by a person of ordinary skill in the art, a binding domain that competes for binding with a reference binding domain may not necessarily bind to the identical epitope as the reference binding domain, but may sterically block binding of the reference binding domain by binding an overlapping or adjacent epitope. The methods described above to determine competition and epitope binding with an anti-TyrD binding domain can likewise be applied to an anti-TyrD binding domains.

[0085] Two binding domains bind to the same or overlapping epitope if each competitively inhibits (blocks) binding of the other to the antigen. That is, a 1-, 5-, 10-, 20- or 100-fold excess of one binding domain inhibits binding of the other by at least 50%, for example, 75%, 90% or even 99% as measured in a competitive binding assay (see, e.g.. Junghans er al., Cancer Res. 1990 50:1495-1502). Alternatively, two binding domains have the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one binding domain reduce or eliminate binding of the other. Two binding domains have overlapping epitopes if some amino acid mutations that reduce or eliminate binding of one binding domain reduce or eliminate binding of the other.

[0086] Additional routine experimentation {e.g., peptide mutation and binding analyses) can then be carried out to confirm whether the observed lack of binding of the test binding domain is in fact due to binding to the same cpitope as the reference binding domain or if steric blocking (or another phenomenon) is responsible for the lack of observed binding. Experiments of this sort can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry or any other quantitative or qualitative binding assay available in the art. 10087] The present disclosure provides antibodies and CARs with “substantial identity” or “substantial similarity” to the sequences provided herein in the CDR or framework regions. The term "substantial identity" or "substantially identical," when referring to a nucleic acid or fragment thereof, indicates that. when optimally aligned with another nucleic acid (or the complementary strand of the other nucleic acid), there is nucleotide sequence identity in %, for example, at least 80%. at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%. at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST or GAP, as discussed below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule may, in certain instances, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule. 10088] As applied to polypeptides, the term "substantial similarity" or “substantially simifar™ means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity. In some aspects, residue positions, which are not identical, differ by conservative amino acid substitutions. A "conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent or degree of similarity may be adjusted upwards to comect for the conservative nature of the substitution, Means for making this adjustment are well known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24: 307-331, which is herein incorporated by reference. Examples of groups of amino acids that have side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine: 2) aliphatic-hvdroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartate and glutamate, and 7) sulfur-containing side chains: cysteine and methionine. Preferred conservative amino acids substitution groups are: valine- leucine-isoleucine. phenylalanine-tyrosine, lysine-atginine, alanine-valine. glutamate-aspartate, and asparagine-glutamine. Aliematively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet ef al. (1992) Science 256: 1443 45, herein incorporated by reference. A "moderately conservative" replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix.

[0089] Sequence identity and / or similarity for polypeptides is typically measured using sequence analysis software, Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, GCG soflware contains programs such as GAP and BESTFIT which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutein thereof. See, e.g., GCG Version 6.1. Polypeptide sequences also can be compared using FASTA with default or recommended parameters; a program in GCG Version 6.1. FASTA (e.g, FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson (2000) supra). Sequences also can be compared using the Smith-Waterman homology search algorithm using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 2, BLOSUM matrix of 62. Another preferred algorithm when comparing a sequence disclosed herein to a database containing a large number of sequences from different organisms is the computer program BLAST, especially BLASTP or TBLASTN, using default parameters. See, e.g.. Altschul ef al. (1990) J. Mol. Biol. 215: 403-410 and (1997) Nucleic Acids Res. 25:3389-3402, each of which is herein incorporated by reference.

[0090] Provided herein are anti-TyrD or anti-GPC3 CARs comprising variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein having one or more substitutions (e.g.. conservative substitutions). For example, the present disclosure includes anti- TyrD CARs having HCVR, LCVR, and / or CDR amino acid sequences with, e.g., 20 or fewer, 19 or fewer, 18 or fewer, 17 or fewer, 16 or fewer, 15 or fewer, 14 or fewer, 13 or fewer, 12 or fewer. 11 or fewer, 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer. 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or | amino acid substitutions relative to any of the HCVR, LCVR, and / or CDR (e.g.. HCDR1!, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3) amino acid sequences disclosed herein. For example, an anti-TyrD) CAR can comprise 20, 19, 18, 17, 16, 15,14 13,12, 11,10,9.8,7,6, 5,4, 3, 2, or | amino acid substitutions (e.g., conservative amino acid substitutions) relative to any of the HCVR, LCVR, and / or CDR (e.g.. HCDR1, HCDR2, HCDR3. LCDR. L.CDR2, or LCDR3) amino acid sequences disclosed herein.

[0091] Similarly, the present disclosure includes anti-GPC3 CARs having HCVR, LCVR. and / or CDR amino acid sequences with, e.g., 20 or fewer, 19 or fewer, 18 or fewer, 17 or fewer, 16 or fewer, 15 or fewer, 14 or fewer, 13 or fewer, 12 or fewer, 1 or fewer, 10 or fewer, ¢ or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 3 or fewer, 4 or fewer, 3 or fewer, 2 or fewer. or | amino acid substitutions relative 10 any of the HCVR, LCVR, and / or CDR {(e.g., HCDRI1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3} amino acid sequences disclosed herein. For example, an anti-GPC3 CAR can comprise 20, 19, 18, 17,16, 15, 14 13,12, 11, 10,9, 8,7. 6, 5, 4, 3,2, or | amino acid substitutions (e.g., conservative amino acid substitutions) relative to any of the HCVR, LCVR, and / or CDR (e.g, HCDR1, HCDR2, HCDR3, LCDRI, LCDR2, or LCDR3) amino acid sequences disclosed herein. 10092] Exemplary binding domains described herein typically comprise, in order from the amino to carboxy terminus, a heavy chain region followed by a light chain region (VH-VL). Where a certain order of of VH and VL region in the binding domain is explicitly or implicitly described, the present disclosure is also understand to describe the alternate embodiment in which the order of VH and VL regions are reversed, e.g., in an scFV or a CAR comprising an an scFv binding domain.

[6093] Generally, the CAR encoding nucleic acids described herein include an extracellular linker portion that encodes a peptide linker that links the binding domain to a transmembrane domain. Exemplary linker portions include. without limitation, a linker portion that encodes the CD8a hinge domain, 5 SEQ ID NO:1 (PTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY) or SEQ ID NO:2 (TTTPAPRP PTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY). Typically, the region encoding the peptide linker (e.g., CD8a hinge domain) is 3° of the region encoding the binding domain and 5° of a region encoding a transmembrane domain.

[0094] The CAR encoding nucleic acids described herein include a transmembrane domain. The transmembrane domain can link an extracellular antigen binding domain, e.g., and hinge, to one or more intracellular signaling components. For example, the transmembrane domain can link an antigen binding domain, e.g., and hinge. to a CD3( signaling domain and optionally with one or two costimulation endodomains. Exemplary transmembrane domains include without limitation a CD8a transmembrane domain, eg. SEQ ID NO:3 (IWAPLAGTCGVLLLSLVITLYC). Typically, the region encoding the transmembrane domain — e.g; CD8a transmembrane-domain}-is-3* of the region encoding the peptide linker (e.g, CD8a hinge domain) and 5° of a region encoding one or more cytoplasmic domains.

[6095] In some embodiments, the isolated nucleic acid encodes a cytoplasmic region containing one or more cytoplasmic domains. The region encoding the cytoplasmic region is typically 3° of the region encoding the transmembrane domain. The cytoplasmic domains are typically signaling domains that provide an activating signal for v8 T cell proliferation. cytotoxic activity, and / or pro-inflammatory cylokine expression (e.g, TNF-a or IFNy). An exemplary cytoplasmic domain is a CD3( signaling domain. [n some embodiments, the CD3({ signaling domain is or comprises SEQ ID NO:4 (RVKFSRSADAPAYQQGQNQLYNELNLGR REEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGK GITDGLYQGLSTATKDTYDALIIMQALPPR). In some embodiments, the CD3{ signaling domain is or comprises SEQ 1D NO:5 (RVKFSRSADAPAYQQGQONQLYNELNLGRREEYDV LDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLY QGLSTATKDTYDALHMQALPPR). In some embodiments, the cytoplasmic region contains multiple (e.g., 2, 3, 4, 3, or 6) signaling domains, such as multiple (e.g., 2, 3. 4, 5, or 6) CD3{ signaling domains, e.g., each independently selected from SEQ ID NO: 4 and 5. In some embodiments, the cytoplasmic region contains multiple (e.g, 2, 3. 4, 5, or 6) non- CD3{ signaling domains and a CD3{ signaling domain. In some embodiments, the cytoplasmic region contains a non- CN3{ signaling domain and multiple (e.g, 2, 3, 4, 5, or 6) CD3{ signaling domains. Additional or alternative signaling domains include, without limitation.

[0096] The cytoplasmic region can contain one or more costimulation endodomains. A region encoding one or more costimulation endodomains can be 5' or 3” of a region encoding a signaling domain. In some embodiments, the region encoding one or more costimulation endodomains is 5° of the region encoding a signaling domain. In some embodiments, a region encoding one or more costimulation endodomains is 5° of a signaling domain and an additional region encoding one or more costimulation endodomains is 3° of the signaling domain. Exemplary costimulation endodomains include, without limitation, CD28; CD137 (4-1BB): CD278 (ICOS); CD27. CDI34 (0X40). and TLR2 costimulation endodomains, and combinations thereof. 16097] In some embodiments, additional signaling modalities can be included to increase proliferation, persistence, and / or cytotoxic activity of the v8-T cells described herein. For example, in some embodiments, the CAR construct can encode a soluble common gamma chain cytokine at the 3° end of the isolated nucleic acid. The common gamma chain cytokine encoding region can be linked to the 5° portion of the CAR construct via a T2A linker encoding region, such that the common gamma chain cytokine is cleaved from the CAR polypeptide and secreted by the cell.

[0098] In some embodiments, the construct encodes at least one 4-1BB costimulation endodomain, and optionally a second costimulation endodomain selected from a 4-1BB, 1COS, CD28. and CD27 costimulation endodomain. In some embodiments, the construct encodes at least two 4-1BB costimulation endodomains. or two 4-1BB costimulation endodomains in combination with one, two, three, or four. or mare. costimulation endodomains selected from a 4-1BB, 1COS, CD28. and CD27. In some embodiments, the 4-1BB costimulation endodomain comprises SEQ 1D NO: 6 (KRGRKKLLYIFKQPFMRPVQTT QEEDGCSCRFPEEEEGGCEL).

[0099] In some embodiments, the construct encodes one CD27 costimulation endodomain, and optionally a second costimulation endodomain selected from a 4-1BB, ICOS. CD28, and CD27 costimulation endodomain. In some embodiments, the construct encodes a CD27 costimulation endodomain, and a 4-1BB costimufation endodomain. In some embodiments, the construct encodes two CD27 costimulation eadodomains. In some embodiments, the CD27 costimulation endodomain comprises SEQ 1D NO: 7 (QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQED YRKPEPACSP).

[60100] In some embodiments, the construct encodes a secretion signal, e.g., SEQ ID NO: 12 (MALPVTALLLPLALLLHAARP) operably linked to facilitate secretion of a C-terminal polypeptide, such as a cytokine that supports the activation, cytotoxicity, and / or persistence of a T cell (e.g.. CAR-T cell). In some embodiments, the secretion signal is a secretion signal of SEQ ID NO: 26 (MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEA). In some embodiments, the construct encodes a secretion signal, e.g., SEQ 1D NQ: 12 operably linked to facilitate secretion of a common gamma chain cytokine such as IL-13 or an active fragment thereof, e.g., SEQ in NO: 14 (NWVNVISDELKKIEDLIQSMHIDATLYT ESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKEC EELEEKNIKEFLQSFVHIVQMFINTS). Other 1L-15 sequences, including codon optimized nucleic acid sequences encoding sIL 15, are disclosed in WO 2007 / 037780. Exemplary common gamma chain cytokines include 1L-2 and IL-15. In some embodiments, the common gamma chain cytokine is selected from 11-2. IL-7, and {1-15 [00101} In some embodiments, the construct encodes one or more mulli-cistronic linker regions, e.g., between a signaling domain and / or costimulation endodomain and a secretion signal operably linked to facilitate secretion of a cytokine. A multi-cistronic linker region is a region of polypeptide sequence or RNA sequence that facilitates the production of multiple discrete polypeptides from a single transcription product. In some embodiments, the multi- cistronic linker region encodes a cleavage sequence. Suitable cleavage sequences include self- cleavage sequences such as a P2A, F2A, E2A. or T2A cleavage sequence and / or sequences that are cleaved by an endogenous protease, such as furin.

[00102] In some embodiments, the cleavage sequence is a P2A cleavage sequence. In some embodiments, the cleavage sequence is a lurin cleavage sequence. In some embodiments, the cleavage sequences are a P2A and a furin cleavage sequence. In some embodiments, the cleavage sequence is the P2A cleavage sequence of SEQ ID NO: [5 (SGSGATNFSLLKQAGDVEENPGP). In some embodiments, the cleavage sequence is a furin cleavage sequence of SEQ ID NO: 16 (RAKR). In some embodiments, the cleavage sequence is a P2A+{urin cleavage sequence of SEQ ID NO: 17 (RAKRSGSGATNFSLLKQAGDVEENP GP). In some embodiments, the cleavage sequence is the P2A cleavage sequence of SEQ ID NO: 25 (GSGATNFSLLKQAGDVEENPGP).

[00103] In some embodiments, the cleavage sequence is or comprises a P2A cleavage sequence of SEQ ID NO: 27 (ATNFSLLKQAGDVEENPGP). In some embodiments, the cleavage sequence is or comprises an F2A cleavage sequence of SEQ [D NO: 28 (VKQTLNNFDLLKLAGDVESNPGP). [n some embodiments, the cleavage sequence is or comprises an E2A cleavage sequence of SEQ ID NO: 29 (QCTNYALLKLAGDVESNPGP). In some embodiments, the cleavage sequence is or comprises an T2A cleavage sequence of SEQ ID NO: 30 (EGRSLLTCGDVEENPGP). In certain aspects, multiple self-cleavage sequences can be encoded carboxy terminal to a signaling and / or costimulatory domain and amino-terminal to an encoded secreted cytokine (e.g. common gamma chain cytokine such as [1-13), preferably wherein the multiple self cleavage sequences are independently selected from the group consisting of a P2A cleavage sequence, a T2A cleavage sequence, an E2A cleavage sequence, and an F2A cleavage sequence. In certain aspects, one or more self-cleavage sequences and one or more sequences cleaved by an endogenous protease are encoded in a construct described herein. In certain embodiments, a endogenous protease recognition site is encoded amino terminal to a sell cleavage sequence.

[00104] In some embodiments, the muiti-cistronic linker region encodes an internal ribosome entry site. An exemplary internal ribosome entry site is encoded by SEQ ID NO: 31 (CTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGT TATTTTCCACCATATTIGCCOGTCTTITIGGCAATGTGAGGGCCCGOGAAACCTGGCCCTAGT CTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCT GITGAATGTCGTGAAGGAAGCAGTICCTCTGGAAGCTTCTITGAAGACAAACAACGTC TGTAGCCGACCCTTTGCAGGCAGCGGAACCCCCCACCIOGCCGACAGOTGCCTCTGCG GCCAAAAGCCACGTOGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCAC GTTGTGAGTTGGATAGTTGCTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAAC AAGGGGCTGAAGGATGCCCAGAAGGTACCCCATIGTATGGGATCTGATCTGGGGCC TCOGOGTGCACATOGCTTTACATGTGTITTAGTCGAGGTTAAAAAAACGTCTAGGCCCCCC GAACCACGGGGACGTGGTTITTICCTTTGAAAAACACGATGATA),

[00105] Another exemplary internal ribosome eniry site is encoded by SEQ ID NO: 32 (AGCAGGTTTCCCCAACTGACACAAAACGTGCAACTTGAAACTCCGCCTGGTICITTC CAGGTCTAGAGGGGTAACACTTTIGTACTGCOGTTTGGCTCCACGCTCGATCCACTGGC GAGTGTTAGTAACAGCACTGTTGCTTCGTAGCGGAGCATGACGGCCGTGGGAACTCC TCCTTGGTAACAAGGACCCACGGGGCCAAAAGCCACGCCCACACGOGGCCCGTCATG TGTGCAACCCCAGCACGGCGACTTITACTGCGAAACCCACTTTAAAGTGACATTGAAA CTGGTACCCACACACTGGTGACAGGCTAAGGATGCCCTTCAGGTACCCCGAGGTAA CACGCGACACTCGGGATCTGAGAAGGOGACTGOGGCTICTATAAAAGCGCTCGGTT TAAAAAGCTTICTATGCCTGAATAGGTGACCOGGAGGTCGGCACCTITTCCTTTGCAATT ACTGACCAQ).

[00106] Further suitable internal ribosome entry sites include, but are not limited to, those described in Nucleic Acids Res. 2010 Jan:38(Database issue}:D131-6. doi: 10.1093 / nar / gkp981. Epub 2009 Nov 16, those described at iresite.org, those described in WO 2018 / 215787, the sequence described in GenBank accession No. KP(19382.1, and the IRES element disclosed in GenBank accession No, LT1727339.1. the contents of which are incorporated by reference in the entirety and for all purposes and in particular for the internal ribosome entry sites and their use described therein.

[00107] Additional multi-cistronic linker regions, including cleavage self-cleavage, and IRES elements, are disclosed in US 2018 / 0360992 and U.S. 8.863.467. cliements, are disCiosed In Us LV 6 / UL0UYYL and U.s. 8,600,507. MN

[00108] In some embodiments, the isolated nucleic acid encodes SEQ ID NO:8 (MSVPTQVLGLLLLWLTDARCDIQMTQSPSSLSASVGDRVTITCKASQDIHNYIAWYQQ KPGKAPKLLIHYTSTLQPGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCLQYDNLWTFGQ GTKVEIKRGGGGSGGGGSGGGGQITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVS Ww IRQPPGRALEWLAHIY WDDDKRYNPSLKSRLTITRDTSKNQVVLTMINMDPVDTSRY YCARKDYGSSFYAMHYWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAG GAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTT QEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGONQLYNELNLGRREEYDVLDKR RGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLS TATKDTYDALHMQALPPR), a hD11-CD8-BBz polypeptide comprising an hd || anti-TyrD binding domain (anti-1yrDsse.377), a CDS8a hinge and transmembrane region, a 4-1BB costimulation endodomain, and a C3 signaling domain. —

[00109] In some embodiments, the isolated nucleic acid encoding an{anti-TyDgg 7-CDS- — . . . CN =) polypeptide comprises the sequence of SEQ in NO: (ATGTCCGTGCCTACCCAGGTGCTGGGCCTGCTGCTGCTGTGGCTGACCGACGCCAG ATGCGACATCCAGATOGACCCAGTCTCCATCCTCCCTOTCTGCATCTGTAGGAGACAG AGTCACCATCACTTGCAAGGCGAGTCAGGACATTCACAACTATATAGCTTGGTATCA GCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCCACTATACATCCACTITGCAACC AGGOCTCCCATCAAGCOTICAGTGGAAGTCGATCTGGOCACAGATTTTACTTTCACCAT CAGCAGCCTGCAGCCTGAAGATATTGCAACATATTACTOTCTACAGTATGATAATCT CTGGACGTTCGGTCAAGGCACCAAGOGTGGAAATCAAACGGGGTGGAGGTGGATCTG GAGGCGAGGAGGATCCGGTGGAGGAGO TCAGATCACCTTIGAAGGAGTCTGGICCTACG CTGGTGAAACCCACACAGACCCTCACGCTGACCTGCACCTICTCTGGGTTCICACTC AGCACTAGTGGAATGGOGTGTGTCCTGOATCCOGTCAGCCCCCAGGAAAGGCCCTGGA GTGGCTTGCACACATTTATTGGGATGATGATAAGCGCTACAACCCATCTCTGAAGAG CAGGCTCACCATCACCAAGGACACCTCCAAAAACCAGGTGUGTCCTTACAATGACCA ACATGGACCCTGTGGACACAGCCACATATTACTGTGCACGAAAGGACTACGGTAGT AGCTTCTATGCTATGCACTACTGOGOTCAAGGAACCCTAGTCACCOGTGTCGAGTACC ACGACGCCAGCGCCGCGACCACCAACACCGOGGCGCCCACCATOCGCOTCQGCAGCCCCT GTCCCTGCGCCCAGAGGUGTGCCOGUCCAGCGGUOGGGOGGGCGCAGTGCACACGAGGO GGCTGGACTTCGCCTGTGATATCTACATCTGGGOGCCCTTIGGCOCGGGACTIOTGGAGG TCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCC TGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGAT GGCIGTAGCTGCCGATTICCAGAAGAAGAAGAAGGAGGATGOTGAACTGAGAGTGAA GTTCAGCAGGAGCGCACACGCCCCUGCGTACCAGCAGGGCCAGAACCAGCTCTATA ACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTITGGACAAGAGACGTGGC CGGGACCCTGAGATGOGGGOGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCC TGTACAATGAACTGCAGAAAGATAAGATGOGCGGAGGCCTACAGTGAGATTGGGATG AAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTAC AGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA). 100110] In some embodiments, the isolated nucleic acid comprises a codon optimized sequence encoding a CD8a hinge region. Fup Sar spss oe hinge region nucleic acid sequences include, without Himitation, SEQ ID NO: 10 nucleic acid sequences include, without limitation, SEQ ID NO: 10 {ACCACCACCCCTGCACCAAGGCCCCCGACTCCCGCGCCCACCATCGCGTCACA GCCTCTTAGCCTGCGACCGGAAGCATGCAGACCAGCTGCCGGGGGGGCCGTGCATA CGAGAGGTTTGGACTTCGCCTGCGAT). In some embodiments, the CD8a hinge region is encoded by the following sequence SEQ ID NO:11 (ACCACGACGCCAGCG CCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTICCCTGCGC CCA GAGGCOGTGCCGOGCCAGCGGCGOGOGGGCOGCAGTOGCACACGAGGGGGCTGGACTTCG CCTGTGAT). [oo111} Tn some embodiments, the isolated nucleic acid encodes SEQ 1D NO: 18 (MSVPTQVLGLLLLWLTDARCDIQMTQSPSSESASVGDRVTITCKASQDIHNYIAWYQQ KPGKAPKLLIHYTSTLQPGVPSRFSGSGSGTDFTFTISSLOPEDIATYYCLQYDNLWTFGQ GTKVEIKRGGGGSGGGGSGGOGQITLKESGPTLVKPTQTLTLTCTFSGESLSTSGMGVYS WIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMINMDPVYDTATY YCARKDYGSSFYAMHYWGQGTLVTVSSTITPAPRPPTPAPTIASQPLSLRPEACRPAAG GAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPEFMRPVQTT QEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQONQLYNELNLGRREEYDVLDKR RGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGILYQGLS TATKDTYDALHMQALPPRRAKRSGSGATNESLLKQAGDVEENPGPMALPVTALLLPLA LLLHAARPNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISL ESGD, HD TYE NSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS *), 4 hD11-CD8-BBz-slL.15 pplypeptide comprising an anti-TyrD hD11 (anti-TyrDses.377) binding domain, a @ ninge and transmembrane region, a 4-1BB costimulation endodomain, a CD3Y signaling domain, a furin-P2A cleavage sequence, and a secretion signal operably linked to an IL-15 domain. to an IL-15 domain. pd — 00112] In some embodiments, the isolated nucleic acid encoding lpi Conn L1s polypeptide comprises the sequence of SEQ ID NO: 19 (ATGTCCGTGCCTACCCAG GTGCTGGGCCTGCTGCTGCTGTGGCTGACCGACGCCAGATGCGACATCCAGATGACC CAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCAAG GCGAGTCAGGACATTCACAACTATATAGCTTGGTATCAGCAGAAACCAGGGAAAGC CCCTAAGCTCCTGATCCACTATACATCCACTTTGCAACCAGGGGTCCCATCAAGGTT CAGTGGAAGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCCTGA AGATATTGCAACATATTACTGTCTACAGTATGATAATCTCTGGACGTTCGGTCAAGG CACCAAGGTGGAAATCAAACGGGGTGGAGGTGGATCTGGAGGAGGAGGATCCGGT GGAGGAGGTCAGATCACCTTGAAGGAGTCTGGTCCTACGCTGGTGAAACCCACACA GACCCTCACGCTGACCTGCACCTTCTCTGGGTTCTCACTCAGCACTAGTGGAATGGG TGTGTCCTGGATCCGTCAGCCCCCAGGAAAGGCCCTGGAGTGGCTTGCACACATTTA TTGGGATGATGATAAGCGCTACAACCCATCTCTGAAGAGCAGGCTCACCATCACCA AGGACACCTCCAAAAACCAGGTGGTCCTTACAATGACCAACATGGACCCTGTGGAC ACAGCCACATATTACTGTGCACGAAAGGACTACGGTAGTAGCTTCTATGCTATGCAC TACTGGGGTCAAGGAACCCTAGTCACCGTGTCGAGTACCACCACCCCTGCACCAAG GCCCCCCGACTCCCGCGCCCACCATCGCGTCACAGCCTCTTAGCCTGCGACCGGAAGC ATGCAGACCAGCTGCCGGGGGGGCCGTGCATACGAGAGGTTTGGACTTCGCCTGCG ATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGT TATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATICAAACAACC ATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCC AGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGAC GCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACG AAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGG GAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAA AGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGG GGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTA CGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCCGCGCGAAGCGATCAGGCAGCG GGGCGACAAATTTCAGCCTTCTGAAACAAGCAGGCGACGTGGAAGAAAACCCCGGT CCAATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGLTGGCCTTGCTGCTCCACGCC.. GCCAGGCCGAACTGGGTGAATGTAATAAGTGATTTGAAAAAAATTGAAGATCTTAT TCAATCTATGCATATTGATGCTACTTTATATACOGGAAAGTGATGTTCACCCCAGTTGC AAAGTAACAGCAATGAAGTGCTTICTCTTGGAGTTACAAGTTATTITCACTTGAGT CC GGAGATGCAAGTATTCATGATACAGTAGAAAATCTGATCATCCTAGCAAACAACAG TITCTCTTCTAATGGGAATOTAACAGAATCTGCGATGCAAAGAATGTGAGGAACTGG AGGAAAAAAATATTAAAGAATTTTIGCAGAGTTITTGTACATATTGTCCAAATGTTCA TCAACACTTCTTGA).

[00113] In some embodiments, the isolated nucleic acid encodes SEQ ID NO: 20 (MSVPTQVLGLLLLWLTDARCQVQLVQSGAEVKKPGASVKVSCKASGYTFIDYEMHW VRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADKSTSTAYMELSSLTSEDTAV YYCTRFYSYTYWGQGTLVTVSSGGGGSGGGGSGGGGDYVMTQSPLSLPVTPGEPASIS CRSSQSLVHSNRNTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISR VEAEDVGVYYCSQONTHVPPTFGQGTKLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAA GGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQT TQEEDGCSCRFPEFEEGGCELRVKFESRSADAPAYQQGQONQLYNEINLGRREEYDVLDK RRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQG LSTATKDTYDALHMQALPPR*), a polypeptide comprising a GC33 anti-GPC3 binding domain, a CD8a« hinge and transmembrane region, a 4-1BRB costimulation endodomain, and a CD3{ signaling domain.

[60114] [n some embodiments, the isolated nucleic acid encoding an GC33-CD8-BBz polypeptide comprises the sequence of SEQ ID NO: 21 (ATGTCCGTGCCTACCCAGG TGCTGGGCCTGCTOCTGCTGTGGCTCGACCGACGCCAGATGCCAAGTGCAGCTGGTCC AGAGCGGCGCCGAGGTGAAAAAGCCTGGCGCCAGCGTGAAGOGTGTCCTGCAAGGCC TCTGGCTACACCTTCACCGACTACGAGATGCACTGGOGTOCOGCAGGCCCCTGGACA GGGCCTGGAATGGATGGGCGCTCTGGACCCCAAGACCGGCGACACCGCTTATAGCC AGAAGTTCAAGGGCAGAGTOGACCCTGACAGCTGATAAGAGCACAAGCACCGCCTAC ATGGAACTGAGCAGCCTGACCAGCGAGGACACCGCCGTIGTACTACTGCACCAGATT CTACAGCTACACCTACTGGGGCCAGGGGACCCTGAGTGACAGTAGTCTAGCGAGTGGAG GTGGATCTGGAGGAGGAGGATCCGGTGGAGGAGGTGATGTGGTGATGACCCAGAGC CCTCTGAGCCOCTGCCTGTGACCCOTGOAGAGCOCTGCCAGCATCAGCTGCAGAAGCAG CCAATCTCTGGTGCACAGCAACCGGAACACATACCTGCACTGGTACCTGCAGAAAC CTGGCCAGAGCCCCCAGCTGCTGATCTACAAGGTGTCCAACAGATICAGCGGCGTG CCTGATAGATTCAGCGGATCTGGCAGCGGCACCGACTICACCCTGAAGATCTCTAGA GITGGAAGCCGAGGACGTGGGCGTGTACTACTGCAGCCAGAACACCCACGTGC CCC CACCTTCGGCCAGGGCACAAAGCTGGAAATCAAGACCACGACGCCAGCGCCGCGAC CACCAACACCGGCGCCCACCATCGCOGTCGCAGCCCCTOTCCCTGCGCCCAGAGGCGT GCOCGGCCAGCGGCAGGGGOGCGCAGTOGCACACGAGGGOGGCTGGACTTCGCOCTGTGAT ATCTACATCTGGGCGCCCTIGGCCGGOGACTTGTGGGGTCCTTICTCCTGTCACTGOGTTA TCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCAT TTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTICCA GAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTICAGCAGGAGCGCAGACG CCCCCGOCGTACCAGCAGGGCCAGAACCAGCTICTATAACGAGCTCAATCTAGGACGA AGAGAGGAGTACGATGTTTTOGGACAAGAGACGTGGCCGGGACCCTGAGATOGGGGG AAAGCCGCAGAGCAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAA GATAAGATGGCGGAGGCCTACAGTGAGATTGGGATOGAAAGGCGAGCGCCGGAGGG GCAAGGGGCACGATAGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTAC GACGCCCTICACATGCAGGCCCTGCCCCCICGCTAA). [00115} In some embodiments, the isolated nucleic acid encodes SEQ ID NO: 22 (MSVPTQVLGLLLLWLTDARCQVQLVQSGALVKKPGASVKVSCKASGYTFIDYEMHW VRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADKSTSTAYMELSSLTSEDTAV YYCTRFYSYTYWGQGTLVTVSSGGGGSGGGGSGGGGDVYVMTQSPLSLPVTPGEPASIS CRSSQSLVHSNRNTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISR VEAEDVGVYYCSQNTHVPPTFGQGTKLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAA GGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQT TQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGONQLYNEILNLGRREEYDVLDK RRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQG LSTATKDTYDALHMQALPPRGSGATNFSLLKQAGDVEENPGPMALPVTALLLPLALLLH AARPNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGD ASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS*), a polypeptide comprising a GC33 anti-GPC3 binding domain, a CD8a. hinge and transmembrane region, a 4-1BB costimulation endodomain, a C13{ signaling domain, a furin and P2A cleavage region, and a secretion signal operably linked to an 1L-15 domain.

[00116] In some embodiments. the isolated nucleic acid encoding a GC33-CD8-BBz-sIL15 polypeptide comprises the sequence of SEQ iD NO: 23 (ATGTCCGTGCCTACCCAGGTGCTGGGCCTGCTGCTGCTGTGGCTGACCGACGCCAG ATGCCAAGTGCAGCTGOTCCAGAGCGOGCGCCGAGOTGAAAAAGCCOTGGCGCCAGCG TGAAGGTGTCCTGCAAGGCCTCTGGOCTACACCTTCACCGACTACGAGATGCACTAGGOG TGCGGCAGGCCCCTGGACAGGGCCTGGAATGGATGGGCGCTCTGGACCCCAAGACC GOCGACACCOGCTTATAGCCAGAAGTTCAAGGGCAGAGTGACCCTGACAGCTGATAA GAGCACAAGCACCGCCTACATGGAACTGAGCAGCCTGACCAGCGAGGCACACCGCCG TGTACTACTGCACCAGATTCTACAGCTACACCTACTGGGGCCAGGGGACCCTGGTGA CAGTGTCTAGCGGTGGAGGTGGATCTGGAGGAGGAGGATCCGGTGGAGGAGGTGAT GTGOTGATGACCCAGAGCCCTICTGAGCCTIGUCCTGTGACCCCTGGAGAQLCCTOGCCAGC ATCAGCTGCAGAAGCAGCCAATCTICTGGTGCACAGCAACCGGAACACATACCTGCA CTGGTACCTGCAGAAACCTGCGCCAGAGCCCCCAGCTGCTGATCTACAAGGTGTCCA ACAGATTICAGCGGCGTGCCTGATAGATTCAGCGCGATCTGGCAGCGGCACCGACTTC ACCCTGAAGATCTCTAGAGTGGAAGCCGAGGACGTOGGCGTGTACTACTGCAGCCA GAACACCCACGTGCCCCCCACCTTCGGCCAGGGCACAAAGCTGGAAATCAAGACCA COGACGCCAGCGCCGCGACCACCAACACCGGCOGCCCACCATCGCGTCGCAGCCCCTG TCCCTGCGCCCAGAGGCGTGCOCGOCCAGCOGCGGOGOGGGCGCAGTGCACACGAGGGG GCTGGACTTICGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGAGGT CCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCT GTATATATTCAAACAACCATITATGAGACCAGTACAAACTACTCAAGAGGAAGATG GCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGCGAGCGATGTGAACTGAGAGTGAAG TTCAGCAGGAGCGCAGACGCCCCOCGCGTACCAGCAGGGCCAGAACCAGCTCTATAA CGAGCTCAATCTAGCACGAAGAGAGGCGAGTACGATGTTTIGGACAAGAGACGTGGCC GGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCT GTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATIGGGATGA AAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTICTCAGTACA GCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTOGLCCCCCTCGCGAOGTAGC GGGGCTACGAACTTCTCCCTTCTTAAACAAGCGGOGAGACGTGGAAGAAAATCCCGG ACCTATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCC GCCAGGCCGAACTGGGOGTGAATGTAATAAGTGATTTGAAAAAAATTGAAGATCTTAT TCAATCTATGCATATTGATGCTACTTTATATACGGAAAGTGATGTTCACCCCAGTTGC AAAGTAACAGCAATGAAGTGCTTICTCTTIGGAGTTACAAGTTATTTCACTTGAGTCC GGAGATGCAAGTATTCATGATACAGTAGAAAATCTGATCATCCTAGCAAACAACAG TITGCTCTTCTAATGGGAATGTAACAGAATCTGGATGCAAAGAATGTGAGGCGAACTGG AGGAAAAAAATATTAAAGAATTTTTGCAGAGTTTTGTACATATTGTCCAAATGTICA TCAACACTTCTTGA).

[60117] In some embodiments, the isolated nucleic acid encoding a GC33-CD8-BBz-sIL15 polypeptide comprises the sequence of SEQ 1D NO: 24 {ATGTCCGTGCCTACCCAGGTGCTGGGCCTGCTGCTGCTGTGGCTGACCGACGCCAG ATGCCAAGTGCAGCTGGTCCAGAGCGGCOGCCGAGGTGAAAAAGCCTGGCGCCAGCG TGAAGGTGTCCTGCAAGGCCTCTGGOTACACCTTICACCGACTACGAGATGCACTAGGG TGCGGCAGGCCCCTGGACAGGGCCTCGAATGCATGGGCGCTCTGGACCCCAAGACC GGCGACACCGCTTATAGCCAGAAGTTCAAGGGCAGAGTGACCCTGACAGCTGATAA GAGCACAAGCACCGCCTACATGGAACTGAGCAGCCTGACCAGCGAGGACATUGCCG TGTACTACTGCACCAGATTICTACAGCTACACCTACTGGGGCCAGGGGACCCTGGTGA CAGTGTCTAGCGGTGGAGGTGGATCTGGAGGAGGAGGATCCGGTGGAGGAGGTGAT GIGOTGATGACCCAGAGCCCTICTGAGCCTGCCTGTGACCCCTIGCAGAGCCTGCCAGC ATCAGCTGCAGAAGCAGCCAATCICTGGTGCACAGCAACCGGAACACATACCTGCA CTGGTACCTGCAGAAACCTGGCCAGAGUCCCCAGCTGCTGATCTACAAGGTGTCCA ACAGATTCAGCGGCGTGCCTGATAGATTCAGCGGATCTOGGCAGCGGCACCGACTTC ACCCTGAAGATCTCTAGAGTGGAAGCCGAGGACGTGGGCGTGTACTACTGCAGCCA GAACACCCACGTGCCCCCCACCTTICGGCCAGGGCACAAAGCTGGAAATCAAGACCA CGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTG TCCCTGCGCCCAGCGAGGCGTOGCCGAGCCAGCOGOCGGGOGGGCGCAGTIGCACACGAGGGOG GCTGGACTTCGCCTGTGATATCTACATCTGOOCGCCOCTTGGCCOGGACTTATGGGOGT CCTTCTICCTGTCACTGOGTTATCACCCTTITACTGCAAACGGGGCAGAAAGAAACTCCT GTATATATTCAAACAACCATTITATGAGACCAGTACAAACTACTCAAGAGGAAGATG GCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAG TTCAGCAGGAGCGCAGACGCCCCCGOCGTACCAGCAGGGCCAGAACCAGCTCTATAA CGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCC GGGACCCTGAGATOGGGGGGCAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCT GTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGA AAGGCGAGCGCCOGGAGGGGCAAGGGGCACGATGGCCTITACCAGGGTCTCAGTACA GCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCLCTCGCGAGTAGC GGGGCTACGAACTTCTCCCTTCTTAAACAAGCGGOGAGACGTGGAAGAAAATCCCGG ACCTATOGCCTTACCAGTGACCGCCTTGCTCCTGCOCGCTGGCCTTGOTGCTCCACGCC GCCAGGCCGAACTGGGTGAATGTGATCAGCGATCTGAAGAAGATCGAGGATCTGAT CCAGTCCATGCACATCGATGCCACCCTGTATACCGAGAGCGATGTGCACCCCAGCTG CAAGGTGACCGCCATGCAAGTGCTTITOTGCTGGAGCTGCAGGTGATCTCCCTGGAGTC CGGAGATGCCAGCATCCACGATACCGTGGAGAATCTGATCATCCTGGCCAACAACA GCCTGICCTCCAATGGCAATGTGACCGAGTCGGUGATGCAAGGAGTGCGAGGAGCTG GAGGAGAAGAATATCAAGGAGTTTCTGCAGAGCTTTGTACATATTGTCCAAATGTIC ATCAACACTTCTTGA). 100118] In some embodiments, the isolated nucleic acid is a linear nucleic acid. In some embodiments, the isolated nucleic acid is a vector. such as a plasmid vector, an adenoviral vector, an adeno-associated viral vector, a viral vector, a retroviral vector (e.g, a gamma retroviral vector), or a lentiviral vector. In some embodiments, the isolated nucleic acid, or an, e.g.. contiguous, portion thereof containing the binding domain transmembrane domain and one or more signaling and / or costimulation endodomains is integrated into the genome of a host cell, such as a host y8 T cell. In an exemplary embodiment, the isolated nucleic acid is retroviral vector. v8 T Cells:

[00119] Aspects of the invention include v3 T cells that functionally express an isolated nucleic acid described herein, and thereby expresses a CAR on the surface of the y8 1 cell. 100120§ Aspects of the invention can additionally or alternatively include v8 T cells having in vitro or in vivo cytotoxic activity against a solid tumor cell that exhibits cell surface expression of the tumor associated antigen (TAA). In some cases, the cytotoxic activily is innate activity. In some cases, the cytotoxicity is at least in part, significantly (> about 25%). or entirely, due to the presence of a CAR construct having a binding domain that specifically binds the TAA expressed on the surface of the solid tumor cell. In some cases, the v8 T cells exhibit solid tumor cell killing activity of said v8 T cell is greater than an innate level of in vitro and / or in vive solid tumor cell killing activity in a control vd '[' cell. In some cases, the control v6 'I cell docs not comprise a CAR construct. Tn some cases, the control ¥y8 T cell comprises a CAR construct lacking a binding domain described herein, a hinge region described herein. a transmembrane domain described herein, a signaling domain described herein, and / or a costimulation endodomain described herein.

[60121] [n some cases, the cytotoxicity is at least in part, significantly (> about 25%). or entirely. due to the presence of a CAR construct having a binding domain that specifically binds TyrD or an epitope within TyrD, such as TyrIieo377. In some cases, the cytotoxicity is at least in part, significantly (> about 25%), or entirely, due fo the presence of a CAR construct having a binding domain that specifically binds TyrD or an epitope within TyrD, such as TyrDago.377 in an HLA restricted (e.g., class 1 HLA-restricted) manner. [n some cases, the cytotoxicity is at least in part, significantly (> about 25%), or entirely, due to the presence of a CAR construct having a binding domain that specifically binds HLA-A2 / TvtDis0.377. In some cases, the v8 T cells functionally express a CAR encoded by an isolated nucleic acid described herein that specifically binds TyrD or a peptide fragment thereof. 160122] In some embodiments, v8 T cells described herein can exhibit HLA-restricted (e.g., HLA class 1 restricted) cytotoxicity. In other embodiments, most (>50%), substantially all (>90%), or all of the cviotoxic activity is not HLA-restricted (e.g, HLA class I restricted). HELA-restricted cytotoxic activity can be assessed by comparing in vitro cytotoxicity against an HLA (e.g., HLA class 1) (null) tumor cell line versus in vitro cytotoxicity against an HLA+ (e.g. HLA class I'} tumor cell line. In some embodiments, the HLA-restricted cytotoxic activity is at least in part, significantly (>25%), or entirely, provided by the use of a T cell Receptor-like binding domain. T cell receptor like binding domains are binding domains that specifically recognize the antigen when presented on the surface of a cell in complex with an MHC molecule. T cell Receptor-like binding domains are further described, e.g., in WO 2016 / 199141. [00123} +43 T cells described herein can exhibit robust and / or persistent solid tumor cell killing activity. In some cases, the solid tumor cell Killing activity can persist for at least about 6 days to 120 days. or for at least about 6 days to 180 days, from first contact with a solid tumor cell, In some cases, the solid tumor cell killing activity of a ¥8 T cell described herein, or a progeny thereof, can persist for at least about 6 days to 120 days, or for at least about 6 days to 180 days, from first contact with a solid tumor cell, or from administration of the v8 T cell described herein. This persistent solid tumor cell killing activity can be exhibited in vitro, in vivo, or both in vitro and in vivo. 100124) Aspects of the invention can additionally or alternatively include y8 T cells that proliferate in response to contact with cells that exhibit cell surface expression, or overexpression, of the tumor associated antigen (TAA). The cells that exhibit cell surface expression of the tumor associated antigen (TAA) can be normal cells, such as normal endothelial cells. The cells that exhibit cell surface expression, or overexpression, of the tumor associated antigen (TAA) can be solid tumor cells. [n some cases, the proliferation is an innate activity. In some cases, the proliferation is at least in part, significantly (> about 20% or > about 25%), or entirely, due to the presence of a CAR construct having a binding domain that specifically binds the TAA expressed on the surface of the cell. In some cases, the v8 T cells exhibit a greater level of in vitro and / or in vive proliferation as compared to a control v8 T cell. In some cases. the control y& T cell does not comprise a CAR construct. In some cases. the control v6 T cell comprises a CAR construct lacking a binding domain described herein, a hinge region described herein, a transmembrane domain described herein, a signaling domain described herein, and / or a costimulation endodomain described herein.

[60125] In some cases, the proliferation is at least in part, significantly (> about 20 or > about 25%), or entirely, due to the presence of a CAR construct having a binding domain that specifically binds TvrD or an epitope within I'ytD. In some cases, y& ‘I cells exhibiting proliferation in response to contact with a cell that exhibits cell surface expression of TyrD functionally express a TyrD-specific CAR encoded by an isolated nucleic acid described herein.

[00126] v8 T cells described herein can exhibit robust and / or persistent proliferation in a host organism that comprises the cell that exhibits cell surface expression, or overexpression. of the tumor associated antigen (TAA). In some cases, the proliferation can persist for at least about 6 days to 120 days, or for at least about 6 days to [80 days, from first contact with a cell that exhibits ceil surface expression, or averexpression, of the tumor associated antigen (TAA) or from a date of administration of the ¥8 T cell to the host organism. In some cases, the proliferation of a v8 1 cell described herein, or a progeny thereof, in the host organism that comprises the cell that exhibits cell surface expression, or overexpression, of the tumor associated antigen (TAA) can persist for at least about 6 days to 120 days. or for at least about 6 days to 180 days, from first contact with the cell or from the date of first administration of the v8 T cell to the host organism. In some cases, the proliferation in the host organism is at least in part, significantly (> about 20% or > about 25%), ot entirely, due to the presence of a CAR construct having a binding domain that specifically binds TyrD or an epitope within TyrD. In some cases, vo T cells exhibiting proliferation in the host organism comprising a cell that exhibits cell surface expression of TyrD functionally express a TyrD specific CAR encoded by an isolated nucleic acid described herein. 100127} Tn some embodiments, the yd T cells described herein express, or persistently express, pro-inflammatory cytokines such as tumor necrosis factor alpha or interferon gamma afier surface of the cell. In some embodiments, the v8 T cells described herein, or progeny thereof, express, or persistently express, pro-inflammatory cytokines such as tumor necrosis factor alpha or interferon gamma after contact with the cell that expresses or over expresses TyrD or a peptide fragment thereof on the surface of the cell. e.g., in a host organism comprising the cell that expresses or over expresses TyrD or a peptide fragment thereof on the surface of the cell.

[00128] In some embodiments, the v8 T cell. or a pharmaceutical composition containing the v8 T cell, exhibits essentially no, or no graft versus host response when introduced into an allogeneic host. In some embodiments, the v8 T cell, or a pharmaceutical composition containing the v8 T cell, exhibits a clinically acceptable level of graft versus host response when introduced into an allogeneic host. In some embodiments, a clinically acceptable level is an amount of graft versus host response that does not require cessation of a yd I cell treatment to achieve a therapeutically effective treatment. In some embodiments, a clinically acceptable level of graft versus host response (GvHD) is an acute response that is less severe than Grade C according to an applicable IBMTR grading scale. The severity of acute graft versus host response is determined by an assessment of the degree of involvement of the skin, liver, and gastrointestinal tract. The stages of individual organ involvement are combined to produce an overall grade, which has prognostic significance. Grade 1(A) GvHD is characterized as mild disease, grade 11(B) GvHD as moderate, grade 11[{C) as severe, and grade [V(D) life-threatening. The [BMTR grading system defines the severity of acute GvHD as follows (Rowlings et al., Br J Haematol 1997; 97:855): oGrade A — Stage | skin involvement alone (maculopapular rash over 25 percent of the body) with no liver or gastrointestinal involvement eGrade B — Stage 2 skin involvement; Stage 1 to 2 gut or liver involvement oGrade C — Stage 3 involvement of any organ system (generalized erythroderma; bilirubin 6.1 to 15.0 mg / dL; diarrhea 500 to 2000 ml. / day) oGrade D — Stage 4 involvement of any organ system {generalized erythroderma with bullous formation; bilirubin >15 mg / dL; diarrhea >2000 mL / day OR pain OR ileus) See also, Schoemans et al., Bone Marrow Transplantation volume 53, pages!401-1415 (2018), e.g., at Tables 1 and 2, which discloses criteria for assessing and grading acute GVHD.

[60129] In some embodinients, the ¥3 T cell. or a pharmaceutical composition containing the v3 T cell, exhibits reduced or substantially reduced grafl versus host response when introduced into an allogeneic host as compared to a graft versus host response exhibited by control aff T cells, or a control pharmaceutical composition comprising the control af} T cells, administered to an allogeneic host. Tn some cases, the control afd T cell is an allogeneic non-engineered control afd T cell. In some cases, the control off T cell does not comprise a CAR or does not comprise the same CAR as a reference yd T cell 100130] The v8 T cells described herein can be 81, 82, 83, ar 54 v8 T cells, or combinations thereof. In some cases, the vd T cells are mostly (50%), substantially (>90%). essentially all, or entirely 82° v8 T cells. In some cases, the vd T cells are mostly (>50%), substantially (>90%), essentially all, or entirely 81 v8 T cells.

[00131] +8 T cells can be obtained from an allogencic or an autologous donor. The v8 1 cells can be, partially or entirely purified. or not purified, and expanded ex vivo. Methods and compositions for ex vivo expansion include, without limitation, those described in WO 2017 / 197347 The expansion may be performed before or afier, or before and after, a CAR construct is introduced into the v8 T cell(s).

[00132] +d T cells described herein can be stored, e.g.. cryopreserved, for use in adoptive cell transfer. Methods of Inhibiting or Killing Tumor Cells

[00133] One or multiple non-engineered, v8 T-cell populations. engineered, y§ T- cell populations, and / or admixtures thereof, having cytotoxic activity against a solid tumor cell can be administered to a subject in any order or simultaneously. Tf simultaneously, the multiple non- ehgineered, v8 T-cell population, engineered, y8 T-cell population, and / or admixtures thereof, of the invention can be provided in a single. unified form, such as an intravenous injection, or in multiple forms, for example, as multiple intravenous infusions, s.c, injections or pills. The non- engineered. v6 T-cell population, engineered, v8 T-cell population, and / or admixtures thereof, of’ the invention can be packed together or separately, in a single package or in a plurality of packages. One or all of the non-engincered v8 T-cell population, engineered v8 T-cell population, and / or admixtures thereof, of the invention can be given in multiple doses. If not simultaneous, the timing between the multiple doses may vary to as much as about a week, a month, two months, three months, four months, five months, six months. or about a year. In some cases, a non~engineered, enriched vad T-cell population, an engineered, enriched v8 T-cell population, and / or admixtures thereof, of the invention can proliferate within a subject's body, in vivo, atter administration to a subject. One or more non-engineered v& T-cell populations, one or more engineered y§ T-cell populations, and / or admixtures thereof, can be frozen to provide cells for multiple treatments with the same cell preparation. One or more nan-engincered v8 T-cell populations, one or more engineered yd T-cell populations, and / or admixtures thereof, of the disclosure, and pharmaceutical compositions comprising the same, can be packaged as a kit. A kit may include instruclions (e.g. written instructions) on the use ol the non-engineered v8 T-cell population, the engineered v8 T-cell population, and / or admixtures thereof, and compositions comprising the same.

[00134] [n some cases, a method of treating a solid cancer comprises administering to a subject a therapeutically-effective amount of a non-engineered y8 T-cell population, an engincered yd T-cell population, and / or admixtures thereof, wherein the administration treats the solid cancer. In some embodiments the therapeutically-effective amount of the non-engineered, vd T-cell population, the engineered ¥3 T-cell population, and / or admixtures thereof, is administered for at least about 10 seconds, 30 seconds, 1 minute, 10 minutes, 30 minutes, 1 hour. 2 hours, 3 hours, 4 hours, 5 hours, 3 hours, 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, § days, 1 week, 2 weeks, 3 weeks, | month, 2 months, 3 months, 4 months, 5 months, 6 months, or 1 year. In some embodiments the therapeutically-effective amount of the non-engineered yd T- cell population. the engineered ¥8 T-cell population, and / or admixtures thereof, is administered for at least one week. In some embodiments the therapeutically-effective amount of the non- engineered v8 T-cell population, the engineered v8 T-cell population, and / or admixtures thereof, is administered for at least two weeks.

[00135] A non-engineered v8 T-cell population, an engineered v8 T-cell popwlation, and / or admixtures thereof. described herein can be administered before, during, or after the occurrence of a disease or condition, and the timing of administering a pharmaceutical composition containing the y3 T-cell population can vary. For example, the v8 T-cell population can be used as a prophylactic and can be administered continuously to subjects with a propensity to conditions or diseases in order to lessen a likelihood of the occurrence of the disease or condition. The initial administration can be via any route practical. such as by any route described herein using any formulation described herein. In some examples, the administration of a v8 T-cell population of the disclosure is an intravenous administration. One or multiple dosages of the y8 T-cell population can be administered as soon as is practicable after the onset of a solid cancer and for a length of time necessary for the treatment of the immune disease, such as, for example, from about 24 hours to about 48 hours, from about 48 hours to about 1 week, from about 1 week to about 2 weeks, from about 2 weeks to about | month. from about 1 month 10 about 3 months. In some embodiments. one ur multiple dosages of the v8 T-cell population can be administered years afier onset of the cancer and before or after other treatments.

[00136] In some embodiments, the y8 T-cell population is administered simultaneously or sequentially with one or more methods to elevate common gamma chain cytokine(s). As used herein, “one or more methods to elevate common gamma chain cytokine(s): refers to a method, or combination of methods. that alters the physiological state of a subject. such that at least one common gamma chain cytokine level is elevated in the subject. In some embodiments, the method elevates the level of one or more common gamma chain cytokine(s) selected from the group consisting of 11-2, 11.-7, and IL-1, preferably wherein the method elevates the level of [L-15 in the subject. In some embodiments. the method comprises lymphodepletion. In some embodiments, the method comprises administering one or more common gamma chain cytokine(s) to the subject. In some cases, IL-2, IL-7, and / or 1L-15, preferably 1L-15, are administered, In some embodiments, the method comprises secreting common gamma chain cytokine(s) from an administered, e.g. v8 T, cell. In some cases, II.-2, I.-7, and / or IL-15, preferably IL-135, are secreted. 100137] In some embodiments, the administering one or more niethods to elevate common gamma chain eytokine(s) comprises lymphodepletion before introducing the ¥8 T cell(s). In some embodiments, the administering one or more methods to elevate common gamma chain cytokine(s) comprises administering simultaneously with introducing the y8 T cells) or sequentially an amount of common gamma chain cytokine(s) effective to increase proliferation, cytotoxic activity, persistence, or the combination thereof of the introduced v8 T cell(s), preferably wherein the method comprises administering 1L-2 or one or more mimetics thereof, more preferably wherein the method comprises administering IL-15 or one or more mimetics thereof. The amount of administered common gamma chain cvtokine(s) can be an amount effective to increase proliferation, cytotoxic activity, persistence, or the combination thereof of the introduced v8 T cell(s) before and / or after introducing the v& T cell(s). Exemplary amounts of IL-15 include, without limitation between 0.01 — 10 pg / kg / dose every 24 hours. Exemplary amounts of IL-2 include, without limitation. between about 3x 108 and about 22x 10¢ units every 8 - 48 hours. For example, the dosing regimen for IL2 in RCC is 600,000 Internationa! Units / kg (0.037 mg / kg) 1V g8hr infused over 15 minutes for a maximum 14 doses.

[60138] In some embodiments, the administering one or more methods to elevate common gamma chain cytokine(s) comprises lymphndepletion before administering the v8 1 cell(s) and administering simultaneously with introducing the v8 T cell(s) or sequentially an amount of common gamma chain cytokine(s) effective to increase proliferation, cytotoxic activity, persistence, or the combination thereof of the introduced v8 T celi(s) EXAMPLES

[00139] Example 1

[00140] Human PBMCs at 1x105mL in a modified culture media were activated on pre- coated with anti=-V81 antibody D1-08 or D1-35 for 5 days in the presence of IL-2 (100 U / mL) in 24-well plates (Costar). On day 3, cell cultures were transduced with y-retroviral constructs encoding an anti-TyrD chimeric antigen receptor (SEQ ID NO:8) in the presence of retronectin. On day 6 cells were returned to the modified culture media and further expanded with feeding and IL-2 replacement as needed. On days 17, 18 or 19, cells were harvested, and remaining aff T cells were depleted using AutoMACS® kit (Miltenyi Biotee). Purity of 8 cell population and transduction efficiency was assessed by FACS. In parallel, untransduced cell cultures were expanded in the same manner. without adding the retroviral supernatant. As shown in Fig. 2, untransduced expanded V31 cells elicited some degree of cytotoxicity against 526 and WM266.1-Luc melanoma cel line that are known 10 express Tyrosinase and present the Tyr369- 377 peptide. This cytotoxicity was augmented by introduction of an anti-TyrD CAR. Cytotoxicity was determined by total luminescence measurement in 96-well plates, by adding luminescent substrate D-Luciferin (Perkin Elmer) after 18 hr co-incubation at inidcated E / T ratios [00141} Example 2 [00142} WM266.4-Luc cells (4x10° per animal) were subcutaneously implanted into NSG mice (Jackson Labs). When tumors reached 100-200 mm? size, animals were treated with 6x10¢ anti-TyrD CAR+ Vil cells. Animals were dosed concomitantly with TL-2 (60,000 1J / dose) 3 times a week throughout the study. The results are illustrated in Fig. 3. As shown in Fig. 3. the animals adminisicred the anti-TyrD CAR+ V81 cells exhibited robust control of tumor burden,

[60143] Example 3

[00144] Tyr CAR constructs were introduced into V81 T cells as described above and cells were expanded and tested in cytotoxicity assay against WM266.4-Luc cells. Control, non-TyrD targeting CAR constructs were used as control, The results are illustrated in Fig. 5 and show the increased cytotoxicity afforded by the anti-TyrD) CAR constructs. 100145] Example d

[00146] Human PBMCs at | x 10° / mL. in growth media were activated in a 24-well plate (Costar) pre-coated with anti-V8! antibady D1-08 or D1-35 for 5 days in the presence of IL-2 (100 U / mL). On day 3, cell cultures were transduced with y-retroviral constructs encoding anti- GPC3 chimeric antigen receptor (SEQ ID NO: 20 (GC33 CAR) or SEQ ID NO: 22 (GC33 CAR + sIL.15 and GC33 CAR + CO slIL15) in the presence of retronectin. GC33 CAR is encoded by the nucleic acid sequence of SEQ ID NO:21; GC33 CAR + sll. 13 is encoded by the nucleic acid sequence SEQ ID NO: 23; GC33 CAR + CO sIL15 includes a codon optimized sIL15 encoding region and is encoded by the nucleic acid sequence of SEQ ID NO: 24, On day 6 cells were returned to growth media and further expanded with feeding and 11.-2 replacement as needed. On days 17, 18 or 19, cells were harvested, and remaining off T cells were depleted using AutoMACS® kit (Miltenyi Biotec). Purity of vd cell population and transduction efficiency was assessed by FACS (Fig. 6). Briefly, CAR-T cells were stained by incubating cells with 1 pg / mL of soluble recombinant biotinylated GPC3 (R&D Systems). Detection of binding was performed using streptavidin-PE at the manufacturer-suggested dilution of 1:500.

[00147] In parallel, untransduced cell cultures were expanded in the same manner, without adding the retroviral supernatant. Expanded cells were tested in the in vitro cytotoxicity assay on GPC3 positive (HepG2, Hep3B, PLC / PRF / 5). As shown in Fig. 7, untransduced expanded V31 cells elicit some degree of cytotoxicity against liver cancer cell lines that are known to express GPC3. This cytotoxicity is potentiated by introduction of GPC3 CAR, with or without sIL15 cytokine engineered to he expressed in tandem. Cytotoxicity was determined by total luminescence measurement in 96-well plates, by adding luminescent substrate D-Luciferin (Perkin Elmer) after 18 h co-incubation at indicated E / T ratios. * % %

[00148] The preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles and aspects of the invention as well as specitic examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary aspects shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims.

Claims

Claims:

1. An isolated nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises a. a binding domain that specifically binds 10 a protein-peptide complex comprising a tumor associated antigen (TAA) peptide and an MHC protein, wherein the complex is expressed on a surface of a solid tumor cell, optionally wherein the binding domain binds the complex in an HLA restricted manner; b. a CD8a hinge domain; £s a CD8o transmembrane demain; d. a costimulatory signaling region selected from a 4-1BB costimulatory signaling region and a CD27 costimulatory signaling region; and e. a CD3{ signaling domain 2. The isolated nucleic acid sequence of claim 1, wherein the (a)-(e) are in 5" to 3° order.

3. The isolated nucleic acid sequence of claim 1 or 2, wherein the TAA comprises a contiguous region of TyrD.

4. The isolated nucleic acid sequence of claim 3, wherein the contiguous region of TyrD comprises at least, or at least about. 4 and no more than, or no more than about, 12 contiguous amine acids of TyrD, preferably, or preferably about. 7, 8, or 9 contiguous amino acids of TyrD.

5. The isolated nucleic acid sequence of claim 4, wherein the contiguous region of TyrD is TyrDs3eo-377.

6. The isolated nucleic acid sequence of any one of claims 1 to 5. wherein the binding domain that specifically binds the TAA peptide MHC complex specifically binds HLA- A2 / TyrDiso.

377. 7 The isolated nucleic acid sequence of any one of claims 1 to 6, wherein the binding domain specifically binds to the epitope bound by, or competes with, an antibody comprising: a. a CDRHI comprising TSGMGVS (SEQ ID NO: 33); b. a CDRH2 comprising HIYWDDDKRYNPSLKS (SEQ ID NO: 34): Cc. a CDRH3 comprising KDYGSSFYAMHY (SEQ ID NO: 35); d. a CDRL comprising KASQDIHNYIA (SEQ 1D NO: 36); e. a CDRL1 comprising YTSTLQP (SEQ ID NO: 37); and f. a CDRL2 comprising LQYDNLWT (SEQ 1D NO: 38).

8. An isolated nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises a. a binding domain that specifically binds to a tumor associated antigen (TAA) expressed on a surface of a solid tumor cell, optionally wherein the antigen is a protein-peptide complex, wherein the protein is an MHC protein, wherein the binding domain binds the protein-peptide complex in an HLA restricted manner; b. a CD8u hinge domain; Cc. 4 CD&u transmembrane domain; d. a costimulatory signaling region selected from a 4-1BB costimulatory signaling region and a CD27 costimulatory signaling region; and e. a CN3{ signaling domain 3. The isolated nucleic acid sequence of claim 8. wherein the binding domain specifically binds an epitope within GPC3 expressed on the surface of a solid tumor cell.

10. The isolated nucleic acid sequence of claim 9, wherein the binding domain comprises the following complementarity determining regions (CDRs) binds the same GPC3 epitope as an antibody comprising the following CDRs, and / or competes for binding to an epitope of GPC3 with an antibody comprising the following CDRs: a. a CDRH] comprising a sequence of DYEMH (SEQ ID NO: 39) (or GYTFTDYEMH (SEQ ID NQ: 40): b. a CDRH2 comprising a sequence of ALDPKTGDTAYSQKFKG (SEQ ID NO: 41); c. a CDRH3 comprising a sequence of FYSYTY (SEQ ID NO: 42); d. a CDRLIcomprising a sequence of RSSQSLVHSNRNTYLH (SEQ ID NO: 43); e. a CDRL2 comprising a sequence of KVSNRFS (SEQ 1D NO: 44); and / or a f. CDRI.3 comprising a sequence of SQNTHVPPT (SEQ ID NO: 45), 11. The isolated nucleic acid sequence of any one of claims 1 to 10, wherein the CAR comprises: a. a CD8a hinge domain comprising SEQ ID NO: | {PTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY) or SEQ ID NO:2 (TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY); b. a CD8¢ transmembrane domain comprising SEQ ID NO:3 (IWAPLAGTCGVLLLSLVITLYC); and / or Cc. a CD3L signaling domain comprising: (i) SEQIDNO:4 (RVKFSRSADAPAYQQGONQLYNELNLGRREEYDVL DKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMA EAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDA LHMQALPPR); or (iy SEQIDNO:5 (RVKFSRSADAPAYQQGQONQLYNELNLGRREEYDVL DKRRGRDPEMGGKPRRENPQEGLYNELQKDKMAE AYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDAL HMQALPPR).

12. The isolated nucleic acid sequence of claim 11, wherein the CAR comprises: a. a 4-1BB costimulatory signaling region comprising SEQ [D NO:6 (KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL): or b. a CD27 costimulatory signaling region comprising SEQ ID NO:

7. (QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP), or wherein the isolated nucleic acid encodes the 4-1BDB costimulatory signaling region comprising SEQ 1D NO:6 and the CD27 costimulatory signaling region comprising SEQ ID NO:

7.

13. The isolated nucleic acid sequence of any one of claims 1 to 12, wherein the nucleic acid further encodes: a. a secreted cytokine: or b. a secreted common gamma chain interleukin, or Bs a secreted 11-15, preferably wherein the 1-15 comprises the sequence of SEQ ID NO:

14. more preferably wherein the IL-15 comprises the sequence of SEQ ID NO: 14 operably linked to a secretion signal sequence of SEQ ID NO: 12, or wherein the IL-5 comprises the sequence of SEQ ID NO: 14 operably linked to a secretion signal sequence of SEQ ID NO: 26; or d. a secreted common gamma chain interleukin, preferably [L-15, and a multi-cistronic linker region amino terminal to the interleukin or interteukin secretion signal, preferably wherein the multicistronic linker region comprises a sequence of any one of SEQ ID NOs: 15- 17,25, or 27-30, or a combination thereof, or encodes an internal ribosome entry site, e.g., SEQ 1D NO: 31 or 32.

14. The isolated nucleic acid sequence of claim 13, whetein: a. the secretion signal comprises a sequence of SEQ ID NO: 12 or SEQ ID NO:

26. preferably SEQ 11) NO:12; and / or b. the siL.15 domain comprises a sequence of SEQ 1D NO: 14; and / or &. the P2A cleavage sequence comprises a sequence of SEQ 1D NO: 15 or SEQ ID NO:25; and / or d. the furin cleavage sequence comprises a sequence of SEQ ID NO: 16: and / or e. the CAR comprises, in amino to carboxy order, a sequence of SEQ ID NO:

17. SEQ ID NO:

12. and SEQ ID NO:

14.

15. The isolated nucleic acid sequence of any one of claims 1 to 14, wherein: a. the binding domain specifically binds to HLA-A2 / TyrDsg9.377 and the nucleic acid encodes SEQ ID NO: 8, or SEQ ID NO: 18; b. the binding domain specifically binds te-GPC3 and the nucleic acid encodes SEQ ID NO: 20, or 22.

16. The isolated nucleic acid sequence of claim 15, wherein the nucleic acid comprises the sequence of SEQ 11D NO: 9, SEQ 1D NO: 19, SEQ 1D NO: 21, 23, or 24.

17. A polypeptide comprising a chimeric antigen receptor comprising an amino acid sequence encoded by any one of the preceding isolated nucleic acids of claims 1 to 16.

18. A vdT cell comprising a polypeptide according to claim 17, or comprising a nucleic acid encoding a CAR construct according to any one of claims 1 to 16, wherein the v8 T cell functionally expresses the a binding domain of the polypeptide or nucleic acid encoded CAR on the surface of the vo T cell.

19. The v8 T cell of claim 18, wherein the v8 T cell exhibits in vitro and / or in vivo cell killing activity against a solid tumor cell that exhibits cell surface expression of the tumor associated antigen (TAA).

20. The vd T cell of claim 19, wherein the solid tumor cell killing activity of said v3 T cell is greater than an innate level of in vitro and / or in vive solid tumor cell killing activity in a control v8 T cell that does nol comprise a CAR construct.

21. The v8 T cell of claim 20, wherein the v8 T cell exhibils the increased solid tumor cell killing activity against HLA class I* solid tumor cells.

2. The y8 T cell of any one of claims 19 to 21, wherein the solid tumor cell killing activity or increased solid tumor cell killing activity persists for, for about, for at least, or for at least about, 6 days to 180 days after first contact with the solid tumor cell.

23. The vd T cell of any onc of claims 18 to 22, wherein the y8 T cell proliferates in response to contact with a solid tumor cell that exhibits cell surface expression of the tumor associated antigen (TAA).

24. Thevyd T cell of any one of claims 18 to 22, wherein the v8 T cell exhibits increased proliferation in response to contact with a solid tumor cell that exhibits cell surface expression of the tumor associated antigen (TAA) as compared to a control v8 T cell that does not functionally express the nucleic acid encoded CAR on the surface of the v8 T cell.

25. The v8 T cell of any onc of claims 18 to 24, wherein the ¥8 T ccll proliferates in a host organism that comprises the solid tumor cell that exhibits cell surface expression of the tumor associated antigen (TAA).

26. The v3 T cell of any one of claims 18 to 25, wherein the v3 T cell proliferation or increased yd T cell proliferation persists for, for abow, for at least, or for at least about, 6 days to 180 days after first contact with the solid tumor cell.

27. The y8 T cell of any one of claims 18 to 26, wherein the ¥8 T cell expresses pro- inflammatory cytokines comprising tumor necrosis factor alpha or interferon gamma after contact with the solid tumor cell.

28. The v8 T cell of any one of claims 18 to 27, wherein the v8 T cell exhibits reduced, substantially reduced, essentially none. or no graft versus host response when introduced into an allogeneic host in comparison to a graft versus host response exhibited by an af3 T cell administered to an allogeneic host.

29. The v8 T cell of any one of claims 18 to 28, wherein the y3 T cell isa 81, a 82, 4 83, or a 84 v8 T cell, preferably a 82° v8 T cell, more preferably a 81 y8 T cell.

30. A plurality of ¥8 T cells according to any one of claims [8 to 29.

31. The plurality of vd T cells of claim 30. wherein the plurality comprises at least about 108 v8 T cells, preferably from about 10% v8 T cells to about 10'! v8 T cells.

32. The plurality of v8 T cells of claim 30 or 31, wherein the plurality comprises a composition that is at least 60%, 80%, or from about 60% or 80% to about 90% or 95% 81, 82, 83, or 84 v8 T cells, preferably 81 or 82 v8 T cells, more preferably 82798 T cells, most preferably 81 v8 T cells 33. A method of making the v8 T cell of any onc of claims 18 10 29 or a plurality of v8 T cells of any one of claims 30 to 32, wherein the method comprises transfecting v3 T cell(s) with a construct comprising an isolated nucleic acid sequence according to any one of claims 1 to 16.

34. The method of claim 33, wherein the method comprises retroviral transduction, preferably gammaretroviral transduction.

35. The method of claim 33 or 34, wherein the method comprises ex vivo expansion of the v8 T cell(s), wherein the ex vivo expansion is performed before transfection and / or after transfection of the isolated nucleic acid sequence.

36. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and a y3 T cell of any one of ¢laims 18 to 29 or a plurality of y8 T cells of any one of claims 30 to 32.

37. A method of killing a solid tumor cel, the method comprising contacting the solid tumor cell with a rumor cell killing effective amount of a y8 T cell of any one of claims 18 to 29; a plurality of v5 T cells of any one of claims 30 to 32; or a pharmaceutical composition of claim 36.

38. The method of claim 37, wherein the method comprises introducing a therapeutically effective amount of the ¥8 T cellts) or the pharmaceutical composition into a host organism comprising the solid tumor ceil.

39. The method of claim 38, wherein the methed comprises introducing into a host organism comprising the solid tumor cell a therapeutically effective amount of the v5 T cell(s) or the pharmaceutical composition and simultaneously or sequentially administering one or more methods to elevate common gamma chain cytokine(s).

40. The method of claim 39. wherein the administering one or more methods to elevate common gamma chain cytokine(s) comprises administering simultaneously with introducing the v8 1 cell(s) or sequentially an amount of common gamma chain cytokine(s) effective to increase proliferation, cytotoxic activity, persistence, or the combination thereof of the introduced v8 T cell(s), preferably wherein the method comprises administering IL-2, more preferably wherein the method comprises administering 1L-15.

41. The method of claim 40, wherein the one or more methods to elevate common gamma chain cytokine(s) comprise administering an amount of common gamma chain cytokine(s) effective to increase proliferation, cytotoxic activity, persistence, or the combination thereof of the introduced v3 T cell(s) before and / or after introducing the v8 T cell(s).

42. The method of any one of claims 39 to 41, wherein the one or more methods to elevate common gamma chain cytokine(s) comprises lymphodepletion before introducing the v8 T cells).

43. The method of any one of claims 39 to 41, wherein the one or more methods to elevate common gamma chain cytokine(s) comprises secretion of one or more common gamma chain cytokine(s) from the introduced v8 T cell(s). 44, The method of any one of claims 38 to 43, wherein the method reduces the in vive tumor burden in the host organism, and / or increases the mean survival time of the host organism as compared to a control organism, wherein the control organism is not treated with the v8 T cell(s) or the pharmaceutical composition.

45. The method of any one of claims 37 to 44, wherein the method is a method of treating cancer in a subject in need thereof.

46. Usc of a tumor cell killing effective amount of a v8 T cell of any onc of claims 18 to 29; a plurality of ¥8 T cells of any one of claims 30 to 32; or a pharmaceutical composition of claim 36 in the manufacture of a medicament for the weatment of a solid tumor cell cancer in a subject in need thereof.

47. A method of treating cancer in a subject in need thereof, the method comprising administering a therapeutically effective amount of v8 T cells, wherein the cancer comprises solid tumor cells that exhibit cell surface expression of TyrD or GPC3.

48. The method of claim 47, wherein the method comprises simultaneously with the administering of 8 T cells or sequentially, administering one or more methads to elevate common gamma chain cytokine(s).

49. The method of claim 47 or 48, wherein the method comprises performing a plurality of administrations of the v8 T cells, wherein the interval between the plurality of administrations is at least about a week, preferably at least about 2, 3, 4, 5, 6, 7, 8, or 12 weeks, and / or no more than once every 6 or 12 months.

50. A pharmaceutical composition for use in any one of the methods of claims 47 to 49.

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