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

Abstract

To provide compositions and methods to improve the specificity or selectivity of cells.SOLUTION: Aspects of the present invention include compositions and methods for treatment of solid tumors with engineered or non-engineered γδ-T cells. In some embodiments, the γδ-T cells comprise a chimeric antigen receptor (CAR) construct. The CAR construct can comprise an anti-TryD 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 comprise an anti-GPC3 binding domain, a CD8α hinge and transmembrane domain, a costimulatory domain, a CD3ζ signalling domain, a combination thereof, or all thereof.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 739,826, filed October 1, 2018, the contents of which are incorporated herein in their entirety for all purposes.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The above ASCII copy, created on January 8, 2020, is named ADC-0006-PCT_SL.txt and is 48406 bytes in size. [Background technology]

[0003] Adoptive immune cell therapy has undergone continuous iterations for over 30 years, from early approaches focused on basal lymphokine activation and / or tumor infiltration to more recent strategies such as genetically engineering these immune cells to express engineered antigen receptors, such as chimeric antigen receptors (CARs). While there have been some hints and indications of the curative potential of these approaches, much remains to be done. In particular, successful tumor eradication by CAR-T lymphocytes depends on the persistence and effector function of CAR-T cells, and an excess of either can induce graft-versus-host effects in patients. Furthermore, solid tissues, in particular, present challenges due to the lack of available positive stimuli and the presence of an inhibitory environment. Therefore, the art is testing numerous costimulatory strategies for both T cells and NK cells, particularly αβ T cells, with the aim of balancing efficacy and safety. Notably, given the current lack of understanding regarding the costimulatory requirements of γδ T cells compared to αβ T cells, the actual translation of any of these various approaches to γδ T cells is uncertain at best. See, e.g., Ribot et al., "Searching for 'signal 2': costimulation requirements of γδ T cells," Cell. Mol. Life Sci. (2011) 68:2345-2355.

[0004] Thus, there remains a need for improved strategies to improve cell specificity or selectivity, to improve cell safety, e.g., by reducing or avoiding the graft-versus-host (GVH) effect, to improve efficacy against solid tumor cells, e.g., by avoiding suppression of effector functions, and to improve cell activity and / or survival upon administration to a subject. Methods, cells, compositions, kits, and systems that meet such needs are provided. Summary of the Invention

[0005]

[0010] Embodiments of the present invention include an isolated nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises a binding domain that specifically binds a protein-peptide complex comprising a tumor-associated antigen (TAA) peptide and an MHC protein, wherein the complex is expressed on the surface of a solid tumor cell, and optionally, wherein the binding domain comprises an isolated nucleic acid sequence that binds to the complex in an HLA-restricted manner, a CD8α hinge domain, a CD8α transmembrane domain, a costimulatory signaling region selected from a 4-1BB costimulatory signaling region and a CD27 costimulatory signaling region, and a CD3ζ signaling domain.

[0011] Embodiments of the present invention further include unengineered γδ T-cells described herein, as well as engineered γδ T-cells comprising a nucleic acid encoding a CAR construct described herein, wherein the γδ T-cells functionally express the nucleic acid encoding the CAR on their surface.

[0006] Embodiments of the invention further include a plurality of γδ T cells as described herein. Embodiments of the invention further include methods of making a γδ T cell or a plurality of γδ T cells as described herein. Embodiments of the invention further include a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a γδ T cell or a plurality of γδ T cells as described herein. Embodiments of the invention further include contacting solid tumor cells with a γδ T cell as described herein or a plurality of γδ T cells as described herein in an amount effective for tumor cell cytotoxicity.

[0007] 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 the surface of a solid tumor cell, optionally wherein the binding domain binds to the complex in an HLA-restricted manner; (b) a hinge domain, such as a CD8α hinge domain; (c) a transmembrane domain, such as a CD8α transmembrane domain; (d) a costimulatory signaling region or combination of costimulatory signaling regions, optionally wherein the costimulatory signaling region(s) are selected from the 4-1BB (CD137) costimulatory signaling region and the CD27 costimulatory signaling region; and (e) a signaling domain, such as a CD3ζ signaling domain. In some embodiments, the foregoing elements (a) through (e) are encoded in 5' to 3' order on the sense strand of the isolated nucleic acid.

[0008] In some embodiments, the TAA comprises a flanking region of TyrD. In some embodiments, the flanking region of TyrD comprises at least 4, or at least about 4, and no more than 12, or no more than about 12, contiguous amino acids of TyrD, preferably 7, 8, or 9, or preferably about 7, 8, or 9, contiguous amino acids ... 369-377 In some embodiments, the binding domain that specifically binds to a TAA peptide-MHC complex is HLA-A2 / TyrD 369-377 It specifically binds to

[0009] In some embodiments, the binding domain specifically binds to or competes with an epitope bound by an antibody comprising a CDRH1 comprising TSGMGVS (SEQ ID NO: 33), a CDRH2 comprising HIYWDDKRYNPSLKS (SEQ ID NO: 34), a CDRH3 comprising KDYGSSFYAMHY (SEQ ID NO: 35), a CDRL1 comprising KASQDIHNYIA (SEQ ID NO: 36), a CDRL1 comprising YTSTLQP (SEQ ID NO: 37), and a CDRL2 comprising LQYDNLWT (SEQ ID NO: 38).

[0010] In another aspect, the binding domain specifically binds to a tumor-associated antigen (TAA) expressed on the surface of a solid tumor cell, optionally wherein the antigen is a protein-peptide complex, wherein the protein is an MHC protein, and wherein the binding domain binds to 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 CD8α hinge domain; (c) a transmembrane domain, such as a CD8α transmembrane domain; (d) a costimulatory signaling region or a combination of costimulatory signaling regions, optionally wherein the costimulatory signaling region is selected from a 4-1BB (CD137) costimulatory signaling region and a CD27 costimulatory signaling region; and (e) a signaling domain, such as a CD3ζ signaling domain. In some embodiments, the foregoing elements (a)-(e) are encoded in 5' to 3' order on the sense strand of the isolated nucleic acid.

[0011] In some embodiments, the binding domain specifically binds to an epitope in GPC3 expressed on the surface of solid tumor cells. In some embodiments, the binding domain comprises the following complementarity-determining regions (CDRs) that bind to the same GPC3 epitope as an antibody comprising the following CDRs, and / or competes for binding to the GPC3 epitope with an antibody comprising the following CDRs: CDRH1 comprising the sequence of DYEMH (SEQ ID NO: 39) (or GYTFTDYEMH (SEQ ID NO: 40)), CDRH2 comprising the sequence of ALDPKTGDTAYSQKFKG (SEQ ID NO: 41), CDRH3 comprising the sequence of FYSYTY (SEQ ID NO: 42), CDRL1 comprising the sequence of RSSQSLVHSNRNTYLH (SEQ ID NO: 43), CDRL2 comprising the sequence of KVSNRFS (SEQ ID NO: 44), and / or CDRL3 comprising the sequence of SQNTHVPPT (SEQ ID NO: 45).

[0012] In some embodiments of any one of the aforementioned aspects or embodiments of a CAR-encoding nucleic acid described herein, the encoded CAR comprises a CD8α hinge domain comprising SEQ ID NO: 1 (PTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY) or SEQ ID NO: 2 (TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY) or a CD8α transmembrane domain and / or a CD3ζ signaling domain comprising SEQ ID NO: 3 (IWAPLAGTCGVLLLSLVITLYC). In some cases, the CD3 zeta signaling domain comprises the sequence of SEQ ID NO: 4 (RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMG GKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR) or SEQ ID NO: 5 (RVKFSRSADAPAYQQGQNQLYNELNLGRREEYD VLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR).

[0013] In some embodiments, the CAR comprises a nucleic acid sequence encoding 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 a 4-1BB costimulatory signaling region comprising SEQ ID NO: 6 and a CD27 costimulatory signaling region comprising SEQ ID NO: 7.

[0014] In some embodiments as described herein, the isolated nucleic acid encodes a secreted cytokine, or a secreted common gamma chain interleukin, or a secreted common gamma chain interleukin, such as IL-15, preferably wherein the secreted common gamma chain interleukin, such as IL-15, comprises an interleukin polypeptide sequence operably linked to a secretory signal sequence (e.g., a secretory signal of SEQ ID NO: 12 or 26). In some embodiments, the isolated nucleic acid encodes secreted IL-15, preferably wherein the IL-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 the secretory signal sequence of SEQ ID NO: 12, or wherein the IL-15 comprises the sequence of SEQ ID NO: 14 operably linked to the secretory signal sequence of SEQ ID NO: 26. In some cases, the secreted cytokine, common gamma chain interleukin, and / or IL-15 is carboxy-terminally encoded by a binding region, a hinge and transmembrane domain, a signaling domain, and / or a costimulatory endodomain. In some cases, the secreted cytokine, commonly gamma chain interleukin and / or IL-15, is encoded on the sense strand 3' of the binding region, hinge and transmembrane domain, signaling domain and / or costimulatory endodomain.

[0015] In some embodiments, the nucleic acid encodes a multicistronic linker region configured to facilitate translation of the CAR and the secreted cytokine, general gamma chain cytokine, or IL-15 as separate polypeptides. In some embodiments, the multicistronic linker region encodes a self-cleaving sequence and / or a cleavage polypeptide sequence. Optionally, the self-cleaving sequence is a P2A, F2A, T2A, or E2A self-cleaving sequence. Optionally, the cleavage sequence is a furin cleavage sequence. Optionally, the cleavage sequence (e.g., a furin cleavage sequence) is amino-terminal to the self-cleaving sequence. In some embodiments, the multicistronic linker region encodes an internal ribosome entry site. In some embodiments, the nucleic acid encodes an interleukin or cytokine or an interleukin or cytokine secretion signal amino-terminal to the multicistronic linker region, preferably wherein the multicistronic linker region comprises the 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 ID NO: 31 or 32.

[0016] In some embodiments, the secretion signal comprises the sequence of SEQ ID NO: 12 or SEQ ID NO: 26, preferably SEQ ID NO: 12, and / or the sIL15 domain comprises the sequence of SEQ ID NO: 14, and / or the P2A cleavage sequence comprises the sequence of SEQ ID NO: 15 or SEQ ID NO: 25, and / or the furin cleavage sequence comprises the sequence of SEQ ID NO: 16, and / or the CAR comprises, in amino to carboxy order, the sequences of SEQ ID NO: 17, SEQ ID NO: 12, and SEQ ID NO: 14.

[0017] In some embodiments, the binding domain is HLA-A2 / TyrD 369-377and the nucleic acid encodes SEQ ID 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.

[0018] In another aspect, the invention provides a polypeptide comprising a CAR binding domain, such as one of the polypeptides encoded by any one of the preceding nucleic acids, or a polypeptide described herein.

[0019] In another aspect, the invention provides γδ T cells, e.g., comprising a polypeptide as described above or comprising a nucleic acid encoding a CAR described herein, wherein the cells functionally express the binding domain of the polypeptide or the nucleic acid encoding the CAR on the surface of the cell. In some embodiments, the cells exhibit in vitro and / or in vivo cytotoxic activity against solid tumor cells that exhibit cell surface expression of a tumor-associated antigen (TAA). In some embodiments, the solid tumor cytotoxic activity of these cells is greater than the native level of in vitro and / or in vivo solid tumor cytotoxic activity in control cells that do not contain the CAR construct. In some embodiments, the cells are HLA class I + The solid tumor cytotoxic activity against solid tumor cells is increased. In some embodiments, the solid tumor cytotoxic activity or increased solid tumor cytotoxic activity persists for about, at least, or at least about 6 days to 180 days after initial contact with the solid tumor cells.

[0020] In some embodiments, the cells proliferate in response to contact with solid tumor cells that exhibit cell surface expression of a tumor-associated antigen (TAA). In some embodiments, the cells experience increased proliferation in response to contact with solid tumor cells that exhibit cell surface expression of a tumor-associated antigen (TAA) compared to control cells that do not functionally express a nucleic acid encoding a CAR on their surface. In some embodiments, the cells are grown in a host organism comprising solid tumor cells that exhibit cell surface expression of a tumor-associated antigen (TAA). In some embodiments, the cell proliferation or increased cell proliferation of the cells persists for about, at least, or at least about 6 days to 180 days after initial contact with the solid tumor cells. In some embodiments, the cells express one or more proinflammatory cytokines, optionally, wherein the one or more proinflammatory cytokines include tumor necrosis factor alpha and / or interferon gamma, preferably in greater amounts than control cells that do not functionally express a nucleic acid encoding a CAR on their cell surface after contact with the solid tumor cells.

[0021] In some embodiments, the T cells exhibit a reduced, substantially reduced, substantially absent, or abolished graft-versus-host response when introduced into an allogeneic host compared to the graft-versus-host response exhibited by αβ T cells administered to an allogeneic host. In some embodiments, the T cells exhibit a reduced, substantially reduced, substantially absent, or abolished graft-versus-host response when introduced into an allogeneic host compared to the graft-versus-host response exhibited by αβ T cells administered to an allogeneic host. In some embodiments, the T cells are γ T cells. In some embodiments, the T cells are δ T cells. In some embodiments, the T cells are γδ T cells. In some embodiments, the T cells are δ1, δ2, δ3, or δ4 T cells, preferably δ2 - δT cells, more preferably δ1δT cells. In some embodiments, the T cells are δ1, δ2, δ3 or δ4γδT cells, preferably δ2 - γδ T cells, more preferably δ1γδ T cells.

[0022] In another aspect, the invention provides a plurality of any one of the foregoing cells, e.g., γδ, T cells, or a plurality of cells, e.g., γδ, T cells, as described herein. In some embodiments, the plurality is, e.g., 10 8 at least about 10 γδ T cells, etc. 8 of cells, preferably about 10 of e.g., γδ T cells 8 cells, e.g., about 10 of γδ T cells 11 In some embodiments, the plurality comprises at least 60%, 80%, or about 60% or 80% to about 90% or 95% δ1, δ2, δ3, or δ4 cells, e.g., γδ T cells, preferably δ1 or δ2 γδ T cells, more preferably δ2 - The composition comprises γδ T cells, most preferably δ1 γδ T cells.

[0023] In some embodiments, the invention provides methods of producing a cell, e.g., a γδ, T cell, or a plurality of cells, e.g., γδ, T cells, as described herein, wherein the method comprises transfecting a cell(s) with a construct comprising an isolated nucleic acid sequence as described herein. Optionally, the method comprises, e.g., gamma, retroviral transduction. Optionally, the method comprises expanding the cell(s) ex vivo, wherein the ex vivo expansion is performed before and / or after transfection with the isolated nucleic acid sequence. Optionally, the method comprises expanding the cell(s) ex vivo, wherein the ex vivo expansion is performed before and after transfection with the isolated nucleic acid sequence. Optionally, the method comprises expanding the cell(s) ex vivo, wherein the ex vivo expansion is performed after transfection with the isolated nucleic acid sequence. In some embodiments, the method produces approximately 10 T cells, e.g., γδ, T cells, etc., that functionally express a CAR described herein within about 30 days of transfection. 8From cells, for example, γδ and T cells, about 10 11 The method includes producing cells of the present invention.

[0024] In another aspect, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a cell or a plurality of cells as described herein, e.g., a γδ, T cell(s) described herein.

[0025] In another aspect, the invention provides a method of killing solid tumor cells, the method comprising contacting a solid tumor cell with a tumor cell-killing effective amount of the cell or cells or pharmaceutical composition described above, or a cell or cells or pharmaceutical composition as described herein. In some cases, the cell or cells are, for example, gamma delta, T cell(s).

[0026] In some embodiments, the method comprises introducing a therapeutically effective amount of cells, e.g., γδ, T cell(s), or a pharmaceutical composition into a host organism that contains solid tumor cells. In some embodiments, the method comprises introducing a therapeutically effective amount of cells, e.g., γδ, T cell(s), or a pharmaceutical composition thereof into a host organism that contains solid tumor cells and simultaneously or sequentially administering one or more methods that elevate general gamma chain cytokine(s).

[0027] In some embodiments, the one or more administration methods for increasing general gamma chain cytokine(s) include administering an effective amount of general gamma chain cytokine(s) simultaneously with or consecutively with the introduction of the cell(s) to increase the proliferation, cytotoxic activity, persistence, or a combination thereof, of the introduced cell(s), preferably wherein the method comprises administering IL-2, and more preferably wherein the method comprises administering IL-15. In some embodiments, the one or more administration methods for increasing general gamma chain cytokine(s) include administering an effective amount of general gamma chain cytokine(s) before and / or after the introduction of the cell(s) to increase the proliferation, cytotoxic activity, persistence, or a combination thereof, of the introduced cell(s).

[0028] In some embodiments, the one or more methods of elevating general gamma chain cytokine(s) comprise lymphodepletion prior to introducing the γδ T cell(s). In some embodiments, the one or more methods of elevating general gamma chain cytokine(s) comprise secretion of one or more general gamma chain cytokine(s) from the introduced cell(s). In some embodiments, the method reduces in vivo tumor burden and / or increases mean survival time of the host organism compared to a control organism, wherein the control organism is not treated with the cell(s) or pharmaceutical composition. In some embodiments, the method is a method of treating cancer in a subject in need of treatment.

[0029] In another aspect, the invention provides the use of any one of the above-described cells or cells as described herein (e.g., γδ T cells), a plurality of such cells, or a pharmaceutical composition comprising such cells in the manufacture of a medicament for treating solid tumor cell cancer in a subject in need thereof, in an amount effective for tumor cell killing. In another aspect, the invention provides a method of treating cancer in a subject in need thereof, the method comprising administering a therapeutically effective amount of cells, wherein the cancer comprises solid tumor cells that exhibit cell surface expression of TyrD or GPC3.

[0030] In some embodiments, the method comprises administering one or more methods that elevate general gamma chain cytokine(s) simultaneously with or sequentially with administration of the cells. In some embodiments, the method comprises administering multiple administrations of the cells, wherein the interval between the multiple administrations is at least about 1 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.

[0031] In another embodiment, the present invention provides a pharmaceutical composition for use in any one of the foregoing methods or methods described herein.

[0032] Incorporation by Reference 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 individually and specifically indicated to be incorporated by reference. [Brief explanation of the drawings]

[0033] [Figure 1]Schematic diagrams of chimeric antigen receptor (CAR) embodiments containing one costimulatory signaling endodomain (left) or two costimulatory signaling endodomains (right) are shown. 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, but are not limited to, CD28, CD137 (4-1BB), CD278 (ICOS), CD27, CD134 (OX40), TLR2, and combinations thereof. [Figure 2] 1 shows the in vitro cytotoxicity of engineered and non-engineered γδ T cells described herein against 526 and WM266.1-Luc melanoma cell lines. [Figure 3] 1 shows the in vivo therapeutic efficacy of γδ T cells described herein in a subcutaneous WM266.4 cell NOD scid gamma (NSG) mouse model. [Figure 4] 1 shows the manufacturing process for producing engineered and non-engineered γδ CAR-T cells, e.g., for treating solid tumors. [Figure 5] Cytotoxic activity of V51 T cells transduced with a control CAR construct or a construct targeting a tyrosinase polypeptide is shown. [Figure 6] Figure 1 shows the transduction efficiency of V51 cells with anti-glypican 3 (GPC3) as soluble IL-15 (sIL15) (SEQ ID NO: 14) and codon-optimized (WO2007 / 037780A2) sIL15. [Figure 7] Figure 1 shows the cytotoxic activity of V51 T cells either untransduced or transduced with an anti-GPC3CAR construct against a panel of liver cancer cell lines with different levels of GPC3 expression. DETAILED DESCRIPTION OF THE INVENTION

[0034] Definition: For purposes of interpreting this specification, the following definitions shall apply, except that where appropriate, terms used in the singular shall 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 control. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0035] As used herein, the term "about," when referring to a measurable value such as an amount, temporal duration, and the like, is intended to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the particular value, as such variations are appropriate for performing the disclosed methods.

[0036] As used herein, the term "γδ T cells (gamma delta T cells)" refers to a subset of T cells that express a distinct T cell receptor (TCR), i.e., γδ TCR, on their surface, which is composed of one γ chain and one δ chain. The term "γδ T cells" includes all subsets of γδ T cells, particularly, but not limited to, Vδ1, Vδ2, and Vδ3 γδ T cells, as well as naive, effector memory, central memory, and terminally differentiated γδ T cells. As a further example, the term "γδ T cells" includes Vδ4, Vδ5, Vδ7, and Vδ8 γδ T cells, as well as Vγ2, Vγ3, Vγ5, Vγ8, Vγ9, Vγ10, and Vγ11 γδ T cells. In some embodiments, γδ T cells are Vδ1 - , Vδ2 - or Vδ1 - and Vδ2 -Compositions and methods for making and using engineered and non-engineered γδ T cells and / or their subtypes include, but are not limited to, those described in US2016 / 0175358, WO2017 / 197347, US9499788, US2018 / 0169147, US9907820, US2018 / 0125889, and US2017 / 0196910, the contents of each of which are incorporated by reference for all purposes, including compositions and methods for making and using engineered and non-engineered γδ T cells and / or their subtypes. The present application further contemplates T cells, or other engineered leukocytes or lymphocytes, that express one gamma chain or one delta chain, optionally in combination with a second polypeptide to form a functional TCR. Such genetically engineered leukocytes or lymphocytes that express one gamma chain or one delta chain may be used in the methods or may be present in the compositions described herein.

[0037] 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. T cells that "express" CD3 and TCR have been genetically engineered to eliminate CD3 and / or TCR cell surface expression.

[0038] As used herein, the term "TCR" or "T cell receptor" refers to dimeric heterologous cell surface signaling proteins that form alpha-beta or gamma-delta receptors, or combinations thereof. αβTCRs recognize antigens presented by MHC molecules, whereas γδTCRs are capable of recognizing antigens independently of MHC presentation.

[0039] The term "MHC" (major histocompatibility complex) refers to a subset of genes that code for cell surface antigen-presenting proteins. In humans, these genes are called human leukocyte antigen (HLA) genes. The abbreviations MHC or HLA are used interchangeably herein.

[0040] As used herein, "activated" refers to a state of T cells that have been sufficiently stimulated to induce detectable cell proliferation. Activation may also be associated with induced cytokine production and detectable effector function. The term "activated T cells" refers, inter alia, to T cells that are undergoing cell division.

[0041] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to an antigen. Antibodies can be intact immunoglobulins derived from natural or recombinant sources, or can be immunoreactive portions of intact immunoglobulins. Typically, antibodies are tetramers of immunoglobulin molecules. Antibodies of the invention can exist in a variety of forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab, and F(ab)2, 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, NY; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

[0042] The term "antibody fragment" refers to a portion of an intact antibody and refers to the antigen-determining variable region of the intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies formed from antibody fragments.

[0043] As used herein, "antibody heavy chain" refers to the larger of the two polypeptide chains present in antibody molecules in their naturally occurring conformation.

[0044] As used herein, "antibody light chain" refers to the smaller of the two polypeptide chains present in antibody molecules in their naturally occurring conformation. Kappa and lambda light chains refer to the two major antibody light chain isotypes.

[0045] As used herein, the term "synthetic antibody" refers to an antibody made using recombinant DNA techniques, such as, for example, antibodies expressed by bacteriophage as described herein. The term should also be construed to mean an antibody made by synthesizing a DNA molecule encoding the antibody, which DNA molecule expresses an antibody protein or amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence is obtained using synthetic DNA or amino acid sequence techniques available and well known in the art.

[0046] As used herein, the term "antigen" or "Ag" is defined as a molecule that elicits an immune response. This immune response may include either antibody production or activation of specific immunocompetent cells, or both. Those skilled in the art will understand that any macromolecule, including proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. Thus, those skilled in the art will understand that any DNA containing a nucleotide sequence or partial nucleotide sequence encoding a protein that elicits an immune response encodes an "antigen" as the term is used herein. Furthermore, those skilled in the art will understand that an antigen need not be encoded solely by the full-length nucleotide sequence of a gene. It will be readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of one or more genes, and that these nucleotide sequences may be arranged in various combinations to elicit a desired immune response. Furthermore, those skilled in the art will understand that an antigen need not be encoded by a "gene" at all. It will be readily apparent that an antigen can be produced, synthesized, or derived from a biological sample. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0047] The term "epitope" includes any protein, 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, lipid, or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. The equilibrium dissociation constant (K D ) is 10 -6 ~10 -12 If the antibody is in the range of M, then the antibody is said to specifically bind to the antigen.

[0048] As used herein, the term "chimeric antigen receptor (CAR)" may refer to, for example, an artificial T cell receptor, T body, single-chain immunoreceptor, chimeric T cell receptor, or chimeric immunoreceptor, and encompasses genetically engineered receptors that graft artificial specificity onto specific immune effector cells. CARs may be used to confer the specificity of a monoclonal antibody to T cells, thereby enabling the generation of large numbers of specific T cells, for example, for use in adoptive cell therapy. In certain embodiments, CARs direct the specificity of cells to, for example, a tumor-associated antigen. In some embodiments, CARs comprise an intracellular activation domain (which enables T cells to be activated upon binding of the targeting moiety to a target cell, such as a targeted tumor cell), a transmembrane domain, and an extracellular domain that can vary in length, and further comprise a disease or disorder-associated, e.g., tumor-antigen-binding region. In certain aspects, CARs comprise a fusion of a single-chain variable fragment (scFv) derived from a monoclonal antibody fused to a CD3-zeta transmembrane domain and endodomain. Other CAR design specificities may be derived from the receptor's ligand (e.g., peptide) or pattern recognition receptors such as dectin. In certain cases, the spacing of the antigen recognition domain can be altered to reduce activation-induced cell death. In certain cases, the CAR includes domains for adding costimulatory signaling, such as CD3ζ, FcR, CD27, CD28, CD137, DAP10 / 12, and / or OX40, ICOS, TLRs (e.g., TLR2), and the like. In some cases, molecules may be coexpressed with the CAR, such as costimulatory molecules, reporter genes for imaging (e.g., positron emission tomography), gene products that conditionally ablate T cells upon the addition of prodrugs, homing receptors, chemokines, chemokine receptors, cytokines, and cytokine receptors. Furthermore, those skilled in the art will understand that the costimulatory domain need not be encoded solely by the 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 one or more genes, and that these nucleotide sequences may be arranged in various combinations to elicit a desired immune response.

[0049] As used herein, the term "anti-tumor effect" refers to a biological effect that may be manifested by a reduction in tumor volume, a reduction in tumor cell number, a reduction in the number of metastases, an increase in life expectancy, or an improvement in various physiological symptoms associated with a cancerous condition. An "anti-tumor effect" may also be manifested by the ability of the peptides, polynucleotides, cells, and antibodies of the present invention to prevent the development of tumors in the first place.

[0050] The term "autoantigen" as used herein refers to any self-antigen that is mistakenly recognized as foreign by the immune system. Autoantigens include, but are not limited to, cellular proteins, phosphorylated proteins, cell surface proteins, cellular lipids, nucleic acids, and glycoproteins, including cell surface receptors.

[0051] As used herein, the term "autologous" is intended to refer to any material that originates from the same individual into which it is subsequently reintroduced.

[0052] As used herein, the term "allogeneic" is intended to refer to any material derived from an animal that is subsequently introduced into another animal of the same species.

[0053] The term "therapeutically effective amount" refers to an amount of a composition that elicits the biological or medical response of a tissue, system, or subject desired by a researcher, veterinarian, physician, or other clinician. The term "therapeutically effective amount" includes an amount of a composition that, when administered, is sufficient to prevent the onset of, or to alleviate to some extent, one or more of the signs or symptoms of, the disease or disorder (e.g., solid cancer) 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 being treated.

[0054] As used herein, the term "treating" a disease means reducing the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.

[0055] Administration "in combination with" one or more other therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.

[0056] As used herein, the term "pharmaceutically acceptable" refers to a material, such as, but not limited to, a salt, carrier, or diluent, that does not abrogate the biological activity or properties of the compound and that is relatively non-toxic. In other words, the material may be administered to an individual without causing undesired biological effects or interacting in a deleterious manner with any of the components of the composition in which it is included.

[0057] "Encode" refers to the inherent property of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for synthesizing other polymers and macromolecules in a biological process, either having 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 the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, which is the nucleotide sequence identical to that of the mRNA and usually provided in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be said to encode the protein or other product of that gene or cDNA.

[0058] "Isolated" means changed or removed from its natural state. For example, a nucleic acid or 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 a substantially purified form, or it can exist in a non-native environment, such as, for example, a host cell.

[0059] 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 contain introns.

[0060] 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.

[0061] As used herein, the term "specifically binds" with respect to antibodies refers to an antibody that recognizes a specific antigen but does not substantially recognize or bind to other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more other species. However, such cross-species reactivity does not in itself change the classification of the antibody according to its characteristics. In another example, an antibody that specifically binds to an antigen may bind to various alleles of the antigen. However, such cross-reactivity does not in itself change the classification of the antibody according to its characteristics. In some cases, the terms "specific binding" or "specifically binding" may be used in reference to the interaction of an antibody, protein, or peptide with a second chemical species, but this interaction depends on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody is meant to recognize and bind to a specific protein structure rather than a general protein. If an antibody is specific for epitope "A," then in a reaction containing labeled "A" and the antibody, the presence of a molecule containing epitope A (or free, unlabeled A) will reduce the amount of labeled A that binds to the antibody.

[0062] In some embodiments, specific binding is at least about 1x10 -8 M or less (e.g., K D(The smaller the , the stronger the 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. Furthermore, multispecific antibodies that bind to a first antigen and one or more additional antigens, or bispecific antibodies that bind to two different regions of an antigen, are nevertheless considered to be "specifically binding" antibodies as used herein.

[0063] A solid tumor is a tumor that comprises a tumor mass of at least about 10 or at least about 100 tumor cells. Solid tumors include soft tissue tumors, primary solid tumors, or metastatic lesions.

[0064] Examples of solid tumors include sarcomas, adenocarcinomas, and cellular carcinomas of various organ systems, such as those affecting the liver, lung, breast, lymphatic, gastrointestinal (e.g., colon), genitourinary tract (e.g., kidney, urothelial cells), prostate, and pharynx. Adenocarcinomas include malignant tumors such as most colon cancers, rectal cancer, renal cell carcinoma, liver cancer, non-small cell carcinoma of the lung, small intestine cancer, and esophageal cancer. In one embodiment, the cancer is melanoma, such as advanced-stage melanoma. Metastatic lesions of the aforementioned cancers can also be treated or prevented using the methods and compositions of the present invention. Examples of other cancers that may be treated include bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, gastric cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenocortical carcinoma, soft tissue sarcoma, urethral cancer, penile cancer, childhood solid tumors, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) tumors, primary CNS lymphoma, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epithelioid carcinoma, squamous cell carcinoma, environmental cancers including those caused by asbestos, and combinations of these cancers. In preferred embodiments, solid tumor cells express or overexpress TyrD or a fragment thereof. In some embodiments, solid tumor cells express or overexpress HLA:peptide complexes containing a TyrD fragment. In some embodiments, the TyrD fragment is TyrD 369-377In some embodiments, the HLA is a class I HLA, such as HLA-A2. In some embodiments, the solid tumor cells are HLA-A2 / TyrD 369-377 express or overexpress.

[0065] In some embodiments, the solid tumor cells express or overexpress glypican 3 (GPC3). In some embodiments, the solid tumor cells are selected from the group consisting of US7919086, WO2014 / 180306, WO2018 / 019772, WO2016 / 049459, WO2003 / 000883, WO2006 / 046751, WO2007 / 047291, WO2016 / 086813, WO2016 / 047722, WO2016 / 036973, Cancer Res. 2008;68:9832-9838, Proc Natl Acad Sci USA. 2013 Mar 19;110(12):E1083-91, the contents of each of which are incorporated by reference in their entirety for all purposes, particularly the binding domains, antibodies, antibody fragments, complementarity-determining regions, polypeptides comprising the complementarity-determining regions, nucleic acids encoding the complementarity-determining regions, and epitope specificity, as well as assays for determining epitope specificity described therein. In some embodiments, solid tumor cells express or overexpress an epitope of glypican 3 specifically bound by the anti-GPC3 antibody GC33. In some embodiments, the solid tumor expresses or overexpresses an HLA:peptide complex comprising a GPC3 fragment. In some embodiments, the HLA is a class I HLA, such as HLA-A2. In some embodiments, the solid tumor expresses or overexpresses an HLA:peptide complex comprising a GPC3 fragment. In some embodiments, the HLA is a class I HLA, such as HLA-A2. In some embodiments, the solid tumor expresses or overexpresses an epitope of GPC3. 144-152 In some embodiments, the solid tumor expresses or overexpresses an HLA:peptide complex that includes a GPC3 peptide. 298-306 Express or overexpress peptide-containing HLA:peptide complexes. See Oncoimmunology. 2012 Nov 1;1(8):1448-1450.

[0066] An "expression cassette" refers to a nucleic acid comprising expression control sequences operably linked to a nucleic acid encoding a transcript or polypeptide to be expressed. An expression cassette contains sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. An expression cassette can be a component of a vector such as a cosmid, a plasmid (e.g., naked in a liposome or contained in a liposome), or a virus (e.g., a lentivirus, retrovirus, adenovirus, and adeno-associated virus). An expression cassette can be present in a host cell, such as a γδ T cell.

[0067] Ranges: Throughout this disclosure, various aspects of the invention may 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 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within the range such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0068] Chimeric antigen receptor constructs: Aspects of the invention include nucleic acids encoding CARs, as well as constructs and vectors comprising such nucleic acids. In some cases, the nucleic acid is a component of, e.g., a heterologous expression cassette. In some embodiments, the nucleic acid is a component of, e.g., a heterologous retroviral vector. In some embodiments, the nucleic acid is a component of, e.g., a heterologous αβ or γδ T cell, preferably a γδ T cell. In some embodiments, the nucleic acid is a component of, e.g., a heterologous γ + T cells and / or delta +In some embodiments, the nucleic acid is a heterologous, α - T cells and / or β - It is a component of T cells.

[0069] Described herein are nucleic acids encoding a CAR binding domain that specifically binds to a tumor-associated antigen (TAA) expressed on the surface of solid tumor cells. An exemplary TAA is tyrosinase (TyrD) or a peptide fragment thereof. Optionally, the TAA is glypican 3 or a peptide fragment thereof. Optionally, the TAA is a peptide bound to an HLA molecule, such as a class I HLA molecule. Tyrosinase peptides that bind to class I HLA molecules (also referred to interchangeably herein as HLA-restricted tyrosinase epitopes, HLA-restricted tyrosinase epitopes, and MHC-restricted tyrosinase antigens) are derived from the tyrosinase enzyme (Genebank Accession No: NP_000363.1), are typically 8-10 amino acids in length, and bind to the α1-α2 groove of the heavy chain via two or three anchor residues that interact with the corresponding binding pocket in the HLA molecule.

[0070] Tyrosinase, a membrane-bound N-linked glycoprotein, is a key enzyme in melanin synthesis. It is expressed in all normal melanocytes and in nearly all melanoma tumor specimens (H. Takeuchi, et al., 2003; S. Reinke, et al., 2005). Peptides derived from this enzyme are presented on MHC class I molecules and recognized by autologous cytolytic T lymphocytes from 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 on the Istituto Nazionale per lo Studio e la Cura dei Tumori website (www.istitutotumori.mi.it).

[0071] Non-limiting examples of MHC class I restricted tyrosinase antigenic peptides are provided in WO2008 / 120202, which is incorporated herein by reference in its entirety, including Table 139 of WO2008 / 120202. According to some embodiments of the present invention, the tyrosinase antigenic peptide is TyrD 369-377 Binding domains that specifically bind to TyrD, an epitope within TyrD, include, but are not limited to, those that bind in an HLA-restricted manner (e.g., class I HLA), including, but not limited to, those described in WO2016 / 199140, WO2016 / 199141, US9688739, and co-pending application PCT / IB2017 / 053539, the contents of each of which are incorporated by reference in their entirety for all purposes, including, but not limited to, compositions and methods for identifying, making, and using binding domains that specifically bind to TyrD or an epitope within TyrD, in an HLA-restricted or HLA-independent manner.

[0072] GPC3 peptides that bind to class I HLA molecules (also referred to herein as HLA-restricted GPC3 epitopes, HLA-restricted GPC3 epitopes, and MHC-restricted GPC3 antigens) are derived from the glypican 3 protein (Genebank Accession No: NM_001164617.2), are typically 8 to 10 amino acids long, and bind to the α1-α2 groove of the heavy chain via two or three anchor residues that interact with the corresponding binding pocket in the HLA molecule.

[0073] As used herein, a binding domain, CAR, or CAR T cell that specifically binds to TyrD and / or an epitope within TyrD includes, but is not limited to, a binding domain, CAR, or CAR T cell that specifically binds to a TyrD peptide fragment. A binding domain, CAR, or CAR T cell that specifically binds to a TyrD peptide fragment is capable of specifically binding to a reference TyrD peptide fragment in an HLA-restricted manner. Similarly, as used herein, a cell that expresses TyrD on its surface includes a cell that expresses or overexpresses a TyrD peptide fragment on its surface, such as a peptide:HLA complex.

[0074] As used herein, a binding domain, CAR, or CAR T cell that specifically binds to GPC3 and / or an epitope within GPC3 includes, but is not limited to, a binding domain, CAR, or CAR T cell that specifically binds to a GPC3 peptide fragment. A binding domain, CAR, or CAR T cell that specifically binds to a GPC3 peptide fragment is capable of specifically binding to a reference GPC3 peptide fragment in an HLA-restricted manner. Similarly, as used herein, a cell that expresses TyrD on its surface includes a cell that expresses or overexpresses a GPC3 peptide fragment on its surface, such as in a peptide:HLA complex.

[0075] In some embodiments, the binding domain binds to an antigen as expressed as a full-length functional polypeptide on the surface of a cell. In some embodiments, the binding domain binds to an antigen as presented in an MHC:antigen complex. In some embodiments, the binding domain binds to an antigen in an HLA-restricted manner. Binding domains that exhibit specificity for MHC:antigen complexes are described, for example, in WO / 2016 / 199140 and WO / 2016 / 199141.

[0076] In some embodiments, the isolated nucleic acid encodes an anti-TyrD binding domain having a CDRH1 comprising TSGMGVS (SEQ ID NO: 33), a CDRH2 comprising HIYWDDKRYNPSLKS (SEQ ID NO: 34), a CDRH3 comprising KDYGSSFYAMHY (SEQ ID NO: 35), a CDRL1 comprising KASQDIHNYIA (SEQ ID NO: 36), a CDRL1 comprising YTSTLQP (SEQ ID NO: 37), and / or a CDRL2 comprising LQYDNLWT (SEQ ID NO: 38).

[0077] In some embodiments, the isolated nucleic acid encodes an anti-GPC3 binding domain having a CDRH1 comprising DYEMH (SEQ ID NO: 39) (or GYTFTDYEMH (SEQ ID NO: 40)), a CDRH2 comprising ALDPKTGDTAYSQKFKG (SEQ ID NO: 41), a CDRH3 comprising FYSYTY (SEQ ID NO: 42), a CDRL1 comprising RSSQSLVHSNRNTYLH (SEQ ID NO: 43), a CDRL2 comprising KVSNRFS (SEQ ID NO: 44), and / or a CDRL3 comprising SQNTHVPPT (SEQ ID NO: 45).

[0078] The present disclosure also contemplates anti-TyrD binding domains or anti-GPC3 binding domains that compete for binding with the sequences provided herein. Using known methods, it is possible to determine whether an anti-TyrD binding domain binds to the same epitope as a reference antibody or binding domain, or competes for binding with the reference antibody or binding domain. For example, to determine whether a test antibody binds to the same epitope as a reference binding domain, the reference binding domain can be bound to TyrD under saturating conditions. The ability of the test binding domain to bind to a TyrD molecule can then be evaluated. 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 cannot bind to TyrD following saturation binding with the reference binding domain, the test binding domain may bind to the same epitope as the epitope bound by the reference binding domain.

[0079] When a binding domain competes for binding with a reference binding domain, the binding methodology described above is performed in two ways. In the first way, the reference binding domain is allowed to bind to TyrD under saturating conditions, followed by assessing the binding of the test binding domain to the TyrD molecule. In the second way, the test binding domain is allowed to bind to TyrD molecules under saturating conditions, followed by assessing the binding of the reference binding domain to the TyrD molecule. If, in both ways, only the first (saturating) binding domain is able to bind to the TyrD molecule, it is concluded that the test and reference binding domains compete for binding to TyrD. As will be understood by those skilled in the art, a binding domain that competes for binding with a reference binding domain may not necessarily be able to bind to the same epitope as the reference binding domain, but may sterically block the binding of the reference binding domain by binding to an overlapping or adjacent epitope. The methods described above for determining competition and epitope binding with an anti-TyrD binding domain can similarly be applied to anti-TyrD binding domains.

[0080] Two binding domains bind to the same or overlapping epitopes if each competitively inhibits (blocks) the binding of the other to the antigen. That is, a 1-fold, 5-fold, 10-fold, 20-fold, or 100-fold excess of one binding domain inhibits the binding of the other by at least 50%, e.g., 75%, 90%, or even 99%, as measured in a competitive binding assay (see, e.g., Junghans et 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 also 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 also reduce or eliminate binding of the other.

[0081] Further routine experiments (e.g., peptide mutations and binding analysis) can be performed to ascertain whether the observed lack of binding of the test binding domain is indeed due to binding to the same epitope as the reference binding domain, or whether steric hindrance (or other phenomena) is responsible for the observed lack of binding. These types of experiments can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative binding assay available in the art.

[0082] The present disclosure provides antibodies and CARs that have "substantial identity" or "substantial similarity" to the sequences provided herein in the CDR or framework regions. The terms "substantial identity" or "substantially identical," when referring to a nucleic acid or fragment thereof, indicate that when optimally aligned with another nucleic acid (or the complementary strand of another nucleic acid), there is, 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% nucleotide sequence identity as measured by any known sequence identity algorithm, such as FASTA, BLAST, or GAP, as described below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule can, 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.

[0083] When applied to polypeptides, the term "substantial similarity" or "substantially similar" means that two peptide sequences 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 when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights. In some embodiments, residue positions that are not identical differ by conservative amino acid substitutions. A "conservative amino acid substitution" is the replacement of an amino acid residue with another amino acid residue having a side chain (R group) with similar chemical properties (such as charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functional properties of a protein. Where two or more amino acid sequences differ from each other by conservative substitutions, the percent or degree of identity may be adjusted upwards to restore the conservative nature of the substitution. Methods 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 incorporated herein by reference. Examples of amino acid groups having side chains of similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic hydroxyl 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: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine.Instead, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix, as disclosed in Gonnet et al. (1992) Science 256:1443 45, incorporated herein by reference. A "moderately conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0084] Polypeptide sequence identity and / or similarity are typically measured using sequence analysis software. Protein analysis software matches similar sequences, which is used to measure similarity by assigning various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG software includes programs such as GAP and BESTFIT, which can be used with default parameters, to measure 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 its mutant protein. See, for example, GCG Version 6.1. Polypeptide sequences can also be compared using FASTA with default or recommended parameters, i.e., the GCG Version 6.1 program. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the optimal overlap regions between the query and search sequences (Pearson (2000) supra). Sequences can be compared using the Smith-Waterman homology search algorithm, using an affine gap search with a gap opening penalty of 12, a gap extension penalty of 2, and a BLOSUM matrix of 62. Another preferred algorithm for comparing the sequences disclosed herein to databases containing a large number of sequences from various organisms is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-410 and (1997) Nucleic Acids Res. 25:3389-3402, the contents of each of which are incorporated by reference.

[0085] Provided herein are anti-TyrD CARs or anti-GPC3 CARs comprising variants of any of the HCVR, LCVR and / or CDR amino acid sequences disclosed herein with one or more substitutions (such as conservative substitutions). For example, the disclosure includes anti-TyrD CARs having HCVR, LCVR and / or CDR amino acid sequences with, for example, 20 or less, 19 or less, 18 or less, 17 or less, 16 or less, 15 or less, 14 or less, 13 or less, 12 or less, 11 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, or 1 amino acid substitution compared 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 may include 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitution (such as a conservative amino acid substitution) compared to any of the HCVR, LCVR, and / or CDR (e.g., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3) amino acid sequences disclosed herein.

[0086] Similarly, the present disclosure includes anti-GPC3CARs having HCVR, LCVR and / or CDR amino acid sequences with, for example, 20 or less, 19 or less, 18 or less, 17 or less, 16 or less, 15 or less, 14 or less, 13 or less, 12 or less, 11 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, or 1 amino acid substitution compared 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-GPC3CAR may include 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution (such as a conservative amino acid substitution) compared to any of the HCVR, LCVR, and / or CDR (e.g., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3) amino acid sequences disclosed herein.

[0087] Exemplary binding domains described herein typically comprise, from amino-terminus to carboxy-terminus, a heavy chain region followed by a light chain region (VH-VL). Where a particular order of VH and VL regions in a binding domain is explicitly or implicitly described, the disclosure is also understood to describe alternative embodiments in which the order of VH and VL regions is reversed, for example, in a CAR comprising an scFV or scFv binding domain. Thus, a description of a VH-VL order also describes alternative VL-VH orders, for example, in a CAR comprising an scFV or scFv binding domain. Furthermore, a description of a VL-VH order also describes alternative VH-VL orders, for example, in a CAR comprising an scFV or scFv binding domain.

[0088] Generally, the nucleic acids encoding the CARs described herein include an extracellular linker portion that encodes a peptide linker that connects the binding domain to the transmembrane domain. Exemplary linker portions include, but are not limited to, a linker portion encoding a CD8α hinge domain, e.g., SEQ ID NO: 1 (PTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY) or SEQ ID NO: 2 (TTTPAPRP PTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY). Typically, the region encoding the peptide linker (e.g., the CD8α hinge domain) is 3' to the region encoding the binding domain and 5' to the region encoding the transmembrane domain.

[0089] The nucleic acids encoding the CARs described herein comprise a transmembrane domain. The transmembrane domain can link an extracellular antigen-binding domain, e.g., a hinge, to one or more intracellular signaling components. For example, the transmembrane domain can link an antigen-binding domain, e.g., a hinge, to a CD3ζ signaling domain and, optionally, one or two costimulatory endodomains. Exemplary transmembrane domains include, but are not limited to, the CD8α transmembrane domain, e.g., SEQ ID NO: 3 (IWAPLAGTCGVLLLSLVITLYC). Typically, the region encoding the transmembrane domain (e.g., the CD8α transmembrane domain) is 3' to a region encoding a peptide linker (e.g., the CD8α hinge domain) and 5' to a region encoding one or more cytoplasmic domains.

[0090] In some embodiments, the isolated nucleic acid encodes a cytoplasmic region comprising one or more cytoplasmic domains. The region encoding the cytoplasmic region is typically 3' to the region encoding the transmembrane domain. The cytoplasmic domain is typically a signaling domain that provides an activation signal for γδ T cell proliferation, cytotoxic activity, and / or proinflammatory cytokine expression (e.g., TNF-α or IFNγ). An exemplary cytoplasmic domain is the CD3ζ signaling domain. In some embodiments, the CD3ζ signaling domain is or comprises SEQ ID NO: 4 (RVKFSRSADAPAYQQGQNQLYNELNLGR REEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR). In some embodiments, the CD3 zeta signaling domain is or comprises SEQ ID NO: 5 (RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDV LDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR). In some embodiments, the cytoplasmic region comprises multiple (e.g., 2, 3, 4, 5, or 6) signaling domains, such as multiple (e.g., 2, 3, 4, 5, or 6) CD3 zeta signaling domains, each independently selected from SEQ ID NOs: 4 and 5. In some embodiments, the cytoplasmic region comprises multiple (e.g., 2, 3, 4, 5, or 6) non-CD3 zeta signaling domains and a CD3 zeta signaling domain. In some embodiments, the cytoplasmic region comprises a non-CD3 zeta signaling domain and multiple (e.g., 2, 3, 4, 5, or 6) CD3 zeta signaling domains. This includes, but is not limited to, alternative or additional signaling domains.

[0091] The cytoplasmic region may comprise one or more costimulatory endodomains. The region encoding the one or more costimulatory endodomains may be 5' or 3' to the region encoding the signaling domain. In some embodiments, the region encoding the one or more costimulatory endodomains may be 5' to the region encoding the signaling domain. In some embodiments, the region encoding the one or more costimulatory endodomains is 5' to the region of the signaling domain, and the additional region encoding the one or more costimulatory endodomains is 3' to the signaling domain. Exemplary costimulatory endodomains include, but are not limited to, CD28, CD137 (4-1BB), CD278 (ICOS), CD27, CD134 (OX40), and TLR2 costimulatory endodomains, and combinations thereof.

[0092] In some embodiments, additional signaling modalities can be included to increase the proliferation, persistence, and / or cytotoxic activity of the γδ 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 coding region can be linked to the 5' portion of the CAR construct via a T2A linker coding region, such that the common gamma chain cytokine is cleaved from the CAR polypeptide and secreted by the cell.

[0093] In some embodiments, the construct encodes at least one 4-1BB costimulatory endodomain and, optionally, a second costimulatory endodomain selected from a 4-1BB, ICOS, CD28, and CD27 costimulatory endodomain. In some embodiments, the construct encodes at least two 4-1BB costimulatory endodomains or two 4-1BB costimulatory endodomains in combination with one, two, three, or four or more costimulatory endodomains selected from 4-1BB, ICOS, CD28, and CD27. In some embodiments, the 4-1BB costimulatory endodomain comprises SEQ ID NO: 6 (KRGRKKLLYIFKQPFMRPVQTT QEEDGCSCRFPEEEEGGCEL).

[0094] In some embodiments, the construct encodes one CD27 costimulatory endodomain and, optionally, a second costimulatory endodomain selected from 4-1BB, ICOS, CD28, and CD27 costimulatory endodomains. In some embodiments, the construct encodes a CD27 costimulatory endodomain and a 4-1BB costimulatory endodomain. In some embodiments, the construct encodes two CD27 costimulatory endodomains. In some embodiments, the CD27 costimulatory endodomain comprises SEQ ID NO: 7 (QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQED YRKPEPACSP).

[0095] 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 T cell (e.g., CAR-T cell) activation, cytotoxicity, and / or persistence. In some embodiments, the secretion signal is the secretion signal of SEQ ID NO: 26 (MRISKPHLRSISIQKYLCLLNSHFLTEAGIHVFILGCFSAGLPKTEA). In some embodiments, the construct encodes a secretion signal, such as SEQ ID NO: 12, operably linked to facilitate secretion of a general gamma chain cytokine, such as IL-15, or an active fragment thereof, e.g., SEQ ID NO: 14 (NWVNVISDLKKIEDLIQSMHIDATLYT ESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS). Other IL-15 sequences, including codon-optimized nucleic acid sequences encoding sIL15, are disclosed in WO 2007 / 037780. Exemplary general gamma chain cytokines include IL-2 and IL-15. In some embodiments, the general gamma chain cytokine is selected from IL-2, IL-7, and IL-15.

[0096] In some embodiments, the construct encodes one or more multicistronic linker regions, for example, between the signaling domain and / or costimulatory endodomain and the operably linked secretion signal to promote cytokine secretion. A multicistronic linker region is a region of a polypeptide or RNA sequence that facilitates the production of multiple distinct polypeptides from a single transcription product. In some embodiments, the multicistronic linker region encodes a cleavage sequence. Suitable cleavage sequences include self-cleaving sequences, such as P2A, F2A, E2A, or T2A cleavage sequences, and / or sequences that are cleaved by endogenous proteases, such as furin.

[0097] In some embodiments, the cleavage sequence is a P2A cleavage sequence. In some embodiments, the cleavage sequence is a furin cleavage sequence. In some embodiments, the cleavage sequence is a P2A and furin cleavage sequence. In some embodiments, the cleavage sequence is a P2A cleavage sequence of SEQ ID NO: 15 (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 + furin cleavage sequence of SEQ ID NO: 17 (RAKRSGSGATNFSLLKQAGDVEENP GP). In some embodiments, the cleavage sequence is a P2A cleavage sequence of SEQ ID NO: 25 (GSGATNFSLLKQAGDVEENPGP).

[0098] In some embodiments, the cleavage sequence is or comprises the P2A cleavage sequence of SEQ ID NO: 27 (ATNFSLLKQAGDVEENPGP). In some embodiments, the cleavage sequence is or comprises the F2A cleavage sequence of SEQ ID NO: 28 (VKQTLNNFDLLKLAGDVESNPGP). In some embodiments, the cleavage sequence is or comprises the E2A cleavage sequence of SEQ ID NO: 29 (QCTNYALLKLAGDVESNPGP). In some embodiments, the cleavage sequence is or comprises the T2A cleavage sequence of SEQ ID NO: 30 (EGRSLLTCGDVEENPGP). In certain embodiments, multiple self-cleavage sequences may be encoded at the carboxy terminus of the signaling and / or costimulatory domain and the amino terminus of the encoded secreted cytokine (e.g., a general gamma chain cytokine such as IL-15), wherein the multiple self-cleavage sequences are preferably 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-cleaving sequences and one or more sequences cleaved by an endogenous protease are encoded in the constructs described herein, hi certain embodiments, the endogenous protease recognition site is encoded amino-terminally to the self-cleaving sequence.

[0099] In some embodiments, the multicistronic linker region encodes an internal ribosome entry site. An exemplary internal ribosome entry site is encoded by SEQ ID NO: 31().

[0100] Another exemplary internal ribosome entry site is encoded by SEQ ID NO: 32 (AGCAGGTTTCCCCAACTGACACAAAACGTGCAACTTGAAACTCCGCCTGGTCTTTCCAGGTCTAGAGGGGTAACACTTTGTACTGCGTTTGGCTCCACGCTCGATCCACTGGCGAGTGTTAGTAACAGCACTGTTGCTTCGTAGCGGAGCATGACGGCCGTGGGAACTCCTCCTTGGTAACAAGGACCCACGGGGCCAAAAGCCACGCCCACACGGGCCCGTCATGTGTGCAACCCCAGCACGGCGACTTTACTGCGAAACCCACTTTAAAGTGACATTGAAACTGGTACCCACACACTGGTGACAGGCTAAGGATGCCCTTCAGGTACCCCGAGGTAACACGCGACACTCGGGATCTGAGAAGGGGACTGGGGCTTCTATAAAAGCGCTCGGTTTAAAAAGCTTCTATGCCTGAATAGGTGACCGGAGGTCGGCACCTTTCCTTTGCAATTACTGACCAC).

[0101] 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 in iresite.org, those described in WO2018 / 215787, the sequences described in GenBank accession No. KP019382.1, and the IRES elements described in GenBank accession No. LT727339.1, the contents of which are incorporated by reference in their entirety for all purposes, and in particular the internal ribosome entry sites and uses thereof described herein.

[0102] Additional multicistronic linker regions, such as self-cleaving and IRES elements, are disclosed in US2018 / 0360992 and US8865467.

[0103] In some embodiments, the isolated nucleic acid is SEQ ID NO: 8 (MSVPTQVLGLLLLWLTDARCDIQMTQSPSSLSASVGDRVTITCKASQDIHNYIAWYQQKPGKAPKLLIHYTSTLQPGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCLQYDNLWTFGQGTKVEIKRGGGGSGGGGSGGGGQITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCARK DYGSSFYAMHYWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELR hD11 anti-TyrD binding domain (anti-TyrD 369-377 ), which encodes the hD11-CD8-BBz polypeptide, which contains the CD8α hinge and transmembrane region, the 4-1BB costimulatory endodomain, and the CD3ζ signaling domain.

[0104] In some embodiments, anti-TyrD 369-377

[0105] In some embodiments, the isolated nucleic acid comprises a codon-optimized sequence encoding a CD8α hinge region. Exemplary codon-optimized CD8α hinge region nucleic acid sequences include, but are not limited to, SEQ ID NO: 10 (ACCACCACCCCTGCACCAAGGCCCCCGACTCCCGCGCCCACCATCGCGTCACA GCCTCTTAGCCTGCGACCGGAAGCATGCAGACCAGCTGCCGGGGGGGCCGTGCATACGAGAGGTTTGGACTTCGCCTGCGAT). In some embodiments, the CD8α hinge region is encoded by the following SEQ ID NO: 11 (ACCACGACGCCAGCG CCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT).

[0106] In some embodiments, the isolated nucleic acid is SEQ ID NO: 18(*), anti-TyrD hD11 (anti-TyrD 369-377 ) binding domain, CD8α hinge and transmembrane region, 4-1BB costimulatory endodomain, CD3ζ signaling domain, furin-P2A cleavage sequence, and encodes a hD11-CD8-BBz-sIL15 polypeptide comprising a secretion signal operably linked to the IL-15 domain.

[0107]

[0108] In some embodiments, the isolated nucleic acid is SEQ ID NO: 20 (MSVPTQVLGLLLLWLTDARCQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADKSTSTAYMELSSLTSEDTAVYYCTRFYSYTYWGQGTLVTVSSGGGGSGGGGSGGGGDVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNRNTYLHWYLQKPGQSPQLLIYKVSNRFSSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIKTTTPAPRP PTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR*), encoding a polypeptide containing the GC33 anti-GPC3 binding domain, CD8α hinge and transmembrane regions, 4-1BB costimulatory endodomain, and CD3ζ signaling domain.

[0109]

[0110] In some embodiments, the isolated nucleic acid encodes a polypeptide comprising a secretion signal operably linked to SEQ ID NO: 22(*), a GC33 anti-GPC3 binding domain, a CD8α hinge and transmembrane region, a 4-1BB costimulatory endodomain, a CD3ζ signaling domain, a furin and P2A cleavage region, and an IL15 domain.

[0111]

[0112]

[0113] 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 (such as a gammaretroviral vector), or a lentiviral vector. In some embodiments, the isolated nucleic acid, or a contiguous portion of the isolated nucleic acid, e.g., comprising a binding domain, a transmembrane domain, and one or more signaling and / or costimulatory endodomains, is integrated into the genome of a host cell, such as a host γδ T cell. In an exemplary embodiment, the isolated nucleic acid is a retroviral vector.

[0114] γδT cells: An embodiment of the invention includes a γδ T cell that functionally expresses an isolated nucleic acid described herein, thereby expressing a CAR on the surface of the γδ T cell.

[0115] Embodiments of the invention may alternatively or additionally include γδ T-cells that have in vitro or in vivo cytotoxic activity against solid tumor cells that exhibit cell surface expression of a tumor-associated antigen (TAA). In some cases, the cytotoxic activity is innate. In some cases, the cytotoxicity is at least partially, significantly (>about 25%), or entirely due to the presence of a CAR construct having a binding domain that specifically binds to a TAA expressed on the surface of the solid tumor cells. In some cases, the γδ T-cells exhibit solid tumor cytotoxic activity that is greater than the innate level of in vitro and / or in vivo solid tumor cytotoxic activity of control γδ T-cells. In some cases, the control γδ T-cells do not comprise a CAR construct. In some cases, the control γδ T-cells comprise 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 costimulatory endodomain described herein.

[0116] In some cases, cytotoxicity is mediated by TyrD or TyrD 369-377 In some cases, cytotoxicity is at least partially, significantly (> about 25%), or entirely due to the presence of a CAR construct having a binding domain that specifically binds to an epitope within TyrD, such as TyrD. In some cases, cytotoxicity is HLA-restricted (e.g., class I HLA-restricted) and / or TyrD. 369-377 In some cases, cytotoxicity is at least partially, significantly (> about 25%), or entirely due to the presence of a CAR construct having a binding domain that specifically binds to an epitope within TyrD, such as HLA-A2 / TyrD. 369-377 In some cases, the γδ T cells functionally express a CAR encoded by an isolated nucleic acid described herein that specifically binds to TyrD or a peptide fragment thereof.

[0117] In some embodiments, the γδ T cells described herein may exhibit HLA-restricted (e.g., HLA class I-restricted) cytotoxicity. In other embodiments, most (>50%), substantially all (>90%), or all cytotoxic activity is not HLA-restricted (e.g., HLA class I-restricted). HLA-restricted cytotoxic activity can be assessed by comparing in vitro cytotoxicity against HLA (e.g., HLA class I) (null) tumor cell lines with in vitro cytotoxicity against HLA+ (e.g., HLA class I+) tumor cell lines. In some embodiments, HLA-restricted cytotoxic activity is at least partially, significantly (>25%), or completely provided by the use of a T cell receptor-like binding domain. T cell receptor-like binding domains are binding domains that specifically recognize antigens when presented on the surface of cells in complex with MHC molecules. T cell receptor-like binding domains are further described, for example, in WO2016 / 199141.

[0118] The γδ T cells described herein can exhibit potent and / or durable solid tumor cytotoxic activity. In some cases, the solid tumor cytotoxic activity can persist for at least about 6 to 120 days, or at least about 6 to 180 days, from initial contact with solid tumor cells. In some cases, the solid tumor cytotoxic activity of the γδ T cells described herein or their progeny can persist for at least about 6 to 120 days, or at least about 6 to 180 days, from initial contact with solid tumor cells or from administration of the γδ T cells described herein. This durable solid tumor cytotoxic activity can be demonstrated in vitro, in vivo, or both in vitro and in vivo.

[0119] Embodiments of the present invention may alternatively or additionally include γδ T cells that proliferate in response to contact with cells that exhibit cell surface expression or overexpression of a tumor-associated antigen (TAA). The cells that exhibit cell surface expression or overexpression of a tumor-associated antigen (TAA) may be normal cells, such as normal endothelial cells. The cells that exhibit cell surface expression or overexpression of a tumor-associated antigen (TAA) may be solid tumor cells. In some cases, the proliferation is an innate activity. In some cases, the proliferation is at least partially, significantly (>about 20% or >about 25%), or entirely due to the presence of a CAR construct having a binding domain that specifically binds to a TAA expressed on the surface of the cell. In some cases, the γδ T cells exhibit higher levels of proliferation in vitro and / or in vivo compared to control γδ T cells. In some cases, the control γδ T cells do not comprise a CAR construct. In some cases, the control γδ T cells comprise 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 costimulatory endodomain described herein.

[0120] In some cases, proliferation is at least partially, significantly (>about 20% or >about 25%), or entirely due to the presence of a CAR construct having a binding domain that specifically binds to TyrD or an epitope within TyrD. In some cases, γδ T cells that exhibit proliferation in response to contact with cells that exhibit cell surface expression of TyrD functionally express a TyrD-specific CAR encoded by an isolated nucleic acid described herein.

[0121] The γδ T cells described herein can exhibit robust and / or sustained proliferation in a host organism comprising cells exhibiting cell surface expression or overexpression of a tumor-associated antigen (TAA). In some cases, proliferation can persist for at least about 6 to 120 days, or at least about 6 to 180 days, from initial contact with cells exhibiting cell surface expression or overexpression of a tumor-associated antigen (TAA) or from the date of administration of the γδ T cells to the host organism. In some cases, proliferation of the γδ T cells described herein or their progeny in a host organism comprising cells exhibiting cell surface expression or overexpression of a tumor-associated antigen (TAA) can persist for at least about 6 to 120 days, or at least about 6 to 180 days, from initial contact with the cells or from the date of initial administration of the γδ T cells to the host organism. In some cases, proliferation in the host organism is at least partially, significantly (>about 20% or >about 25%), or entirely due to the presence of a CAR construct having a binding domain that specifically binds to TyrD or an epitope within TyrD. In some cases, γδ T cells exhibiting proliferation in a host organism comprising cells exhibiting cell surface expression of TyrD functionally express a TyrD-specific CAR encoded by an isolated nucleic acid described herein.

[0122] In some embodiments, a γδ T cell described herein expresses or persistently expresses a proinflammatory cytokine, such as tumor necrosis factor alpha or interferon gamma, after contact with cells that express or overexpress cell surface TyrD or a peptide fragment thereof. In some embodiments, a γδ T cell described herein expresses or persistently expresses a proinflammatory cytokine, such as tumor necrosis factor alpha or interferon gamma, after contact with cells that express or overexpress cell surface TyrD or a peptide fragment thereof, e.g., in a host organism comprising cells that express or overexpress cell surface TyrD or a peptide fragment thereof.

[0123] In some embodiments, γδ T cells or pharmaceutical compositions comprising γδ T cells, when introduced into an allogeneic host, exhibit substantially no or no graft-versus-host response. In some embodiments, γδ T cells or pharmaceutical compositions comprising γδ T cells, when introduced into an allogeneic host, exhibit a clinically acceptable level of graft-versus-host response. In some embodiments, a clinically acceptable level is an amount of graft-versus-host response that does not require cessation of γδ T cell therapy to achieve therapeutically effective treatment. In some embodiments, a clinically acceptable level of graft-versus-host response (GvHD) is an acute response less severe than Grade C according to the applicable IBMTR grading scale. The severity of acute graft-versus-host response is determined by assessing the degree of skin, liver, and gastrointestinal involvement. The stages of involvement in individual organs are combined to create an overall grade that has prognostic significance. Grade I (A) GvHD is considered mild disease, Grade II (B) GvHD is moderate, Grade III (C) is severe, and Grade IV (D) is life-threatening. The IBMTR grading system defines the severity of acute GvHD as follows (Rowlings et al., Br J Haematol 1997;97:855): Grade A - No liver or gastrointestinal involvement, only stage 1 skin involvement (maculopapular rash on <25% of the body) Grade B - Stage 2 skin lesions, Stage 1 to 2 intestinal or liver lesions Grade C - Stage 3 disease in any organ system (generalized erythroderma, bilirubin 6.1-15.0 mg / dL, diarrhea 1500-2000 mL / day) • Grade D - Stage 4 involvement of any organ system (generalized erythroderma with blister formation, bilirubin >15 mg / dL, diarrhea >2000 mL / day or pain or ileus). See also Tables 1 and 2 in Schoemans et al., Bone Marrow Transplantation volume 53, pages 1401-1415 (2018), which also discloses criteria for assessing and grading acute GvHD.

[0124] In some embodiments, the γδ T cells, or pharmaceutical compositions comprising γδ T cells, exhibit a reduced or substantially reduced graft-versus-host response when introduced into an allogeneic host compared to the graft-versus-host response exhibited by control αβ T cells, or a control pharmaceutical composition comprising control αβ T cells, administered to the allogeneic host. Optionally, the control αβ T cells are allogeneic, non-genetically engineered control αβ T cells. Optionally, the control αβ T cells do not comprise a CAR or do not comprise the same CAR as the reference γδ T cells.

[0125] The γδ T cells described herein can be δ1, δ2, δ3, or δ4 γδ T cells, or a combination thereof. In some cases, the γδ T cells are mostly (>50%), substantially (>90%), essentially all, or entirely δ2 - In some cases, the γδ T cells are mostly (>50%), substantially (>90%), essentially all, or entirely δ1 γδ T cells.

[0126] γδ T cells can be obtained from allogeneic or autologous donors. γδ T cells can be partially or completely purified or unpurified and expanded ex vivo. Methods and compositions for ex vivo expansion include, but are not limited to, those described in WO2017 / 197347. This expansion can be performed before or after, or both, introducing a CAR construct into the γδ T cell(s).

[0127] The γδ T cells described herein can be stored, for example, cryopreserved, for use in adoptive cell transfer.

[0128] Methods for inhibiting or killing tumor cells One or more non-engineered γδ T cell populations, genetically engineered γδ T cell populations, and / or mixtures thereof having cytotoxic activity against solid tumor cells can be administered to a subject in any order or simultaneously. When administered simultaneously, multiple non-engineered γδ T cell populations, genetically engineered γδ T cell populations, and / or mixtures thereof of the present invention can be provided in a single, unified form, such as an intravenous injection, or in multiple forms, such as multiple intravenous infusions, subcutaneous injections, or tablets. Non-engineered γδ T cell populations, genetically engineered γδ T cell populations, and / or mixtures thereof of the present invention can be packaged together or separately in a single package or multiple packages. One or all of the non-engineered γδ T cell populations, genetically engineered γδ T cell populations, and / or mixtures thereof of the present invention can be given in multiple doses. When not administered simultaneously, the timing between multiple doses can vary by about one week, one month, two months, three months, four months, five months, six months, or up to about one year. In some cases, the non-engineered enriched γδ T cell population, the genetically engineered enriched γδ T cell population, and / or a mixture thereof of the present invention may be expanded in vivo within the subject's body after administration to the subject. One or more non-engineered γδ T cell populations, one or more genetically engineered γδ T cell populations, and / or a mixture thereof can be frozen to provide cells for multiple treatments with the same cell preparation. One or more non-engineered γδ T cell populations, one or more genetically engineered γδ T cell populations, and / or a mixture thereof of the present disclosure, as well as pharmaceutical compositions comprising the same, can be packaged as a kit. The kit may include instructions (e.g., written instructions) for using the non-engineered γδ T cell population, the genetically engineered γδ T cell population, and / or a mixture thereof, as well as compositions comprising the same.

[0129] In some cases, the method for treating a solid tumor comprises administering to a subject a therapeutically effective amount of a non-engineered γδ T cell population, a genetically engineered γδ T cell population, and / or a mixture thereof, wherein administering treats the solid tumor. In some embodiments, the therapeutically effective amount of a non-engineered γδ T cell population, a genetically engineered γδ T cell population, and / or a mixture 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, 6 hours, 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or 1 year. In some embodiments, the therapeutically effective amount of a non-engineered γδ T cell population, a genetically engineered γδ T cell population, and / or a mixture thereof is administered for at least 1 week. In some embodiments, a therapeutically effective amount of a non-engineered γδ T cell population, an engineered γδ T cell population and / or a mixture thereof is administered for at least two weeks.

[0130] The non-genetically engineered γδ T cell populations, genetically engineered γδ T cell populations, and / or mixtures thereof described herein can be administered before, during, or after the onset of a disease or symptom, and the timing of administration of a pharmaceutical composition comprising a γδ T cell population can vary. For example, the γδ T cell populations can be used as prophylactic agents and can be administered continuously to subjects exhibiting symptoms or a tendency toward a disease to reduce the likelihood of the disease or symptom occurring. The initial administration can be via any practical route, such as by any route described herein using any formulation described herein. In some embodiments, the γδ T cell populations of the present disclosure are administered intravenously. One or more doses of the γδ T cell populations can be administered as soon as practicable after the onset of a solid tumor, and for a period of time necessary to treat the immune disease, 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 1 month, or from about 1 month to about 3 months. In some embodiments, one or more doses of the γδ T cell population may be administered years after the onset of cancer, and before and after other treatments.

[0131] In some embodiments, the γδ T cell population is administered simultaneously or sequentially with one or more methods for elevating common gamma chain cytokine(s). As used herein, "one or more methods for elevating common gamma chain cytokine(s)" refers to a method or combination of methods for altering the physiological state of a subject such that the level of at least one common gamma chain cytokine 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 IL-2, IL-7, and IL-15, preferably wherein the method elevates the level of IL-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. Optionally, IL-2, IL-7, and / or IL-15, preferably IL-15, are administered. In some embodiments, the method comprises secreting the common gamma chain cytokine(s), such as from the administered γδ T cells. In some cases, IL-2, IL-7 and / or IL-15, preferably IL-15, is secreted.

[0132] In some embodiments, the one or more administration methods for increasing general gamma chain cytokine(s) include lymphodepletion before introducing γδ T cell(s). In some embodiments, the one or more administration methods for increasing general gamma chain cytokine(s) include administering an effective amount of general gamma chain cytokine(s) simultaneously with or consecutively with the introduction of γδ T cell(s) to increase the proliferation, cytotoxic activity, persistence, or a combination thereof, of the introduced γδ T cell(s), preferably wherein the method comprises administering IL-2 or one or more mimetics thereof, more preferably wherein the method comprises administering IL-15 or one or more mimetics thereof. The administration of the general gamma chain cytokine(s) may increase the proliferation, cytotoxic activity, persistence, or a combination thereof, of the introduced γδ T cell(s) before and / or after introducing γδ T cell(s). Exemplary amounts of IL-15 include, but are not limited to, between 0.01 and 10 μg / kg / dose every 24 hours. Exemplary amounts of IL-2 include, but are not limited to, between about 3×10 6 and approximately 22 x 10 6 For example, the dosing regimen for IL2 in RCC is 600,000 International Units / kg (0.037 mg / kg) IV every 8 hours infused over 15 minutes for up to 14 doses.

[0133] In some embodiments, the one or more administration methods that elevate general gamma chain cytokine(s) comprise lymphodepletion prior to administration of γδ T-cell(s), prior to simultaneous administration with the introduced γδ T-cell(s), or prior to sequential administration of general gamma chain cytokine(s) effective to increase proliferation, cytotoxic activity, persistence, or a combination thereof, of the introduced γδ T-cell(s). [Example]

[0134] Example 1 1x106 Human PBMCs (PBMCs) were precoated in 24-well plates (Costar) with anti-Vδ1 antibodies D1-08 or D1-35 at 100 U / mL for 5 days in the presence of IL-2 (100 U / mL) and activated with modified culture medium. On day 5, cell cultures were transduced with a γ-retroviral construct encoding an anti-TyrD chimeric antigen receptor (SEQ ID NO: 8) in the presence of retronectin. On day 6, cells were returned to modified culture medium and further expanded with feeding and IL-2 supplementation as needed. On days 17, 18, or 19, cells were harvested and depleted of remaining αβ T cells using an AutoMACS® kit (Miltenyi Biotec). The purity and transduction efficiency of the γδ cell population were assessed by FACS. In parallel, untransduced cell cultures were grown in the same manner without the addition of retroviral supernatant. As shown in Figure 2, untransduced proliferating Vδ1 cells express tyrosinase and Tyr 369-377 The peptide induced some degree of cytotoxicity in 526 and WM266.1-Luc melanoma cell lines, which are known to present the peptide. This cytotoxicity was enhanced by the introduction of the anti-TyrD CAR. Cytotoxicity was measured by total luminescence measurement in 96-well plates after 18 hours of co-culture at the indicated E / T ratios and addition of the luminescent substrate D-luciferin (Perkin Elmer).

[0135] Example 2 WM266.4-Luc cells (4 x 10 per animal) 6 ) were subcutaneously implanted into NSG mice (Jackson Labs). Tumors were 100–200 mm 3 Once the animal reaches the size of 6x10 6 The animals were treated with anti-TyrD CAR+Vδ1 cells. Animals were simultaneously administered IL-2 (60,000 U / dose) three times a week until the end of the study. The results are shown in Figure 3. As shown in Figure 3, animals receiving anti-TyrD CAR+Vδ1 cells demonstrated robust control of tumor burden.

[0136] Example 3 The Tyr CAR construct was transfected into Vδ1 T cells as described above, and the cells were expanded and tested in a cytotoxicity assay against WM266.4-Luc cells. A control, non-TyrD-targeted CAR construct was used as a control. The results are shown in Figure 5, demonstrating increased cytotoxicity mediated by the anti-TyrD CAR construct.

[0137] Example 4 1x10 cells in growth medium in 24-well plates (Costar) precoated with anti-Vδ1 antibody D1-08 or D1-35 for 5 days in the presence of IL-2 (100 U / mL). 6 1 mL of human PBMCs were activated. On day 5, the cell cultures were transduced with a gamma-retroviral construct encoding an anti-GPC3 chimeric antigen receptor (SEQ ID NO: 20 (GC33CAR) or SEQ ID NO: 22 (GC33CAR+sIL15 and GC33CAR+CO sIL15)) in the presence of retronectin. GC33CAR is encoded by the nucleic acid sequence of SEQ ID NO: 21. GC33CAR+sIL15 is encoded by the nucleic acid sequence of SEQ ID NO: 23, and GC33CAR+CO sIL15 contains a codon-optimized sIL15 coding region and is encoded by the nucleic acid sequence of SEQ ID NO: 24. On day 6, the cells were returned to growth medium and further expanded by feeding and IL-2 supplementation as needed. On days 17, 18, or 19, the cells were harvested and residual αβ T cells were depleted using an AutoMACS® kit (Miltenyi Biotec). The purity and transduction efficiency of the γδ cell population were assessed by FACS (Figure 6). Briefly, CAR-T cells were stained by incubating the cells with 1 μg / mL of soluble recombinant biotinylated GPC3 (R&D Systems). Binding detection was performed using streptavidin-PE at the manufacturer's recommended dilution of 1:500.

[0138] In parallel, untransduced cell cultures were grown in the same manner without the addition of retroviral supernatant. The expanded cells were tested in an in vitro cytotoxicity assay of GPC3-positive cells (HepG2, Hep3B, PLC / PRF / 5). As shown in Figure 7, untransduced expanded Vδ1 cells induce some degree of cytotoxicity against liver cancer cells known to express GPC3. This cytotoxicity is enhanced by the introduction of GPC3CAR, with or without the tandemly engineered sIL15 cytokine. Cytotoxicity was measured after 18 hours of coculture at the indicated E / T ratios, followed by total luminescence measurement in 96-well plates after the addition of the luminescent substrate D-luciferin (Perkin Elmer).

[0139] The foregoing merely illustrates the principles of the present invention. It will be appreciated by those skilled in the art that, although not explicitly described or shown herein, they will be able to devise various configurations which embody the principles of the present invention and are within its spirit and scope. Furthermore, all examples and conditional language recited herein are intended primarily to aid the reader's understanding of the principles of the present invention and the concepts contributed by the inventors to further this technology, and should not be construed as being limited to such specifically recited examples and conditions. Furthermore, all statements herein reciting principles, aspects, and specific examples thereof are intended to encompass both structural and functional equivalents. Furthermore, such equivalents are intended to include both currently known equivalents and equivalents developed in the future, i.e., any elements developed to perform the same function, regardless of structure. Therefore, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention are embodied by the appended claims. SEQUENCE LISTING <110> ADICET BIO, INC. <120> COMPOSITIONS AND METHODS REGARDING ENGINEERED AND NON-ENGINEERED GAMMA-DELTA-T CELLS FOR TREATMENT OF SOLID TUMORS <130> ADC-0006-PCT <140> PCT / US2019 / 054144 <141> 2019-10-01 <150> 62 / 739,826 <151> 2018-10-01 <160> 45 <170> PatentIn version 3.5 <210> 1 <211> 39 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 1 Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro 1 5 10 15 Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu 20 25 30 Asp Phe Ala Cys Asp Ile Tyr 35 <210> 2 <211> 47 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 2 Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 1 5 10 15 Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 20 25 30 Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr 35 40 45 <210> 3 <211> 22 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 3 Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu 1 5 10 15 Val Ile Thr Leu Tyr Cys 20 <210> 4 <211> 113 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 4 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 1 5 10 15 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln 50 55 60 Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu 65 70 75 80 Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr 85 90 95 Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro 100 105 110 Arg <210> 5 <211> 112 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 5 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 1 5 10 15 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 50 55 60 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 65 70 75 80 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 85 90 95 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 110 <210> 6 <211> 42 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 6 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 1 5 10 15 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 20 25 30 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 35 40 <210> 7 <211> 48 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 7 Gln Arg Arg Lys Tyr Arg Ser Asn Lys Gly Glu Ser Pro Val Glu Pro 1 5 10 15 Ala Glu Pro Cys His Tyr Ser Cys Pro Arg Glu Glu Glu Gly Ser Thr 20 25 30 Ile Pro Ile Gln Glu Asp Tyr Arg Lys Pro Glu Pro Ala Cys Ser Pro 35 40 45 <210> 8 <211> 487 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 8 Met Ser Val Pro Thr Gln Val Leu Gly Leu Leu Leu Leu Trp Leu Thr 1 5 10 15 Asp Ala Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser 20 25 30 Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp 35 40 45 Ile His Asn Tyr Ile Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 50 55 60 Lys Leu Leu Ile His Tyr Thr Ser Thr Leu Gln Pro Gly Val Pro Ser 65 70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser 85 90 95 Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp 100 105 110 Asn Leu Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly 115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gln Ile Thr 130 135 140 Leu Lys Glu Ser Gly Pro Thr Leu Val Lys Pro Thr Gln Thr Leu Thr 145 150 155 160 Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser Gly Met Gly 165 170 175 Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu Ala 180 185 190 His Ile Tyr Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ser Leu Lys Ser 195 200 205 Arg Leu Thr Ile Thr Lys Asp Thr Ser Lys Asn Gln Val Val Leu Thr 210 215 220 Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala Arg 225 230 235 240 Lys Asp Tyr Gly Ser Ser Phe Tyr Ala Met His Tyr Trp Gly Gln Gly 245 250 255 Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro 260 265 270 Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu 275 280 285 Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp 290 295 300 Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly 305 310 315 320 Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg 325 330 335 Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln 340 345 350 Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu 355 360 365 Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala 370 375 380 Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu 385 390 395 400 Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp 405 410 415 Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly 420 425 430 Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu 435 440 445 Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu 450 455 460 Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His 465 470 475 480 Met Gln Ala Leu Pro Pro Arg 485 <210> 9 <211> 1464 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 9 atgtccgtgc ctacccaggt gctgggcctg ctgctgctgt ggctgaccga cgccagatgc 60 gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 120 atcacttgca aggcgagtca ggacattcac aactatatag cttggtatca gcagaaacca 180 gggaaagccc ctaagctcct gatccactat acatccactt tgcaaccagg ggtcccatca 240 aggttcagtg gaagtggatc tgggacagat tttactttca ccatcagcag cctgcagcct 300 gaagatattg caacatatta ctgtctacag tatgataatc tctggacgtt cggtcaaggc 360 accaaggtgg aaatcaaacg gggtggaggt ggatctggag gaggaggatc cggtggagga 420 ggtcagatca ccttgaagga gtctggtcct acgctggtga aacccacaca gaccctcacg 480 ctgacctgca ccttctctgg gttctcactc agcactagtg gaatgggtgt gtcctggatc 540 cgtcagcccc caggaaaggc cctggagtgg cttgcacaca tttattggga tgatgataag 600 cgctacaacc catctctgaa gagcaggctc accatcacca aggacacctc caaaaaccag 660 gtggtcctta caatgaccaa catggaccct gtggacacag ccacatatta ctgtgcacga 720 aaggactacg gtagtagctt ctatgctatg cactactggg gtcaaggaac cctagtcacc 780 gtgtcgagta ccacgacgcc agcgccgcga ccaccaacac cggcgcccac catcgcgtcg 840 cagcccctgt ccctgcgccc agaggcgtgc cggccagcgg cggggggcgc agtgcacacg 900 agggggctgg acttcgcctg tgatatctac atctgggcgc ccttggccgg gacttgtggg 960 gtccttctcc tgtcactggt tatcaccctt tactgcaaac ggggcagaaa gaaactcctg 1020 tatatattca aaaaccatt tatgagacca gtacaacta ctcaagagga agatggctgt 1080 1140 agcgcagacg cccccgcgta ccagcagggc cagaaccagc tctataacga gctcaatcta 1200 ggacgaag aggagtacga tgttttggac aagacgtg gccgggaccc tgagatgggg 1260 ggaaagccgc agaaaggaa gaaccctcag gaagcctgt acaatgaact gcagaaagat 1320 aagatggcgg aggcctacag tgagattggg atgaaaggcg agcgccggag gggcaagggg 1380 cacgatggcc tttaccaggg tctcagtaca gccaccaagg acacctacga cgcccttcac 1440 atgcaggccc tgccccctcg ctaa 1464 <210> 10 <211> 135 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 10 accaccaccc ctgcaccaag gcccccgact cccgcgccca ccatcgcgtc acagcctctt 60 agcctgcgac cggaagcatg cagaccagct gccggggggg ccgtgcatac gagaggtttg 120 gacttcgcct gcgat 135 <210> 11 <211> 135 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 11 accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60 tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120 gacttcgcct gtgat 135 <210> 12 <211> 21 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 12 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro 20 <210> 13 <400> 13 000 <210> 14 <211> 114 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 14 Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile 1 5 10 15 Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His 20 25 30 Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln 35 40 45 Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu 50 55 60 Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val 65 70 75 80 Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile 85 90 95 Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn 100 105 110 Thr Ser <210> 15 <211> 23 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 15 Ser Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp 1 5 10 15 Val Glu Glu Asn Pro Gly Pro 20 <210> 16 <211> 4 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 16 Arg Ala Lys Arg 1 <210> 17 <211> 27 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 17 Arg Ala Lys Arg Ser Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys 1 5 10 15 Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro 20 25 <210> 18 <211> 649 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 18 Met Ser Val Pro Thr Gln Val Leu Gly Leu Leu Leu Leu Trp Leu Thr 1 5 10 15 Asp Ala Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser 20 25 30 Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp 35 40 45 Ile His Asn Tyr Ile Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 50 55 60 Lys Leu Leu Ile His Tyr Thr Ser Thr Leu Gln Pro Gly Val Pro Ser 65 70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser 85 90 95 Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp 100 105 110 Asn Leu Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly 115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gln Ile Thr 130 135 140 Leu Lys Glu Ser Gly Pro Thr Leu Val Lys Pro Thr Gln Thr Leu Thr 145 150 155 160 Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser Gly Met Gly 165 170 175 Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu Ala 180 185 190 His Ile Tyr Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ser Leu Lys Ser 195 200 205 Arg Leu Thr Ile Thr Lys Asp Thr Ser Lys Asn Gln Val Val Leu Thr 210 215 220 Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala Arg 225 230 235 240 Lys Asp Tyr Gly Ser Ser Phe Tyr Ala Met His Tyr Trp Gly Gln Gly 245 250 255 Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro 260 265 270 Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu 275 280 285 Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp 290 295 300 Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly 305 310 315 320 Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg 325 330 335 Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln 340 345 350 Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu 355 360 365 Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala 370 375 380 Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu 385 390 395 400 Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp 405 410 415 Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly 420 425 430 Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu 435 440 445 Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu 450 455 460 Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His 465 470 475 480 Met Gln Ala Leu Pro Pro Arg Arg Ala Lys Arg Ser Gly Ser Gly Ala 485 490 495 Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro 500 505 510 Gly Pro Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu 515 520 525 Leu Leu His Ala Ala Arg Pro Asn Trp Val Asn Val Ile Ser Asp Leu 530 535 540 Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu 545 550 555 560 Tyr Thr Glu Ser Asp Val His Pro Ser Cys Lys Val Thr Ala Met Lys 565 570 575 Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala 580 585 590 Ser Ile His Asp Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser 595 600 605 Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu 610 615 620 Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His 625 630 635 640 Ile Val Gln Met Phe Ile Asn Thr Ser 645 <210> 19 <211> 1950 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 19 atgtccgtgc ctacccaggt gctgggcctg ctgctgctgt ggctgaccga cgccagatgc 60 gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 120 atcacttgca aggcgagtca ggacattcac aactatatag cttggtatca gcagaaacca 180 gggaaagccc ctaagctcct gatccactat acatccactt tgcaaccagg ggtcccatca 240 aggttcagtg gaagtggatc tgggacagat tttactttca ccatcagcag cctgcagcct 300 gaagatattg caacatatta ctgtctacag tatgataatc tctggacgtt cggtcaaggc 360 accaaggtgg aaatcaaacg gggtggaggt ggatctggag gaggaggatc cggtggagga 420 ggtcagatca ccttgaagga gtctggtcct acgctggtga aacccacaca gaccctcacg 480 ctgacctgca ccttctctgg gttctcactc agcactagtg gaatgggtgt gtcctggatc 540 cgtcagcccc caggaaaggc cctggagtgg cttgcacaca tttattggga tgatgataag 600 cgctacaacc catctctgaa gagcaggctc accatcacca aggacacctc caaaaaccag 660 gtggtcctta caatgaccaa catggaccct gtggacacag ccacatatta ctgtgcacga 720 aaggactacg gtagtagctt ctatgctatg cactactggg gtcaaggaac cctagtcacc 780 gtgcgagta ccaccacccc tgcaccaagg cccccgactc ccgcgcccac catcgcgtca 840 cagcctctta gcctgcgacc ggaagcatgc agaccagctg ccgggggc cgtgcatacg 900 agaggtttgg acttcgcctg cgatatctac atctgggcgc ccttggccgg gacttgtggg 960 gtccttctcc tgtcactggt tatcaccctt tactgcaaac ggggcagaaa gaaactcctg 1020 tatatattca aacaaccatt tatgagacca gtacaaacta ctcaagagga agatggctgt 1080 agctgccgat ttccagaaga agaagaagga ggatgtgaac tgagagtgaa gttcagcagg 1140 agcgcagacg cccccgcgta ccagcagggc cagaaccagc tctataacga gctcaatcta 1200 ggacgaagag aggagtacga tgttttggac aagagacgtg gccgggaccc tgagatgggg 1260 ggaaagccgc agagaagga gaaccctcag gaaggcctgt acaatgaact gcagaaagat aagatggcgg aggcctacag tgagattggg atgaaaggcg agcgccggag gggcaagggg 1440. ccgatggcc tttaccaggg tctcagtaca gccaccaagg acacctacga cgcccttcac atgcaggccc tgccccctcg ccgcgcgag cgatcaggca gcggggcgac aaatttcagc cttctgaaac aagcaggcga cgtggaaga aaccccggtc caatggcctt accagtgacc gccttgctcc tgccgctggc cttgctgctc cacgccgcca ggccgaactg ggtgaatgta 1620 ataagtgatt tgaaaaaaat tgaagatctt attcaatcta tgcatattga tgctacttta father gtgatgttca ccccagttgc aaagtaacag caatgaagtg ctttctcttg gagttacaag ttatttcact tgagtccgga gatgcaagta ttcatgatac agtagaaaat ctgatcatcc tagcaaacaa cagtttgtct tctaatggga atgtaacaga atctggatgc aaagaatgtg aggaactgga ggaaaaaaat attaaagaat ttttgcagag ttttgtacat 1920 attgtccaaa tgttcatcaa cacttcttga 1950 <210> 20 <211> 485 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 20 Met Ser Val Pro Thr Gln Val Leu Gly Leu Leu Leu Leu Trp Leu Thr 1 5 10 15 Asp Ala Arg Cys Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys 20 25 30 Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr 35 40 45 Phe Thr Asp Tyr Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly 50 55 60 Leu Glu Trp Met Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr 65 70 75 80 Ser Gln Lys Phe Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr 85 90 95 Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala 100 105 110 Val Tyr Tyr Cys Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly 115 120 125 Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140 Ser Gly Gly Gly Gly Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu 145 150 155 160 Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln 165 170 175 Ser Leu Val His Ser Asn Arg Asn Thr Tyr Leu His Trp Tyr Leu Gln 180 185 190 Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg 195 200 205 Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 210 215 220 Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr 225 230 235 240 Tyr Cys Ser Gln Asn Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr 245 250 255 Lys Leu Glu Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro 260 265 270 Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys 275 280 285 Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala 290 295 300 Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu 305 310 315 320 Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys 325 330 335 Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr 340 345 350 Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly 355 360 365 Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala 370 375 380 Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg 385 390 395 400 Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu 405 410 415 Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr 420 425 430 Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly 435 440 445 Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln 450 455 460 Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln 465 470 475 480 Ala Leu Pro Pro Arg 485 <210> 21 <211> 1458 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 21 atgtccgtgc ctacccaggt gctgggcctg ctgctgctgt ggctgaccga cgccagatgc 60 caagtgcagc tggtccagag cggcgccgag gtgaaaaagc ctggcgccag cgtgaaggtg 120 tcctgcaagg cctctggcta caccttcacc gactacgaga tgcactgggt gcggcaggcc 180 cctggacagg gcctggaatg gatgggcgct ctggacccca agaccggcga caccgcttat 240 agccagaagt tcaagggcag agtgaccctg acagctgata agagcacaag caccgcctac 300 atggaactga gcagcctgac cagcgaggac accgccgtgt actactgcac cagattctac 360 agctacacct actggggcca ggggaccctg gtgacagtgt ctagcggtgg aggtggatct 420 ggaggaggag gatccggtgg aggaggtgat gtggtgatga cccagagccc tctgagcctg 480 cctgtgaccc ctggagagcc tgccagcatc agctgcagaa gcagccaatc tctggtgcac 540 agcaaccgga acacatacct gcactggtac ctgcagaaac ctggccagag cccccagctg 600 ctgatctaca aggtgtccaa cagattcagc ggcgtgcctg atagattcag cggatctggc 660 agcggcaccg acttcaccct gaagatctct agagtggaag ccgaggacgt gggcgtgtac 720 tactgcagcc agaacaccca cgtgcccccc accttcggcc agggcacaaa gctggaaatc 780 aagaccacga cgccagcgcc gcgaccacca acaccggcgc ccaccacgc gtcgcagcccc 840 ctgtccctgc gcccagaggc gtgccggcca gcggcggggg gcgcagtgca cacgagggggg 900 ctggacttcg cctgtgatat ctacatctgg gcgcccttgg ccggacttg tggggtcctt 960 ctcctgtcac tggttatcac cctttactgc aaacggggca gaagaaact cctgtatatata 1020 ttcaacaac cattttagag accagtacaa actactcaag aggagatgg ctgtagctgc 1080 cgatttccag agagaga aggagatgt gaacgagag tgagttcag caggagcgca 1140 gacgcccccg cgtaccagca gggccagaac cagctctata acgagctca tctaggacga 1200 agagaggagt acgatgtttt ggacaagaga cgtggccggg accctgagat gggggaaag 1260 ccgcagagaa ggaagaaccc tcaggaggc ctgtacaatg aactgcagaa agatagatg 1320 gcggaggcct acagtgagat tgggatgaa ggcgagcgcc ggaggggca ggggcacgat 1380 ggcctttacc agggtctcag tacagccacc aaggacacct acgacgccct tcacatgcag 1440 gccctgcccc ctcgctaa 1458 <210> 22 <211> 642 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 22 Met Ser Val Pro Thr Gln Val Leu Gly Leu Leu Leu Leu Trp Leu Thr 1 5 10 15 Asp Ala Arg Cys Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys 20 25 30 Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr 35 40 45 Phe Thr Asp Tyr Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly 50 55 60 Leu Glu Trp Met Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr 65 70 75 80 Ser Gln Lys Phe Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr 85 90 95 Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala 100 105 110 Val Tyr Tyr Cys Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly 115 120 125 Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140 Ser Gly Gly Gly Gly Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu 145 150 155 160 Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln 165 170 175 Ser Leu Val His Ser Asn Arg Asn Thr Tyr Leu His Trp Tyr Leu Gln 180 185 190 Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg 195 200 205 Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 210 215 220 Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr 225 230 235 240 Tyr Cys Ser Gln Asn Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr 245 250 255 Lys Leu Glu Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro 260 265 270 Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys 275 280 285 Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala 290 295 300 Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu 305 310 315 320 Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys 325 330 335 Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr 340 345 350 Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly 355 360 365 Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala 370 375 380 Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg 385 390 395 400 Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu 405 410 415 Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr 420 425 430 Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly 435 440 445 Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln 450 455 460 Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln 465 470 475 480 Ala Leu Pro Pro Arg Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys 485 490 495 Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Ala Leu Pro Val 500 505 510 Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His Ala Ala Arg Pro 515 520 525 Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile 530 535 540 Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His 545 550 555 560 Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln 565 570 575 Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu 580 585 590 Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val 595 600 605 Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile 610 615 620 Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn 625 630 635 640 Thr Ser <210> 23 <211> 1929 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 23 atgtccgtgc ctacccaggt gctgggcctg ctgctgctgt ggctgaccga cgccagatgc 60 caagtgcagc tggtccagag cggcgccgag gtgaaaaagc ctggcgccag cgtgaaggtg 120 tcctgcaagg cctctggcta caccttcacc gactacgaga tgcactgggt gcggcaggcc 180 cctggacagg gcctggaatg gatgggcgct ctggacccca agaccggcga caccgcttat 240 agccagaagt tcaagggcag agtgaccctg acagctgata agagcacaag caccgcctac 300 atggaactga gcagcctgac cagcgaggac accgccgtgt actactgcac cagattctac 360 agctacacct actggggcca ggggaccctg gtgacagtgt ctagcggtgg aggtggatct 420 ggaggaggag gatccggtgg aggaggtgat gtggtgatga cccagagccc tctgagcctg 480 cctgtgaccc ctggagagcc tgccagcatc agctgcagaa gcagccaatc tctggtgcac 540 agcaaccgga acacatacct gcactggtac ctgcagaaac ctggccagag cccccagctg 600 ctgatctaca aggtgtccaa cagattcagc ggcgtgcctg atagattcag cggatctggc 660 agcggcaccg acttcaccct gaagatctct aggtggaag ccgaggacgt gggcgtgtac 720 tactgcagcc agacaccca cgtgcccccc accttcggcc agggcacaaa gctggaaatc aagaccacga cgccagcgcc gcgaccacca acaccggcgc ccaccatcgc gtcgcagccc 840 ctgtccctgc gcccagaggc gtgccggcca gcggcgggggg gcgcagtgca cacgagggggg900 ctggacttcg cctgtgatat ctacatctgg gcgcccttgg ccgggacttg tggggtcctt 960 1020. ctcctgtcac tggttatcac cctttactgc aaacggggca gaaagaact cctgtatata ttcaaacaac catttatgag accagtacaa actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt gaactgagag tgaagttcag caggagcgca gacgccccg cgtaccagca gggccagaac cagctctata acgagctcaa tctaggacga agagaggagt acgatgtttt ggacaagaga cgtggccggg accctgagat ggggggaag 1260 ccgcagagaa ggaagaaccc tcaggaaggc ctgtacaatg aactgcagaa agataagatg 1320 gcggaggcct acagtgagat tgggatgaaa ggcgagcgcc ggaggggcaa ggggcacgat 1380 ggcctttacc agggtctcag tacagccacc aggacacct acgacgccct tcacatgcag 1440 gccctgcccc ctcgcggtag cggggctacg aacttctccc ttcttaaaca agcgggagac 1500 gtgaagaaa atcccggacc tatggcctta ccagtgaccg ccttgctcct gccgctggcc 1560 ttgctgctcc acgccgccag gccgaactgg gtgaatgtaa taagtgattt gaaaaaaaatt 1620 gaagatctta ttcaatctat gcatattgat gctactttat atacggaaag tgatgttcac 1680 cccagttgca aagtaacagc aatgaagtgc tttctcttgg agttacaagt tatttcactt 1740 gagtccggag atgcaagtat tcatgataca gtagaaaatc tgatcatcct agcaaacaac 1800 agtttgtctt ctaatgggaa tgtaacagaa tctggatgca aagaatgtga ggaactggag 1860 gaaaaaaata ttaaagaatt tttgcagagt tttgtacata ttgtccaaat gttcatcaac 1920 acttcttga 1929 <210> 24 <211> 1929 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 24 atgtccgtgc ctacccaggt gctgggcctg ctgctgctgt ggctgaccga cgccagatgc 60 caagtgcagc tggtccagag cggcgccgag gtgaaaaagc ctggcgccag cgtgaaggtg 120 tcctgcaagg cctctggcta caccttcacc gactacgaga tgcactgggt gcggcaggcc 180 cctggacagg gcctggaatg gatgggcgct ctggacccca agaccggcga caccgcttat 240 agccagaagt tcaagggcag agtgaccctg acagctgata agagcacaag caccgcctac 300 atggaactga gcagcctgac cagcgaggac accgccgtgt actactgcac cagattctac 360 agctacacct actggggcca ggggaccctg gtgacagtgt ctagcggtgg aggtggatct 420 ggaggaggag gatccggtgg aggaggtgat gtggtgatga cccagagccc tctgagcctg 480 cctgtgaccc ctggagagcc tgccagcatc agctgcagaa gcagccaatc tctggtgcac 540 agcaaccgga acacatacct gcactggtac ctgcagaaac ctggccagag cccccagctg 600 ctgatctaca aggtgtccaa cagattcagc ggcgtgcctg atagattcag cggatctggc 660 agcggcaccg acttcaccct gaagatctct agagtggaag ccgaggacgt gggcgtgtac 720 tactgcagcc agaacaccca cgtgcccccc accttcggcc agggcacaaa gctggaaatc 780 aagaccacga cgccagcgcc gcgaccacca acaccggcgc ccaccatcgc gtcgcagccc 840 ctgtccctgc gcccagaggc gtgccggcca gcggcggggg gcgcagtgca cacgaggggg 900 ctggacttcg cctgtgatat ctacatctgg gcgcccttgg ccgggacttg tggggtcctt 960 ctcctgtcac tggttatcac cctttactgc aaacggggca gaagaaact cctgtatatata 1020 ttcaacaac cattttagag accagtacaa actactcaag aggagatgg ctgtagctgc 1080 cgatttccag agagaga aggagatgt gaacgagag tgagttcag caggagcgca 1140 gacgcccccg cgtaccagca gggccagaac cagctctata acgagctca tctaggacga 1200 agagaggagt acgatgtttt ggacaagaga cgtggccggg accctgagat gggggaaag 1260 ccgcagagaa ggaagaaccc tcaggaggc ctgtacaatg aactgcagaa agatagatg 1320 gcggaggcct acagtgagat tgggatgaa ggcgagcgcc ggaggggca ggggcacgat 1380 ggcctttacc agggtctcag tacaccacc aaggacaccct acgaccccct tcacaccag 1440 gccctgcccc ctcgcggtag cggggctacg aacttctccc ttcttaaca agcgggac 1500 gtggaagaaa atcccggacc tatggcctta ccagtgaccg ccttgctcct gccgctggcc 1560 ttgctgctcc acgccgccag gccgaactgg gtgaatgtga tcagcgatct gaagaagatc 1620 gaggatctga tccagtccat gcacatcgat gccaccctgt ataccgagag cgatgtgcac 1680 cccagctgca aggtgaccgc catgaagtgc tttctgctgg agctgcaggt gatctccctg 1740 gagtccggag atgccagcat ccacgatacc gtggagaatc tgatcatcct ggccaacaac 1800 agcctgtcct ccaatggcaa tgtgaccgag tcgggatgca aggagtgcga ggagctggag 1860 gagaagaata tcaaggagtt tctgcagagc tttgtacata ttgtccaaat gttcatcaac 1920 acttcttga 1929 <210> 25 <211> 22 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 25 Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val 1 5 10 15 Glu Glu Asn Pro Gly Pro 20 <210> 26 <211> 48 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 26 Met Arg Ile Ser Lys Pro His Leu Arg Ser Ile Ser Ile Gln Cys Tyr 1 5 10 15 Leu Cys Leu Leu Leu Asn Ser His Phe Leu Thr Glu Ala Gly Ile His 20 25 30 Val Phe Ile Leu Gly Cys Phe Ser Ala Gly Leu Pro Lys Thr Glu Ala 35 40 45 <210> 27 <211> 19 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 27 Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn 1 5 10 15 Pro Gly Pro <210> 28 <211> 23 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 28 Val Lys Gln Thr Leu Asn Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp 1 5 10 15 Val Glu Ser Asn Pro Gly Pro 20 <210> 29 <211> 20 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 29 Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp Val Glu Ser 1 5 10 15 Asn Pro Gly Pro 20 <210> 30 <211> 17 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 30 Glu Gly Arg Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro Gly 1 5 10 15 Pro <210> 31 <211> 553 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 31 ctaacgttac tggccgaagc cgcttggaat aaggccggtg tgcgtttgtc tatatgttat 60 tttccaccat attgccgtct tttggcaatg tgagggcccg gaaacctggc cctgtcttct 120 tgacgagcat tcctaggggt ctttcccctc tcgccaaagg aatgcaaggt ctgttgaatg 180 tcgtgaagga agcagttcct ctggaagctt cttgaagaca aaacgtct gtagcgaccc 240 tttgcaggca gcggaacccc ccacctggcg acaggtgcct ctgcggccaa aagccacgtg 300 tataagatac acctgcaaag gcggcacaac cccagtgcca cgttgtgagt tggatagttg 360 tggaaagagt caaatggctc tcctcaagcg tattcaacaa ggggctgaag gatgcccaga 420 aggtacccca ttgtatggga tctgatctgg ggcctcggtg cacatgcttt acatgtgttt 480 agtcgaggtt aaaaaaacgt ctaggccccc cgaaccacgg ggacgtggtt ttccttttgaa 540 aaacacgatg ata 553 <210> 32 <211> 461 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 32 agcaggtttc cccaactgac acaaaacgtg caacttgaaa ctccgcctgg tctttccagg 60 tctagagggg taacactttg tactgcgttt ggctccacgc tcgatccact ggcgagtgtt 120 agtaacagca ctgttgcttc gtagcggagc atgacggccg tgggaactcc tccttggtaa 180 caaggaccca cggggccaaa agccacgccc acacgggccc gtcatgtgtg caaccccagc 240 acggcgactt tactgcgaaa cccactttaa agtgacattg aaactggtac ccacacactg 300 gtgacaggct aaggatgccc ttcaggtacc ccgaggtaac acgcgacact cgggatctga 360 gaaggggact ggggcttcta taaaagcgct cggtttaaaa agcttctatg cctgaatagg 420 tgaccggagg tcggcacctt tcctttgcaa ttactgacca c 461 <210> 33 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 33 Thr Ser Gly Met Gly Val Ser 1 5 <210> 34 <211> 16 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 34 His Ile Tyr Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 35 <211> 12 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 35 Lys Asp Tyr Gly Ser Ser Phe Tyr Ala Met His Tyr 1 5 10 <210> 36 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 36 Lys Ala Ser Gln Asp Ile His Asn Tyr Ile Ala 1 5 10 <210> 37 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 37 Tyr Thr Ser Thr Leu Gln Pro 1 5 <210> 38 <211> 8 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 38 Leu Gln Tyr Asp Asn Leu Trp Thr 1 5 <210> 39 <211> 5 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 39 Asp Tyr Glu Met His 1 5 <210> 40 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 40 Gly Tyr Thr Phe Thr Asp Tyr Glu Met His 1 5 10 <210> 41 <211> 17 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 41 Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe Lys 1 5 10 15 Gly <210> 42 <211> 6 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 42 Phe Tyr Ser Tyr Thr Tyr 1 5 <210> 43 <211> 16 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 43 Arg Ser Ser Gln Ser Leu Val His Ser Asn Arg Asn Thr Tyr Leu His 1 5 10 15 <210> 44 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 44 Lys Val Ser Asn Arg Phe Ser 1 5 <210> 45 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 45 Ser Gln Asn Thr His Val Pro Pro Thr 1 5

Claims

1. 1. An isolated nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein said 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 the surface of a solid tumor cell, and optionally, wherein the binding domain binds to the complex in an HLA-restricted manner; b. CD8α hinge domain; and c. CD8α transmembrane domain; d. a costimulatory signaling region selected from a 4-1BB costimulatory signaling region and a CD27 costimulatory signaling region; e. a CD3ζ signaling domain.

2. 2. The isolated nucleic acid sequence of claim 1, wherein (a) to (e) are in 5' to 3' order.

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

4. The isolated nucleic acid sequence of claim 3, wherein the flanking region of TyrD comprises at least 4, or at least about 4, and not more than 12, or not more than about 12, flanking amino acids of TyrD, preferably 7, 8, or 9, or preferably about 7, 8, or 9, flanking amino acids of TyrD.

5. The adjacent region of TyrD is ３６９－３７７ 5. The isolated nucleic acid sequence of claim 4, wherein:

6. The binding domain that specifically binds to the TAA peptide-MHC complex is HLA-A2 / TyrD ３６９－３７７ 6. The isolated nucleic acid sequence of claim 1, which specifically binds to

7. the binding domain is a. a CDRH1 comprising TSGMGVS (SEQ ID NO: 33); and b. a CDRH2 comprising HIYWDDDKRYNPSLKS (SEQ ID NO: 34); and c. a CDRH3 comprising KDYGSSFYAMHY (SEQ ID NO: 35); d. CDRL1 comprising KASQDIHNYIA (SEQ ID NO:36); and e. a CDRL1 comprising YTSTLQP (SEQ ID NO: 37); and f. CDRL2 comprising LQYDNLWT (SEQ ID NO: 38); 7. The isolated nucleic acid sequence of any one of claims 1 to 6, which specifically binds to an epitope bound by an antibody comprising:

8. 1. An isolated nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein said CAR comprises: a. a binding domain that specifically binds to a tumor-associated antigen (TAA) expressed on the surface of a solid tumor cell, optionally wherein the antigen is a protein-peptide complex, wherein the protein is an MHC protein, and wherein the binding domain binds to the protein-peptide complex in an HLA-restricted manner; b. CD8α hinge domain; and c. CD8α transmembrane domain; d. a costimulatory signaling region selected from a 4-1BB costimulatory signaling region and a CD27 costimulatory signaling region; e. a CD3ζ signaling domain.

9. 9. The isolated nucleic acid sequence of claim 8, wherein the binding domain specifically binds to an epitope within GPC3 that is expressed on the surface of solid tumor cells.

10. The binding domain comprises the following complementarity determining regions (CDRs): a. CDRH1 comprising the sequence of DYEMH (SEQ ID NO: 39) (or GYTFTDYEMH (SEQ ID NO: 40)); b. CDRH2 comprising the sequence of ALDPKTGDTAYSQKFKG (SEQ ID NO: 41); c. a CDRH3 comprising the sequence of FYSYTY (SEQ ID NO: 42); d. CDRL1 comprising the sequence RSSQSLVHSNRNTYLH (SEQ ID NO: 43); e. CDRL2 comprising the sequence of KVSNRFS (SEQ ID NO: 44) and / or f. CDRL3 comprising the sequence of SQNTHVPPT (SEQ ID NO:45) binds to the same GPC3 epitope as an antibody comprising the compound, and / or competes with said antibody for binding to the epitope of GPC3; 10. The isolated nucleic acid sequence of claim 9, comprising said CDRs.

11. 11. The isolated nucleic acid sequence of any one of claims 1 to 10, wherein the CAR comprises: a. a CD8α hinge domain comprising SEQ ID NO: 1 (PTPAPTIASQPLSLPEACRPAAGGAVHTRGLDFACDIY) or SEQ ID NO: 2 (TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY); b. CD8α transmembrane domain comprising SEQ ID NO: 3 (IWAPLAGTCGVLLLSLVITLYC) and / or c. (i) SEQ ID NO: 4 (RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR) or (ii) a CD3ζ signaling domain comprising SEQ ID NO: 5 (RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR) The nucleic acid sequence comprising:

12. 12. The isolated nucleic acid sequence of claim 11, wherein the CAR comprises: a. a 4-1BB costimulatory signaling region comprising SEQ ID NO: 6 (KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL) or b. A CD27 costimulatory signaling region comprising SEQ ID NO: 7 (QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP); or wherein said isolated nucleic acid encodes said 4-1BB costimulatory signaling region comprising SEQ ID NO:6 and said CD27 costimulatory signaling region comprising SEQ ID NO:

7.

13. 13. The isolated nucleic acid sequence of any one of claims 1 to 12, wherein the nucleic acid further comprises a. secreted cytokines or b. secreted common gamma-chain interleukins or c. Secreted IL-15, preferably wherein said IL-15 comprises the sequence of SEQ ID NO: 14, more preferably wherein said IL-15 comprises the sequence of SEQ ID NO: 14 operably linked to the secretory signal sequence of SEQ ID NO: 12, or wherein said IL-15 comprises the sequence of SEQ ID NO: 14 operably linked to the secretory signal sequence of SEQ ID NO: 26, or d. A secreted common gamma chain interleukin, preferably IL-15 and a multicistronic linker region amino-terminal to said interleukin or interleukin secretion signal, preferably wherein said multicistronic linker region comprises any one of the sequences set forth in SEQ ID NOs: 15-17, 25, or 27-30, or a combination thereof, or encodes an internal ribosome entry site, such as SEQ ID NO: 31 or 32. The nucleic acid sequence encoding

14. 14. The isolated nucleic acid sequence of claim 13, wherein a. the secretion signal comprises the sequence of SEQ ID NO: 12 or SEQ ID NO: 26, preferably SEQ ID NO: 12, and / or b. the sIL15 domain comprises the sequence of SEQ ID NO: 14, and / or c. the P2A cleavage sequence comprises the sequence of SEQ ID NO: 15 or SEQ ID NO: 25, and / or d. the furin cleavage sequence comprises the sequence of SEQ ID NO: 16, and / or e. the CAR comprises, in amino to carboxy order, the sequences of SEQ ID NO: 17, SEQ ID NO: 12, and SEQ ID NO: 14; The nucleic acid sequence.

15. 15. An isolated nucleic acid sequence according to any one of claims 1 to 14, wherein a. The binding domain is HLA-A2 / TyrD ３６９－３７７ wherein the nucleic acid encodes SEQ ID NO: 8 or SEQ ID NO: 18; b. The nucleic acid sequence wherein the binding domain specifically binds to GPC3, and the nucleic acid encodes SEQ ID NO: 20 or 22.

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

17. 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. 18. A γδ T-cell comprising the polypeptide of claim 17 or comprising a nucleic acid encoding the CAR construct of any one of claims 1 to 16, wherein the γδ T-cell comprises a binding domain of the polypeptide or a nucleic acid encoding a CAR on the surface of the γδ T-cell. The γδ T cell functionally expresses a nucleic acid.

19. 19. The γδ T cell of claim 18, wherein the γδ T cell exhibits in vitro and / or in vivo cytotoxic activity against solid tumor cells that exhibit cell surface expression of the tumor-associated antigen (TAA).

20. 20. The γδ T-cell of claim 19, wherein the solid tumor cytotoxic activity of the γδ T-cell is greater than the native level of in vitro and / or in vivo solid tumor cytotoxic activity in control γδ T-cells that do not comprise a CAR construct.

21. 21. The γδ T cell of claim 20, wherein the γδ T cell is HLA class I. ＋ The γδ T cells exhibit increased solid tumor cytotoxic activity against solid tumor cells.

22. 22. The γδ T-cell of any one of claims 19-21, wherein the solid tumor cytotoxic activity or increased solid tumor cytotoxic activity persists for about, at least, or at least about 6 days to 180 days after initial contact with the solid tumor cells.

23. 23. The γδ T-cell of any one of claims 18 to 22, wherein the γδ T-cell proliferates in response to contact with solid tumor cells that exhibit cell surface expression of the tumor-associated antigen (TAA).

24. 23. The γδ T-cell of any one of claims 18 to 22, wherein the γδ T-cell exhibits increased proliferation in response to contact with solid tumor cells that exhibit cell surface expression of the tumor-associated antigen (TAA) compared to control γδ T-cells that do not functionally express the nucleic acid encoding a CAR on the surface of the γδ T-cell.

25. 25. The γδ T-cell of any one of claims 18 to 24, wherein the γδ T-cell is propagated in a host organism comprising the solid tumor cells that exhibit cell surface expression of the tumor-associated antigen (TAA).

26. 26. The γδ T-cell of any one of claims 18 to 25, wherein the proliferation of the γδ T-cell or increased proliferation of the γδ T-cell persists for about, at least, or at least about 6 days to 180 days after initial contact with the solid tumor cells.

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

28. 28. The γδ T-cell of any one of claims 18-27, wherein the γδ T-cell exhibits a reduced, substantially reduced, substantially absent, or abolished graft-versus-host response when introduced into an allogeneic host compared to the graft-versus-host response exhibited by αβ T-cells administered to an allogeneic host.

29. γδ T cell according to any one of claims 18 to 28, wherein the γδ T cell is a δ1, δ2, δ3, or δ4 γδ T cell, preferably a δ2 － The γδ T cell is a γδ T cell, more preferably a δ1 γδ T cell.

30. 30. A plurality of γδ T cells according to any one of claims 18 to 29.

31. 31. The plurality of γδ T-cells of claim 30, wherein the plurality comprises at least about 10 ８ γδ T cells, preferably about 10 ８ Approximately 10 from γδ T cells １１ the plurality of γδ T cells comprising:

32. 32. The plurality of γδ T cells according to claim 30 or 31, wherein the plurality is at least 60%, 80%, or about 60% or 80% to about 90% or 95% δ1, δ2, δ3, or δ4 γδ T cells, preferably δ1 or δ2 γδ T cells, more preferably δ2 － The plurality of γδ T cells comprises a composition that is γδ T cells, most preferably δ1 γδ T cells.

33. 33. A method of generating a γδ T-cell of any one of claims 18-29 or a plurality of γδ T-cells of any one of claims 30-32, wherein the method comprises transfecting a γδ T-cell(s) with a construct comprising the isolated nucleic acid sequence of any one of claims 1-16.

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

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

36. 33. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and a γδ T-cell according to any one of claims 18 to 29, or a plurality of γδ T-cells according to any one of claims 30 to 32.

37. 37. A method of killing a solid tumor cell, the method comprising contacting the solid tumor cell with a γδ T-cell of any one of claims 18-29, a plurality of γδ T-cells of any one of claims 30-32, or the pharmaceutical composition of claim 36, in an amount effective to kill the tumor cell.

38. 38. The method of claim 37, wherein the method comprises introducing a therapeutically effective amount of the γδ T-cell(s) or the pharmaceutical composition into a host organism comprising the solid tumor cells.

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

40. 40. The method of claim 39, wherein the one or more administration methods for increasing the general gamma chain cytokine(s) comprise administering an effective amount of a general gamma chain cytokine(s) simultaneously with or consecutively with the introduction of the γδ T-cell(s) to increase the proliferation, cytotoxic activity, persistence, or a combination thereof, of the introduced γδ T-cell(s), preferably wherein the method comprises administering IL-2, more preferably wherein the method comprises administering IL-15.

41. 41. The method of claim 40, wherein the one or more methods of increasing the common gamma chain cytokine(s) comprises administering an effective amount of common gamma chain cytokine(s) before and / or after transferring the γδ T-cell(s) to increase proliferation, cytotoxic activity, persistence, or a combination thereof, of the transferred γδ T-cell(s).

42. 42. The method of any one of claims 39-41, wherein the one or more methods of elevating the common gamma chain cytokine(s) comprises lymphodepletion prior to introducing the γδ T-cell(s).

43. 42. The method of any one of claims 39-41, wherein the one or more methods of elevating the common gamma chain cytokine(s) comprises secretion of one or more common gamma chain cytokine(s) from the introduced γδ T-cell(s).

44. 44. The method of any one of claims 38-43, wherein the method reduces the in vivo tumor burden of the host organism and / or increases the mean survival time of the host organism compared to a control organism, wherein the control organism is not treated with the γδ T-cell(s) or the pharmaceutical composition.

45. 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. Use of a γδ T-cell of any one of claims 18 to 29, a plurality of γδ T-cells of any one of claims 30 to 32, or a pharmaceutical composition of claim 36 in the manufacture of a medicament for treating solid tumor cell cancer in a subject in need thereof, in an amount effective to kill tumor cells.

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

48. 48. The method of claim 47, wherein the method comprises administering one or more methods that elevate general gamma chain cytokine(s) simultaneously with or sequentially with administration of γδ T-cells.

49. 49. The method of claim 47 or 48, wherein the method comprises administering multiple administrations of the γδ T-cells, wherein the interval between the multiple administrations is at least about 1 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. 50. A pharmaceutical composition for use in the method of any one of claims 47 to 49.

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