Methods and compositions for modulating t cells

Abstract

The current disclosure describes molecules that modulate T cell activity and efficacy and methods of using the molecules. Provided is a method for making T cells comprising contacting the T cell ex vivo with a T cell modulator, wherein the T cell modulator comprises Telmisartan. Also described is a method for making T cells comprising contacting the T cell ex vivo with a T cell modulator, wherein the T cell modulator comprises Telmisartan. Also provided is a T cell made by a method of the disclosure and a population of T cells made by a method of or the disclosure or derived from a cell of the disclosure.

Description

METHODS AND COMPOSITIONS FOR MODULATING T CELLS

[0001] This application claims priority of U.S. Provisional Patent Application No.63 / 763,100, filed February 25, 2025; which is hereby incorporated by reference in its entirety.

[0002] The application contains a Sequence Listing in compliance with ST.26 format and is hereby incorporated by reference in its entirety. Said Sequence Listing, created on February 25, 2026, is named UCLAP0218WO.xml, and is 10,501 bytes in size.I. Field of the Invention

[0003] This invention relates to the field of methods of cancer biology and cellular therapies.II. Background

[0004] Adoptive cellular immunotherapies have demonstrated clinical efficacy against a subset of malignancies by employing genetically modified immune cells. These cells can be engineered to recognize defined antigens and mediate targeted cytotoxic responses against cancerous tissues. However, variability in cell-product quality, functional limitations associated with the modifications, and the immunosuppressive nature of the tumor microenvironment often compromise both the potency and durability of these therapies across diverse patient populations in preclinical and clinical settings. As a result, there is a need in the art for advanced methods to enhance performance and sustain the therapeutic efficacy of adoptively transferred immune cells in a wide array of clinical applications.SUMMARY

[0005] The current disclosure describes molecules that modulate T cell activity and efficacy and methods of using the molecules. Provided is a method for making T cells comprising contacting the T cell ex vivo with a T cell modulator, wherein the T cell modulator comprises Telmisartan or AZD4573. Further described is a method for making T cells and / or for increasing the cytotoxicity of T cells, the method comprising contacting the T cell ex vivo with a T cell modulator, wherein the T cell modulator comprises Telmisartan or AZD4573 and wherein the T cells comprise engineered CAR-T cells or TCR-T cells.1137193686

[0006] Also described is a method for making T cells comprising contacting the T cell ex vivo with a T cell modulator, wherein the T cell modulator comprises Telmisartan or AZD4573. A further method relates to a method for making T cells comprising contacting the T cell ex vivo with a T cell modulator, wherein the T cell modulator comprises Telmisartan, Calcimycin, Calmidazolium, Thapsigargin, a CDK9 inhibitor, Riviciclib, a BET inhibitor, a CDK8 inhibitor, AZD4573, NSC-95397, PMA, or combinations thereof. Also provided is a T cell made by a method of the disclosure and a population of T cells made by a method of the disclosure or derived from a cell of the disclosure.

[0007] Methods include a method for treating a subject comprising administering a cell or cells of the disclosure. Methods also include a method for treating cancer in a subject, the method comprising administering a T cell modulator to the subject, wherein the subject is being treated with one or more immunotherapies and wherein the T cell modulator comprises Telmisartan or AZD4573. Also provided is a method for treating cancer in a subject, the method comprising administering a T cell modulator to the subject, wherein the subject is being treated with one or more immunotherapies and wherein the T cell modulator comprises Telmisartan or AZD4573. Further described is a method for treating cancer in a subject comprising administering a T cell modulator and one or more immunotherapies to the subject, wherein the T cell modulator comprises Telmisartan or AZD4573. Also described is a method for treating cancer in a subject, the method comprising administering a T cell modulator to the subject, wherein the subject is being treated with one or more immunotherapies and wherein the T cell modulator comprises Telmisartan, Calcimycin, Calmidazolium, Thapsigargin, a CDK9 inhibitor, Riviciclib, a BET inhibitor, a CDK8 inhibitor, AZD4573, NSC-95397, PMA, or combinations thereof. Methods further include a method for treating cancer in a subject comprising administering a T cell modulator and one or more immunotherapies to the subject, wherein the T cell modulator comprises Telmisartan, Calcimycin, Calmidazolium, Thapsigargin, a CDK9 inhibitor, Riviciclib, a BET inhibitor, a CDK8 inhibitor, AZD4573, NSC-95397, PMA, or combinations thereof. Further methods include a method for treating an autoimmune disorder in a subject or for suppressing an immune response in a subject, the method comprising administering a T cell modulator to the subject, wherein the T cell modulator comprises Telmisartan or AZD4573. Also included is a method for treating an autoimmune disorder in a subject or for suppressing an immune response in a subject, the method comprising administering a T cell modulator to the subject, wherein the T cell modulator comprises Telmisartan, Calcimycin, Calmidazolium, Thapsigargin, a CDK9 inhibitor, Riviciclib, a BET inhibitor, a CDK8 inhibitor, AZD4573, NSC-95397, PMA, or 2137193686combinations thereof. The immune response may be further described as an unwanted or pathogenic immune response. The immune response may include a toxic immune response associated with one or more immunotherapies. The subject may be one that has received one or more immunotherapies. The method may further comprise administration of one or more immunotherapies. The immune response may be further defined as cytokine release syndrome.

[0008] The disclosure also provides for a method comprising contacting a composition comprising a MHC molecule and a TCR with Telmisartan or AZD4573. Also described is a method comprising contacting a composition comprising a MHC molecule and a TCR with T cell modulator, wherein the T cell modulator comprises Telmisartan, Calcimycin, Calmidazolium, Thapsigargin, a CDK9 inhibitor, Riviciclib, a BET inhibitor, a CDK8 inhibitor, AZD4573, NSC-95397, PMA, or combinations thereof. The method may be for increasing the binding of the TCR and the MHC molecule. The MHC molecule and / or the TCR may be on a cell. The cell may comprise a stem cell or an immune cell. The immune cell may comprise a T cell. The MHC molecule may comprise a MHC tetramer. Also described is a method for performing a tetramer assay comprising contacting a composition comprising a MHC tetramer and T cells with Telmisartan or AZD4573. Methods further include a method for performing a tetramer assay comprising contacting a composition comprising a MHC tetramer and T cells with a T cell modulator, wherein the T cell modulator comprises Telmisartan, Calcimycin, Calmidazolium, Thapsigargin, a CDK9 inhibitor, Riviciclib, a BET inhibitor, a CDK8 inhibitor, AZD4573, NSC-95397, PMA, or combinations thereof. The methods may further comprise isolating T cells that are specifically bound to the MHC tetramer.

[0009] The CDK9 inhibitor may comprise PHA-767491. The CDK8 inhibitor may comprise CDK8-IN-4. The method may comprise increasing the cytotoxicity of the T cells and / or increases the antitumor activity of the cells and wherein the T cell modulator comprises Telmisartan or AZD4573, The concentration of the T cell modulator in contact with the cell may be 1 nM - 1 mM. The concentration of the T cell modulator in contact with the cell may be, be at least, or be at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153,3137193686154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, or 500 pM, nM, pM, or mM, or any derivable range therein.

[0010] Contacting the T cell ex vivo with the T cell modulator may comprise culturing the cell in cell medium comprising the T cell modulator. The concentration of the T cell modulator in the cell medium may be 1 nM - 1 mM. The concentration of the inhibitor in contact with the cell may be InM - 1 mM. The concentration of the T cell modulator in the cell medium may be, be at least, or be at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229,4137193686230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, or 500 pM, nM, pM, or mM, or any derivable range therein. The T cell modulator may comprise Telmisartan at a concentration of 0.1 pM - 100 pM. The T cell modulator may comprise AZD4573 at a concentration of 0.1 pM - 100 pM.

[0011] The T cell may be or may be derived from a CD8+ T cell. The T cell may be or may be derived from a CD4+ T cell. The T cell may be or may be derived from a CD3+ T cell. The T cell may be or may be derived from a naive or memory T cell. The T cell may comprise T cells isolated from a subject. The T cells may comprise tumor infiltrating lymphocytes (TILs).

[0012] The cell may be contacted with the T cell modulator for a period of time of at least 0.1 hours. The period of time may be, be at least, or be at most 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225,5137193686226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, or 250 hours, or any derivable range therein.

[0013] The cell may be one that has been isolated from a patient or subject by leukapheresis. Cells isolated by leukapheresis may be excluded in the methods, compositions, and cells of the disclosure. Leukapheresis is a laboratory procedure in which white blood cells are separated from a sample of blood. It is a specific type of apheresis, the more general term for separating out one particular constituent of blood and returning the remainder to the circulation. Leukapheresis may be performed to decrease a very high white blood cell count, to obtain blood cells from a patient (autologous) or donor (allogeneic) for later transplant into the patient, or to obtain cells for research purposes. Leukapheresis may be performed to obtain the patient's own blood cells for a later transplant. The population of cells comprising T cells may be human cells. The population of cells comprising T cells may be further defined as primary cells.

[0014] The T cell may comprise an engineered T cell. The T cell may comprise a heterologous nucleic acid encoding a T cell receptor (TCR) or chimeric antigen receptor (CAR). The cell may be one that has been transduced with a viral vector comprising a nucleic acid encoding a TCR or CAR. The viral vector may comprise a lentiviral vector or a vector derived from a lentiviral vector. The viral vector may comprise a retroviral vector or a vector derived from a retroviral vector. The viral vector may comprise an adeno-associated virus (AAV) vector or a vector derived from an AAV vector. The virus may be packaged in a packaging cell. The virus may be packaged in HEK-293T cells. Non-limiting examples of lentiviral vectors include those derived from a lentivirus, such as Human Immunodeficiency Virus 1 (HIV-1), HIV-2, an Simian Immunodeficiency Virus (SIV), Human T-lymphotropic virus 1 (HTLV-1), HTLV-2 or equine infection anemia virus (E1AV). The nucleic acid may comprise an epHIV7 vector backbone. Other suitable viral vectors include, for example, , pLenti-III (Applied Biological Materials; cat # LV587); pl56RRLsinppt (Addgene; cat #42795); pFUGW (Addgene; cat #14883); pFUG (Addgene; cat #14882); pHAGE (Addgene; cat #46793); pHRsin (Addgene; cat #12265); pLenti (AMP) (Addgene; cat #61422); pLL3.7 m (Addgene; cat #89362); pLenti7.3 (Thermo Fisher Scientific; cat #V53406); pLenti (OriGene; cat #PS100109); pSF Lenti (Sigma; cat #OGS269); pLV-GFPSpark (Sinobiological; cat #LVCV-01); pLVX (Takara; cat #632164); pALD-Lenti (Aldevron), pLenti (Vigene; cat #P 100020), pLenti CMV (Addgene), and pLV. The viral vector may exclude epHIV7, pLenti-III (Applied Biological Materials; cat # LV587); pl56RRLsinppt (Addgene; cat #42795); pFUGW (Addgene; cat #14883); pFUG (Addgene; cat #14882);6137193686pHAGE (Addgene; cat #46793); pHRsin (Addgene; cat #12265); pLenti (AMP) (Addgene; cat #61422);93); pLL3.7 m (Addgene; cat #89362); pLenti7.3 (Thermo Fisher Scientific; cat #V53406); pLenti (OriGene; cat #PS100109); pSF_Lenti (Sigma; cat #OGS269); pLV-GFPSpark (Sinobiological; cat #LVCV-01); pLVX (Takara; cat #632164); pALD-Lenti (Aldevron), pLenti (Vigene; cat #P 100020), pLenti CMV (Addgene), and / or pLV.

[0015] In the transduced cells, the MOI may be at least, at most, or exactly 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10, or any derivable range therein. The cells may be transduced at a concentration of 1 x io6cells / mL. The transduced cells may be at a concentration of at least, at most, or exactly 1 x 103, 2 * 103, 3 x 103, 4 x 103, 5 x 103, 6 x 103, 7 x 103, 8 x 103, 9 x 103, 1 x io4, 2 x io4, 3 x 104, 4 x 104, 5 x 104, 6 x 104, 7 x 104, 8 x 104, 9 x 104, 1 x 1O5, 2 x IO5, 3 x 1O5, 4 x IO5, 5 x IO5, 6 x IO5, 7 x IO5, 8 x IO5, 9 x IO5, 1 x 106, 2 x 106, 3 x 106, 4 x 106, 5 x 106, 6 x 106, 7 x 106, 8 x 106, 9 x 106, 1 x 107, 2 x 107, 3 x 107, 4 x 107, 5 x 107, 6 x 107, 7 x 107, 8 x 107, 9 x 107, 1 x 108, 2 x 108, 3 x 108, 4 x 108, 5 x 108, 6 x 108, 7 x 108, 8 x 108, 9 x 108, 1 x io9, 2 x 109, 3 x 109, 4 x 109, 5 x 109, 6 x 109, 7 x 109, 8 x 109, 9 x 109, 1 x io10, 2 x io10, 3 x io10, 4 x 1O10, 5 x 1O10, 6 x 1O10, 7 x 1O10, 8 x 1O10, 9 x IO10, 1 x IQ11, 2 x IQ11, 3 x IQ11, 4 x IQ11, 5 x IQ11, 6 x IO11, 7 x IO11, 8 x IO11, 9 x IO11, 1 x 1012, 2 x 1012, 3 x IQ12, 4 x IQ12, 5 x IQ12, 6 x 1012, 7 x 1012, 8 x 1012, 9 x 1012, 1 x 1013, 2 x 1013, 3 x IQ13, 4 x IQ13, 5 x IQ13, 6 x IQ13, 7 x IQ13, 8 x IQ13, 9 x IQ13,Or 1 x IQ14cells / mL (or any derivable range therein). The cells may be expanded after the transduction. The cells may be expanded 1.5-25 folds after transduction. The cells may be expanded to, to at least, or to at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 fold (or any derivable range therein) after transduction. Expansion, splitting, or passage of the cells may be included or excluded in the methods or compositions of the disclosure.

[0016] The cell may be one in which a nucleic acid encoding the TCR or CAR has been transferred into the cell by a non-viral method.

[0017] The nucleic acid encoding the TCR or CAR may also be transferred into the cell by non-viral methods, such as gene editing and transfection procedures described herein.

[0018] The method may further comprise cryopreservation of the cell before or after contacting the cell with the T cell modulator. The cells may be cryopreserved at a concentration of 1 x 106cells / mL-15x 106cells / mL. The cells may be cryopreserved at a concentration of, of at 7137193686least, or of at most 1 x 103, 2 x 103, 3 x 103, 4 x 103, 5 x 103, 6 x 103, 7 x 103, 8 x 103, 9 x 103, 1 x 104, 2 x 1043 x 104, 4 x 104, 5 x 104, 6 x 104, 7 x 104, 8 x 104, 9 x 104, 1 x IO5, 2 x IO5, 3 x IO5, 4 x IO5, 5 x IO5, 6 x IO5, 7 x IO5, 8 x IO5, 9 x IO5, 1 x 106, 2 x 106, 3 x 106, 4 x 106, 5 x 106, 6 x 106, 7 x 106, 8 x 106, 9 x 106, 1 x 107, 2 x 107, 3 x 107, 4 x 107, 5 x IO7, 6 x 107, 7 x 107, 8 x 107, 9 x 107, 1 x io8, 2 x 108, 3 x 108, 4 x 108, 5 x 108, 6 x 108, 7 x 108, 8 x 108, 9 x 108, 1 x io9, 2 x 109, 3 x 109, 4 x 109, 5 x 109, 6 x 109, 7 x 109, 8 x 109, 9 x 109, 1 x io10, 2 x 1O10, 3 x 1O10, 4 x 1O10, 5 x 1O10, 6 x 1O10, 7 x 1O10, 8 x 1O10, 9 x 1O10, 1 x IO11, 2 x IO11, 3 x IO11, 4 x 1O11, 5 x 1O11, 6 x 1O11, 7 x 1O11, 8 x IO11, 9 x IO11, 1 x 1012, 2 x 1012, 3 x 1012, 4 x 1012, 5 x 1012, 6 x 1012, 7 x 1012, 8 x 1012, 9 x 1012, 1 x 1013, 2 x 1013, 3 x 1013, 4 x 1013, 5 x 1013, 6 x 1013, 7 x 1013, 8 x 1013, 9 x 1013, or 1 x 1014cells / mL (or any derivable range therein). The cells may be cryopreserved at a time period of less than 17 days after transduction. The cells may be cryopreserved at a time period of exactly, or of less than 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 days (or any derivable range therein) after transduction. The cells may be filtered prior to cry opreservation. Cry opreservation may also be excluded in the methods of the disclosure.

[0019] The population of cells may comprise CD4+ T cells, CD8+ T cells, or a mixture of CD4+ and CD8+ T cells. 1 x 106- 1 x IO10cells may be administered. The amount of cells administered to a subject may be, may be at least, or may be at most 1 x 102, 2 x 102, 3 x io2, 4 x 102, 5 x 102, 6 x 102, 7 x 102, 8 x 102, 9 x 102, 1 x 103, 2 x 103, 3 x 103, 4 x 103, 5 x 103, 6 x 103, 7 x 103, 8 x 103, 9 x 103, 1 x io4, 2 x 104, 3 x 104, 4 x 104, 5 x 104, 6 x 104, 7 x 104, 8 x 104, 9 x 104, 1 x io5, 2 x 105, 3 x io5, 4 x 105, 5 x 105, 6 x 105, 7 x 105, 8 x 105, 9 x 105, 1 x 106, 2 x 106, 3 x 106, 4 x 106, 5 x 106, 6 x 106, 7 x 106, 8 x 106, 9 x 106, 1 x 107, 2 x 107, 3 x 107, 4 x 107, 5 x 107, 6 x 107, 7 x 107, 8 x 107, 9 x 107, 1 x 108, 2 x 108, 3 x 108, 4 x 108, 5 x 108, 6 x 108, 7 x 108, 8 x 108, 9 x 108, 1 x 109, 2 x 109, 3 x 109, 4 x 109, 5 x 109, 6 x 109, 7 x 109, 8 x 109, 9 x 109, 1 x io10, 2 x 1O10, 3 x 1O10, 4 x 1O10, 5 x 1O10, 6 x 1O10, 7 x 1O10, 8 x 1010, 9 x 1O10, 1 x io11, 2 x 1011, 3 x 1011, 4 x 1011, 5 x IO11, 6 x IO11, 7 x IO11, 8 x IO11, 9 x 1011, 1 x io12, 2 x 1012, 3 x io12, 4 x 1012, 5 x 1012, 6 x 1012, 7 x 1012, 8 x 1012, 9 x 1012, 1 x 1013, 2 x 1013, 3 x io13, 4 x 1013, 5 x io13, 6 x 1013, 7 x 1013, 8 x 1013, 9 x 1013, or 1 x 1014cells (or any derivable range therein). The cells may be determined to be or evaluated as positive for expression of a CAR or TCR. The cells may be autologous cells. The cells may be allogeneic.

[0020] The subject may be one that is being treated with or has been prescribed an additional therapy. The method may further comprise administration of an additional therapy.8137193686The subject may exclude one that is being treated with an additional therapy. The method may exclude administration of an additional therapy. The additional therapy may include or exclude a therapy described herein. The additional therapy may be a chemotherapy. The additional therapy may comprise lymphodepletion. The additional therapy may include fludarabine and / or cyclophosphamide. The chemotherapy may comprise both fludarabine and cyclophosphamide. The additional therapy may be given prior to administration of the cells. The additional therapy may be given after administration of the cells. The additional therapy may be given to the subject at a time period of five days prior to administration of the cells. The additional therapy may be given to the subject at a time period of, of at least, or of at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 (or any derivable range therein) days prior to or after administration of the cells. The additional therapy may be an additional therapy described herein, such as an immunotherapy, inhibition of co-stimulatory molecules, dendritic cell therapy, TCR T cell therapy, CAR-T cell therapy, adoptive T-cell therapy, chemotherapy, radiotherapy, or surgery.

[0021] The methods may be for treating cancer in a subject having cancer. The cancer may include or exclude lung cancer, prostate cancer, ovarian cancer, testicular cancer, brain cancer, neuroblastoma, sarcoma, osteosarcoma, skin cancer, melanoma, colon cancer, rectal cancer, gastric cancer, esophageal cancer, tracheal cancer, head & neck cancer, pancreatic cancer, liver cancer, breast cancer, ovarian cancer, glioblastoma, glioma, a B-cell malignancy, melanoma, leukemia, sarcomas of bone or soft tissue, cervical cancer, and vulvar cancer. The cancer may include or exclude a cancer described herein. The melanoma may be further classified as superficial spreading, nodular, lentigo maligna, acral lentiginous, amelanotic, nevoid, spitzoid, desmoplastic, cutaneous, eye, choroidal, onjunctival, iris, and / or mucosal melanoma. The melanoma may exclude superficial spreading, nodular, lentigo maligna, acral lentiginous, amelanotic, nevoid, spitzoid, desmoplastic, cutaneous, eye, choroidal, onjunctival, iris, and / or mucosal melanoma.

[0022] The method may be for treating an autoimmune disorder. The autoimmune disorder may include or exclude an autoimmune disorder described herein. The autoimmune or inflammatory condition may include or exclude inflammatory bowel disease, idiopathic pulmonary fibrosis, multiple sclerosis, type 1 diabetes, Crohn’s disease, psoriasis, acute inflammation, chronic inflammation, neuroinflammation, arthritis, rheumatoid arthritis, fibrosis, infection, allergy, inflammatory therapy-related adverse events, and -related inflammatory illness.9137193686

[0023] The T cell modulator and immunotherapy may be administered at the same time. The T cell modulator may be administered prior to the immunotherapy. The T cell modulator may be administered after the immunotherapy. The T cell modulator may be administered at a time period of, of at least, or of at most or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 hours, minutes, or days, (or any derivable range therein) before the administration of the immunotherapy. The T cell modulator may be administered at a time period of, of at least, or of at most or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 hours, minutes, or days, (or any derivable range therein) after the administration of the immunotherapy. The T cell modulator may be administered at a time period of, of at least, or of at most or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 hours, minutes, or days, (or any derivable range therein) before or after the administration of the immunotherapy. The T cell modulator may be administered on the same day, within two days, or within a week of administration of the immunotherapy.

[0024] The immunotherapy may comprise or exclude an immune checkpoint inhibitor (ICI), an immune agonist antibody, or nucleic acids encoding for an immune checkpoint inhibitor or an immune agonist antibody. The ICI or immune agonist antibody may comprise an anti-CD40 agonistic antibody, anti-PDl blocking antibody, anti-PDLl blocking antibody, anti-CTLA4 blocking antibody, anti-LAG3 blocking antibody, or combinations thereof. The ICI may comprise one or more of Ipilimumab, Tremelimumab, Nivolumab, Pembrolizumab, Atezolizumab, Avelumab, Durvalumab, Spartalizumab, Cemiplimab, and Opdualag. The immunotherapy may comprise pro-inflammatory molecules or nucleic acids encoding for pro-inflammatory molecules. The pro-inflammatory molecule may comprise IL-2, IL-12, IL-15, or combinations thereof. The immunotherapy may comprise or exclude CAR-T cell therapy. The10137193686immunotherapy may comprise or exclude TCR-T cell therapy. The immunotherapy may comprise or exclude tumor infiltrating lymphocyte (TIL) cell therapy.

[0025] The T cell modulator may be administered systemically. The T cell modulator may be administered by intravenous injection. The T cell modulator may be administered intratum orally. The T cell modulator may be administered by a route of administration described herein.

[0026] The subject may be any animal, in particular a mouse, non-human primate, or human. The subject may have been determined to have or be at risk for cancer.

[0027] The cells or population of cells of the disclosure may be maintained in a cell culture medium throughout the methods of the claims. At any step of the process, the cell culture medium may be serum-free. The process may be one that maintains the cells in serum-free medium throughout all of the steps. The methods may exclude contacting the cells with serum.

[0028] The compositions and methods described herein may be modified so that the method is for preparing a T cell with a certain phenotype. The methods may be for preparing a T cell comprising or excluding the phenotype: CD4+CD8‘ T cells, CD4 CD8+T cells, CD34+CD7+CDla+cells, CD3+ TCRab+, CD3+ TCRgd+, CD3+ TCRab+ CD4+ CD8-, CD3+ TCRab+ CD8+ CD4-, CD3+ TCRab+ CD4+ CD8- CD45RO- CD45RA+, CD3+ TCRab+ CD8+ CD4- CD45RO- CD45RA+, CD3+ TCRab+ CD4+ CD8- CD45RO- CD45RA+ CCR7+, CD3+ TCRab+ CD8+ CD4- CD45RO- CD45RA+ CCR7+, CD3+ TCRab+ CD4+ CD8-CD45RO- CD45RA+ CD27+, CD3+ TCRab+ CD8+ CD4- CD45RO- CD45RA+ CD27+, CD34+CD7+CDla+cells, CD34+CD5+CD7+, CD34+CD5+CD7-, natural killer T cells, regulatory T cells, antigen-specific T cells, intraepithelial lymphocyte T cells, or cells that are CD45+, CDllb+, CDllb-, CD15+, CD15-, CD24+, CD24-, CD114+, CD114-, CD182+, CD182-, CD4+, CD4-, CD14+, CD14-, CDlla+, CD Ila-, CD91+, CD91-, CD16+, CD16-, CD3+, CD3-, CD25+, CD25-, Foxp3+, Fox3p-, CD8+, CD8-, CD19+, CD19-, CD20+, CD20-, CD24+, CD24, CD38+, CD38-, CD22+, CD22-, CD61+, CD61-, CD16+, CD16-, CD56+, CD56-, CD31+, CD31-, CD30+, CD30-, CD38+, and / or CD38- or combinations thereof.

[0029] The cells may be further defined as having or excluding the following phenotype: CD4 CD8' T cells, CD4 CD8+T cells, CD34+CD7+CDla+cells, CD3+ TCRab+, CD3+ TCRgd+, CD3+ TCRab+ CD4+ CD8-, CD3+ TCRab+ CD8+ CD4-, CD3+ TCRab+ CD4+ CD8- CD45RO- CD45RA+, CD3+ TCRab+ CD8+ CD4- CD45RO- CD45RA+, CD3+ TCRab+ CD4+ CD8- CD45RO- CD45RA+ CCR7+, CD3+ TCRab+ CD8+ CD4- CD45RO-CD45RA+ CCR7+, CD3+ TCRab+ CD4+ CD8- CD45RO- CD45RA+ CD27+, CD3+ TCRab+ CD8+ CD4- CD45RO- CD45RA+ CD27+, CD34+CD7+CDla+cells,11137193686CD34+CD5+CD7+, CD34+CD5+CD7-, natural killer T cells, regulatory T cells, antigenspecific T cells, intraepithelial lymphocyte T cells, or cells that are CD45+, CDllb+, CDllb-, CD15+, CD15-, CD24+, CD24-, CD114+, CD114-, CD182+, CD182-, CD4+, CD4-, CD14+, CD14-, CDlla+, CDlla-, CD91+, CD91-, CD16+, CD16-, CD3+, CD3-, CD25+, CD25-, Foxp3+, Fox3p-, CD8+, CD8-, CD19+, CD19-, CD20+, CD20-, CD24+, CD24, CD38+, CD38-, CD22+, CD22-, CD61+, CD61-, CD16+, CD16-, CD56+, CD56-, CD31+, CD31-, CD30+, CD30-, CD38+, and / or CD38- or combinations thereof.

[0030] Throughout this application, the term “about” is used according to its plain and ordinary meaning in the area of cell and molecular biology to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0031] The use of the word “a” or “an” when used in conjunction with the term “comprising” may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” Any term used in singular form also comprise plural form and vice versa.

[0032] As used herein, the terms “or” and “and / or” are utilized to describe multiple components in combination or exclusive of one another. For example, “x, y, and / or z” can refer to “x” alone, “y” alone, “z” alone, “x, y, and z,” “(x and y) or z,” “x or (y and z),” or “x or y or z.” It is specifically contemplated that x, y, or z may be specifically excluded from an embodiment or aspect.

[0033] The words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”), “characterized by” (and any form of including, such as “characterized as”), or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0034] The compositions and methods for their use can “comprise,” “consist essentially of,” or “consist of’ any of the ingredients or steps disclosed throughout the specification. The phrase “consisting of’ excludes any element, step, or ingredient not specified. The phrase “consisting essentially of’ limits the scope of described subject matter to the specified materials or steps and those that do not materially affect its basic and novel characteristics. It is contemplated that embodiments and aspects described in the context of the term “comprising” may also be implemented in the context of the term “consisting of’ or “consisting essentially of.”12137193686

[0035] Any method in the context of a therapeutic, diagnostic, or physiologic purpose or effect may also be described in “use” claim language such as “Use of’ any compound, composition, or agent discussed herein for achieving or implementing a described therapeutic, diagnostic, or physiologic purpose or effect.

[0036] Use of the one or more sequences or compositions may be employed based on any of the methods described herein. Other aspects and embodiments are discussed throughout this application. Any embodiment or aspect discussed with respect to one aspect of the disclosure applies to other aspects of the disclosure as well and vice versa.

[0037] It is specifically contemplated that any limitation discussed with respect to one embodiment or aspect of the invention may apply to any other embodiment or aspect of the invention. Furthermore, any composition of the invention may be used in any method of the invention, and any method of the invention may be used to produce or to utilize any composition of the invention. Aspects of an embodiment set forth in the Examples are also embodiments that may be implemented in the context of embodiments discussed elsewhere in a different Example or elsewhere in the application, such as in the Summary of Invention, Detailed Description of the Embodiments, Claims, and description of Figure Legends.

[0038] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments and aspects of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0040] FIG. 1. Establishing a high-throughput drug screen to pharmacologically improve neoepitope-specific T cell responses.

[0041] FIG. 2A-2B. Functional characterization of TCRs used in high-throughput screen. FIG. 2A: M490 co-culture with T cells engineered to express one of the three different neoantigen-specific TCRs (TCR66, TCR67, TCR68) targeting the same mutations expressed in the M490 cell line. An irrelevant TCR that does not target any mutation in M490 was used13137193686as a negative control. TCR67 (vCP67) showed high cytotoxicity, TCR68 (vCP68) had intermediate cytotoxicity and TCR66 (vCP66) did not shown any cytotoxicity. FIG. 2B: 4- IBB levels in T cells engineered to express TCR66, TCR67, TCR68 after 24 hours co-culture with the autologous M490 melanoma cells. TCR67 and TCR68 present similar levels of 4- IBB activation while TCR66 has reduced 4-1BB expression.

[0042] FIG. 3A-3B Dual luciferase / GFP detection to increase screen sensitivity. Coculture of T cells engineered to express either TCR66, TCR67 or TCR68 with melanoma cells in a 384 well plate. Luciferase activity (FIG. 3 A) was quantified as an endpoint after 48 hours while GFP cell count (FIG. 3B) was monitored every two hours.

[0043] FIG. 4 Screen workflow.

[0044] FIG. 5. Screening platform identifies known T cell regulators and novel hits in low cytotoxic TCRs and cytotoxic TCRs. Total of 7680 drugs tested.

[0045] FIG. 6A-6B Identification of Telmisartan as novel hit that increases T cell cytotoxicity. Graphs showing FDA drug library results for each of the different three TCRs. On top, drug’s z-score derived from luciferase data. Bottom, drug’s z-score derived from GFP readout. Telmisartan is highlighted in yellow. Shown in FIG. 6A are low cytotoxic TCRs and in FIG. 6B are cytotoxic TCRs.

[0046] FIG. 7A-7B. Telmisartan effect on T cell cytotoxicity is antigen-specific. FIG. 7A: M490 co-culture with T cells engineered to express one of the three different neoantigen-specific TCRs (TCR66, TCR67, TCR68) targeting the same mutation expressed in the M490 cell line. Telmisartan increased T cell cytotoxicity in all TCRs only if antigen was present. T cells engineered to express an NY-ESO TCR were used as a negative control, since M490 cells do not express NY-ESO- 1 and no cytotoxicity was observed. FIG. 7B: Representative image at 24 hours timepoint of T cell (TCR66 and NY-ESO) co-culture with melanoma cells (in green) with or without Telmisartan. Only TCR66 with telmisartan is able to kill melanoma cells.

[0047] FIG. 8. Telmisartan does not increase T cell cytotoxicity in mismatched cell lines. Here, the M489 cell line that does not express the antigen or the HLA that TCRs 66, 67 and 68 recognize was used. The inventors performed a M489 co-culture with T cells engineered to express one of the three different neoantigen-specific TCRs (TCR66, TCR67, TCR68) and observed that telmisartan did not lead to unspecific killing of M489 cells.

[0048] FIG. 9. Telmisartan does not increased T cell cytotoxicity in HLA matched cell lines with WT expression of the antigen. Here, the HLA-A2 positive M407 cell line that does not express the mutation recognized by TCRs 66, 67 and 68 was used. The inventors performed 14137193686a M407 co-culture with T cells engineered to express one of the three different neoantigen-specific TCRs (TCR66, TCR67, TCR68) and observed that telmisartan did not lead to unspecific killing of M407 cells.

[0049] FIG. 10 Telmisartan effect on T cell cytotoxicity is antigen-specific and it is not limited to a specific neoantigen or HLA class. Here we show the tumor growth inhibition percentage of M490 cells upon 60h co-culture with TCR-T cells engineered to express one of 11 different neoantigen-specific TCRs with or without 25uM Telmisartan.

[0050] FIG. 11 Telmisartan increased 4-1BB levels in T cells engineered to express either TCR66, TCR67, TCR68 after 24 hours co-culture with the autologous M490 melanoma cells. T cells expressing an irrelevant NY-ESO-1 TCR were used as a negative control (TCR NY).

[0051] FIG. 12 Telmisartan effect is dose-dependent. Showing co-cultures of TCR-engineered T cells expressing TCR66 (top) and TCR68 (bottom) with the M490 cell line and doses of 0.1 pM, 0.25 pM, 0.5 pM, 2.5 pM and 5 pM of Telmisartan.

[0052] FIG. 13. Telmisartan allows the reduction of E:T without compromising cytotoxicity. T cell / melanoma cell co-culture with or without Telmisartan at different E:T ratios: 5:1, 2.5:1, 1:1 and 1:2.5. Showing normalized data for each TCR.

[0053] FIG. 14 Telmisartan bypasses the need of pre-treating M490 cells with IFNg to increase HLA expression. Co-culture of T cells engineered to express either TCR66, TCR67 or TCR68 with melanoma cells that have not been pre-treated with IFNg. Telmisartan allows T cell killing in the absence of melanoma cell IFNg pre-treatment. Of note, killing is slightly slower compared to melanoma cell that have been pre-treated with IFNg.

[0054] FIG. 15. Repetitive antigen challenge experiment. Telmisartan allowed T cells engineered to express TCR66 (vCP66) and TCR68 (vCP68) to maintain its cytotoxic activity through three antigen challenges. T cells engineered to express TCR67 (vCP67), maintained its cytotoxic activity through two antigen challenges.

[0055] FIG. 16A-16B Telmisartan improves ACT efficacy in vivo in a dose-dependent fashion and increases tumor T cell infiltration. FIG. 16 A: Tumor growth curves of M490 tumors treated with TCR68 cells (5M per mice) and 3 doses of 5 pg of systemic IL2 alone (blue) or in combination with Telmisartan (500mg / kg, green) or Telmisartan (150mg / kg, red). FIG.16B: Quantification of the T cells in the tumor (left) and spleen (right) at the end of the experiment. Telmisartan at 500mg / kg increased T cell tumor infiltration.

[0056] FIG. 17A-17B Telmisartan improves the antitumor activity of ICB in vivo. FIG.17A: Tumor growth curves of MC38 tumors treated with anti-PDl (300ug), Telmisartan15137193686(500mg / kg), anti-PD-1 + Telmisartan, and untreated. FIG. 17B. MC38 cell line growth in vitro with or without 25 pM Telmisartan. Telmisartan does not affect MC38 growth in vitro.

[0057] FIG. 18. Telmisartan does not impact functional avidity when using plate-bound peptide MHC. To determine functional avidity, we measured 4-1BB expression levels as a readout of T cell activation 24h after a co-culture with a plate-bound HLA-peptide titration with or without Telmisartan.

[0058] FIG. 19. Schematic of Telmisartan mechanism of action. Telmisartan is an angiotensin receptor 1 blocker with PPARy agonistic activity.

[0059] FIG. 20. AT1R is not expressed in human T cells + no additional ARBs hits. A) Showing AT1R log2FPKMs from human T-cells that have not seen antigen (baseline) or after antigen exposure (RAC1, RAC3). B) Z-score plot for all hypertensive drugs used in FDA drug library. Only Telmisartan increased T cell cytotoxicity.

[0060] FIG. 21. PPARy inhibition does not block Telmisartan effect. T-cell co-culture with melanoma cells with different combinations of drugs: telmisartan alone, PPARy inhibitor alone or combination of both. Blocking telmisartan PPARy agonistic activity does not prevent T cell killing.

[0061] FIG. 22. Few transcriptomic changes upon Telmisartan treatment. Volcano plot derived from the RNA-seq analysis of T cells expressing either TCR68, NYESO or no TCR after 24h co-culture with melanoma cells with or without Telmisartan.

[0062] FIG. 23. Surface proteomics revels effect of Telmisartan in immunological-synapse related proteins. Volcano plot derived from the cell surface protein quantification between untreated vs telmisartan treated T cells. ICAM2, TMIGD2 and MSN among other proteins were up-regulated in telmisartan treated samples.

[0063] FIG. 24 Scatter plot showing z-scores of the co-cultures with melanoma cells + drug vs. melanoma cells + ATP11 A TCR-T cells +drug. Plotted results from GFP readout.

[0064] FIG. 25A-25E. AZD4572 sensitizes melanoma cells to T cell killing. FIG. 25A. Tumor growth inhibition of M490 upon co-culture with neoTCR-T cells engineered to express ATP11 A TCRs with or without AZD4573 at 20nM. FIG. 25B. T-cell activation after 24h coculture with M490 measured by 4- IBB upregulation. FIG. 25C-25E. Quantification of cytokine release: IFNg (FIG. 25C), TNF (FIG. 25D) and IL-2 (FIG. 25E) after 24h coculture with M490.16137193686DETAILED DESCRIPTION

[0065] T cells targeting antigens expressed in the tumor can lead to successful clearance of tumor masses, as shown clinically in several cancer types. The interaction between the T cell and the tumor through the TCR-peptide-HLA axis is key to generating a strong and long-lasting T-cell response. Identifying strategies to modulate this interaction and increase T-cell effector functions could lead to potential new immunotherapeutic approaches to improve the antitumor activity of T-cell-based immunotherapies. The inventors established a high-throughput drug screen using neoantigen-specific patient-derived TCRs together with their autologous melanoma cell lines to identify drugs that improve T-cell cytotoxicity. They tested up to 7680 compounds from five different drug libraries. Importantly, with their screening platform, they successfully identified drugs that had not been previously associated with T-cell cytotoxicity. Interestingly, they found that the FDA-approved drug, Telmisartan and other compounds, increased T-cell cytotoxicity in an antigen-specific manner, with a dose-dependent effect. In addition, Telmisartan increased T-cell activation and allowed the reduction of effector-to-target ratios without compromising T-cell cytotoxicity. Importantly, Telmisartan maintained its activity on T cells through multiple consecutive antigen challenges. Furthermore, the inventors have demonstrated that Telmisartan improved the efficacy of adoptive T-cell therapy in a TCR model in vivo, as well as the activity of anti-PD-1 in the syngeneic murine cancer model MC38. Overall, these results depict the potential of Telmisartan in novel immunotherapy drug combinations.I. Administration of Therapeutic Compositions

[0066] The therapeutic cells of the disclosure may be administered by any route of administration. The cells may be administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. In some embodiments, the T cell modulator is administered intravenously, intramuscularly, intratumorally, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. Any of these may be excluded in an embodiment. The appropriate dosage may be determined based on the type of disease to be treated, severity and course of the disease, the clinical condition of the individual, the individual's clinical history and response to the treatment, and the discretion of the attending physician.17137193686

[0067] The treatments may include various “unit doses.” Unit dose is defined as containing a predetermined-quantity of the therapeutic composition. The quantity to be administered, and the particular route and formulation, is within the skill of determination of those in the clinical arts. A unit dose need not be administered as a single injection but may comprise continuous infusion over a set period of time. A unit dose may comprise a single administrable dose.

[0068] Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the patient, the route of administration, the intended goal of treatment (alleviation of symptoms versus cure) and the potency, stability and toxicity of the particular therapeutic substance or other therapies a subject may be undergoing.

[0069] It will be understood by those skilled in the art and made aware that dosage units of pg / kg or mg / kg of body weight can be converted and expressed in comparable concentration units of pg / ml or mM (blood levels), such as 4 pM to 100 pM. It is also understood that uptake is species and organ / tissue dependent. The applicable conversion factors and physiological assumptions to be made concerning uptake and concentration measurement are well-known and would permit those of skill in the art to convert one concentration measurement to another and make reasonable comparisons and conclusions regarding the doses, efficacies and results described herein.

[0070] It may be desirable to have multiple administrations of the composition, e.g., 2, 3, 4, 5, 6 or more administrations (or any range derivable therein). The administrations can be at 1, 2, 3, 4, 5, 6, 7, 8, to 5, 6, 7, 8, 9, 10, 11, or 12 week intervals, including all ranges there between.

[0071] The phrases “pharmaceutically acceptable” or “pharmacologically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal or human. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, anti-bacterial and anti-fungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients, its use in immunogenic and therapeutic compositions is contemplated. Supplementary active ingredients, such as other anti-infective agents and vaccines, can also be incorporated into the compositions.

[0072] The cells can be formulated for parenteral administration, e.g., formulated for inj ection via the intravenous, intramuscular, subcutaneous, or intraperitoneal routes. Typically,18137193686such compositions can be prepared as either liquid solutions or suspensions; solid forms suitable for use to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and, the preparations can also be emulsified.

[0073] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including, for example, aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that it may be easily injected. It also should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

[0074] The compositions may be formulated into a neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.

[0075] A pharmaceutical composition can include a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various anti-bacterial and anti-fungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0076] Sterile injectable solutions are prepared by incorporating the active components in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filtered sterilization or an equivalent procedure. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable 19137193686solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques, which yield a powder of the active ingredient, plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0077] Administration of the compositions will typically be via any common route. This includes, but is not limited to oral, or intravenous administration. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, or intranasal administration. Such compositions would normally be administered as pharmaceutically acceptable compositions that include physiologically acceptable carriers, buffers or other excipients.

[0078] Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above.II. Cellular TherapiesA. Cell Culture

[0079] Cells may be cultured for at least between about 2 days and about 40 days, for at least between about 7 days and about 35 days, for at least between about 9 days and 21 days, such as for at least about 3, 5, 7, 9, 12 or 15 days. In some aspects, the cells of the disclosure may be cultured for no longer than 60 days, or no longer than 50 days, or no longer than 45 days. The cells may be cultured for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 days (or any range derivable therein). The cells may be cultured in the presence of a liquid culture medium. Typically, the medium may comprise a basal medium formulation as known in the art. Many basal media formulations can be used to culture cells herein, including but not limited to Eagle's Minimum Essential Medium (MEM), Dulbecco's Modified Eagle's Medium (DMEM), alpha modified Minimum Essential Medium (alpha-MEM), Basal Medium Essential (BME), Iscove's Modified Dulbecco's Medium (IMDM), BGJb medium, F-12 Nutrient Mixture (Ham), Liebovitz L-15, DMEM / F-12, Essential Modified Eagle's Medium (EMEM), RPMI-1640, and modifications and / or combinations thereof. Compositions of the above basal media are generally known in the art, and it is within the skill of one in the art to modify or modulate concentrations of media and / or media supplements as necessary for the cells cultured. In some aspects, a culture medium formulation may be explants medium (CEM) which is composed of20137193686IMDM supplemented with 10% fetal bovine serum (FBS), 100 U / ml penicillin G, 100 pg / ml streptomycin and 2 mmol / L L-glutamine. Other aspects may employ further basal media formulations, such as chosen from the ones above.

[0080] Any medium capable of supporting cells in vitro may be used to culture the cells. Media formulations that can support the growth of cells include, but are not limited to, Dulbecco's Modified Eagle's Medium (DMEM), alpha modified Minimal Essential Medium (aMEM), and Roswell Park Memorial Institute Media 1640 (RPMI Media 1640) and the like. Typically, up to 20% fetal bovine serum (FBS) or 1-20% horse serum is added to the above medium in order to support the growth of cells. A defined medium, however, also can be used if the growth factors, cytokines, and hormones necessary for culturing cells are provided at appropriate concentrations in the medium. Media useful in the methods of the disclosure may comprise one or more compounds of interest, including, but not limited to, antibiotics, mitogenic compounds, or differentiation compounds useful for the culturing of cells. The cells may be grown at temperatures between 27° C to 40° C, such as 31° C to 37° C, and may be in a humidified incubator. The carbon dioxide content may be maintained between 2% to 10% and the oxygen content may be maintained between 1% and 22%. The disclosure, however, should in no way be construed to be limited to any one method of isolating and culturing cells. Rather, any method of isolating and culturing cells should be construed to be included in the present disclosure.

[0081] For use in the cell culture, media can be supplied with one or more further components. For example, additional supplements can be used to supply the cells with the necessary trace elements and substances for optimal growth and expansion. Such supplements include insulin, transferrin, selenium salts, and combinations thereof. These components can be included in a salt solution such as, but not limited to, Hanks' Balanced Salt Solution (HBSS), Earle's Salt Solution. Further antioxidant supplements may be added, e.g., P-mercaptoethanol. While many media already contain amino acids, some amino acids may be supplemented later, e.g., L-glutamine, which is known to be less stable when in solution. A medium may be further supplied with antibiotic and / or antimycotic compounds, such as, typically, mixtures of penicillin and streptomycin, and / or other compounds, exemplified but not limited to, amphotericin, ampicillin, gentamicin, bleomycin, hygromycin, kanamycin, mitomycin, mycophenolic acid, nalidixic acid, neomycin, nystatin, paromomycin, polymyxin, puromycin, rifampicin, spectinomycin, tetracycline, tylosin, and zeocin. Also contemplated is supplementation of cell culture medium with mammalian plasma or sera. Plasma or sera often21137193686contain cellular factors and components that are necessary for viability and expansion. The use of suitable serum replacements is also contemplated.

[0082] Reference to particular buffers, media, reagents, cells, culture conditions and the like, or to some subclass of same, is not intended to be limiting, but should be read to include all such related materials that one of ordinary skill in the art would recognize as being of interest or value in the particular context in which that discussion is presented. For example, it is often possible to substitute one buffer system or culture medium for another, such that a different but known way is used to achieve the same goals as those to which the use of a suggested method, material or composition is directed. Cells may be cultured in a cell culture system comprising a cell culture medium, preferably in a culture vessel, in particular a cell culture medium supplemented with a substance suitable and determined for protecting the cells from in vitro aging and / or inducing in an unspecific or specific reprogramming.B. Pharmaceutical Compositions

[0083] In certain aspects, the compositions or agents for use in the methods, such as the cell compositions, are suitably contained in a pharmaceutically acceptable carrier. The carrier is non-toxic, biocompatible and is selected so as not to detrimentally affect the biological activity of the agent. The agents in some aspects of the disclosure may be formulated into preparations for local delivery (i.e. to a specific location of the body) or systemic delivery, in solid, semi-solid, gel, liquid or gaseous forms such as tablets, capsules, powders, granules, ointments, solutions, depositories, inhalants and injections allowing for oral, parenteral or surgical administration. Certain aspects of the disclosure also contemplate local administration of the compositions by coating medical devices and the like.

[0084] Suitable carriers for parenteral delivery via injectable, infusion or irrigation and topical delivery include distilled water, physiological phosphate-buffered saline, normal or lactated Ringer's solutions, dextrose solution, Hank's solution, or propanediol. In addition, sterile, fixed oils may be employed as a solvent or suspending medium. For this purpose any biocompatible oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. The carrier and agent may be compounded as a liquid, suspension, polymerizable or non-polymerizable gel, paste or salve.

[0085] The carrier may also comprise a delivery vehicle to sustain (i.e., extend, delay or regulate) the delivery of the agent(s) or to enhance the delivery, uptake, stability or22137193686pharmacokinetics of the therapeutic agent(s). Such a delivery vehicle may include, by way of non-limiting examples, microparticles, microspheres, nanospheres or nanoparticles composed of proteins, liposomes, carbohydrates, synthetic organic compounds, inorganic compounds, polymeric or copolymeric hydrogels and polymeric micelles.

[0086] In certain aspects, the actual dosage amount of a composition administered to a patient or subject can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.

[0087] Solutions of pharmaceutical compositions can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions also can be prepared in glycerol, liquid polyethylene glycols, mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0088] In certain aspects, the pharmaceutical compositions are advantageously administered in the form of injectable compositions either as liquid solutions or suspensions; solid forms suitable or solution in, or suspension in, liquid prior to injection may also be prepared. These preparations also may be emulsified. A typical composition for such purpose comprises a pharmaceutically acceptable carrier. For instance, the composition may contain 10 mg or less, 25 mg, 50 mg or up to about 100 mg of human serum albumin per milliliter of phosphate buffered saline. Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like.

[0089] Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters such as ethyloleate. Aqueous carriers include water, alcoholic / aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc. Intravenous vehicles include fluid and nutrient replenishers. Preservatives include antimicrobial agents, antgifungal agents, anti-oxidants, chelating agents and inert gases. The pH and exact concentration of the various components the pharmaceutical composition are adjusted according to well-known parameters.

[0090] Additional formulations are suitable for oral administration. Oral formulations include such typical excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. The compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders.23137193686

[0091] In further aspects, the pharmaceutical compositions may include classic pharmaceutical preparations. Administration of pharmaceutical compositions according to certain aspects may be via any common route so long as the target tissue is available via that route. This may include oral, nasal, buccal, rectal, vaginal or topical. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Such compositions would normally be administered as pharmaceutically acceptable compositions that include physiologically acceptable carriers, buffers or other excipients. For treatment of conditions of the lungs, aerosol delivery can be used. Volume of the aerosol may be between about 0.01 ml and 0.5 ml, for example.

[0092] An effective amount of the pharmaceutical composition is determined based on the intended goal. The term “unit dose” or “dosage” refers to physically discrete units suitable for use in a subject, each unit containing a predetermined-quantity of the pharmaceutical composition calculated to produce the desired responses discussed above in association with its administration, i.e., the appropriate route and treatment regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the protection or effect desired.

[0093] Precise amounts of the pharmaceutical composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting the dose include the physical and clinical state of the patient, the route of administration, the intended goal of treatment (e.g., alleviation of symptoms versus cure) and the potency, stability and toxicity of the particular therapeutic substance.C. Cells

[0094] Certain aspects relate to engineered cells that are contacted with modulators to modify the cytotoxicity of the cell. In some aspects the cell is an immune cell or a T cell. “T cell” includes all types of immune cells expressing CD3 including T-helper cells, invariant natural killer T (iNKT) cells, cytotoxic T cells, T-regulatory cells (Treg) gamma-delta T cells, natural-killer (NK) cells, and neutrophils. The T cell may refer to a CD4+ or CD8+ T cell.

[0095] In some instances, the cell is not an immortalized cell line, but is instead a cell (e.g., a primary cell) obtained from an individual. For example, in some cases, the cell is an immune cell obtained from an individual. As an example, the cell is a T lymphocyte obtained from an individual. As another example, the cell is a cytotoxic cell obtained from an individual. As24137193686another example, the cell is a stem cell (e.g., peripheral blood stem cell) or progenitor cell obtained from an individual.III. Methods for modifying genomic DNA

[0096] In certain embodiments, the genomic DNA is modified either to include additional mutations, insertions, or deletions, or to integrate certain molecular constructs of the disclosure so that the constructs are expressed from the genomic DNA. In some embodiments, a nucleic acid encoding a polypeptide of the disclosure is integrated into the genomic DNA of a cell. In some embodiments, a nucleic acid is integrated into a cell via viral transduction, such as gene transfer by lentiviral or retroviral transduction. In some embodiments, genomic DNA is modified by integration of nucleic acid encoding a polypeptide of the present disclosure (e.g., a CAR) into the genome of a host cell via a retroviral vector, a lentiviral vector, or an adeno-associated viral vector.

[0097] In some embodiments, the integration is targeted integration. In some embodiments, targeted integration is achieved through the use of a DNA digesting agent / polynucleotide modification enzyme, such as a site-specific recombinase and / or a targeting endonuclease. The term “DNA digesting agent” refers to an agent that is capable of cleaving bonds (i.e. phosphodiester bonds) between the nucleotide subunits of nucleic acids. One specific target is the TRAC (T cell receptor alpha constant) locus. For instance, cells would first be electroporated with a ribonucleoprotein (RNP) complex consisting of Cas9 protein complexed with a single-guide RNA (sgRNA) targeting the TRAC (T cell receptor alpha constant) locus. Fifteen minutes post electroporation, the cells would be treated with AAV6 carrying the HDR template that encodes for the CAR. In another example, double stranded or single stranded DNA comprises the HDR template and is introduced into the cell via electroporation together with the RNP complex.

[0098] Therefore, one aspect, the current disclosure includes targeted integration. One way of achieving this is through the use of an exogenous nucleic acid sequence (i.e., a landing pad) comprising at least one recognition sequence for at least one polynucleotide modification enzyme, such as a site-specific recombinase and / or a targeting endonuclease. Site-specific recombinases are well known in the art, and may be generally referred to as invertases, resolvases, or integrases. Non-limiting examples of site-specific recombinases may include lambda integrase, Cre recombinase, FLP recombinase, gamma-delta resolvase, Tn3 resolvase, C31 integrase, Bxbl -integrase, and R4 integrase. Site-specific recombinases recognize25137193686specific recognition sequences (or recognition sites) or variants thereof, all of which are well known in the art. For example, Cre recombinases recognize LoxP sites and FLP recombinases recognize FRT sites.

[0099] Contemplated targeting endonucleases include zinc finger nucleases (ZFNs), meganucleases, transcription activator-like effector nucleases (TALENs), CRISPR / Cas-like endonucleases, I-Tevl nucleases or related monomeric hybrids, or artificial targeted DNA double strand break inducing agents. Exemplary targeting endonucleases is further described below. For example, typically, a zinc finger nuclease comprises a DNA binding domain (i.e., zinc finger) and a cleavage domain (i.e., nuclease), both of which are described below. Also included in the definition of polynucleotide modification enzymes are any other useful fusion proteins known to those of skill in the art, such as may comprise a DNA binding domain and a nuclease.

[0100] A landing pad sequence is a nucleotide sequence comprising at least one recognition sequence that is selectively bound and modified by a specific polynucleotide modification enzyme such as a site-specific recombinase and / or a targeting endonuclease. In general, the recognition sequence(s) in the landing pad sequence does not exist endogenously in the genome of the cell to be modified. For example, where the cell to be modified is a CHO cell, the recognition sequence in the landing pad sequence is not present in the endogenous CHO genome. The rate of targeted integration may be improved by selecting a recognition sequence for a high efficiency nucleotide modifying enzyme that does not exist endogenously within the genome of the targeted cell. Selection of a recognition sequence that does not exist endogenously also reduces potential off-target integration. In other aspects, use of a recognition sequence that is native in the cell to be modified may be desirable. For example, where multiple recognition sequences are employed in the landing pad sequence, one or more may be exogenous, and one or more may be native.

[0101] One of ordinary skill in the art can readily determine sequences bound and cut by site-specific recombinases and / or targeting endonucleases.

[0102] Another example of a targeting endonuclease that can be used is an RNA-guided endonuclease comprising at least one nuclear localization signal, which permits entry of the endonuclease into the nuclei of eukaryotic cells. The RNA-guided endonuclease also comprises at least one nuclease domain and at least one domain that interacts with a guiding RNA. An RNA-guided endonuclease is directed to a specific chromosomal sequence by a guiding RNA such that the RNA-guided endonuclease cleaves the specific chromosomal sequence. Since the guiding RNA provides the specificity for the targeted cleavage, the endonuclease of the RNA- 26137193686guided endonuclease is universal and may be used with different guiding RNAs to cleave different target chromosomal sequences. Discussed in further detail below are exemplary RNA-guided endonuclease proteins. For example, the RNA-guided endonuclease can be a CRISPR / Cas protein or a CRISPR / Cas-like fusion protein, an RNA-guided endonuclease derived from a clustered regularly interspersed short palindromic repeats (CRISPR) / CRISPR-associated (Cas) system.

[0103] The targeting endonuclease can also be a meganuclease. Meganucleases are endodeoxyribonucleases characterized by a large recognition site, i.e., the recognition site generally ranges from about 12 base pairs to about 40 base pairs. As a consequence of this requirement, the recognition site generally occurs only once in any given genome. Among meganucleases, the family of homing endonucleases named “LAGLID ADG” has become a valuable tool for the study of genomes and genome engineering. Meganucleases may be targeted to specific chromosomal sequence by modifying their recognition sequence using techniques well known to those skilled in the art. See, for example, Epinat et al., 2003, Nuc. Acid Res., 31(ll):2952-62 and Stoddard, 2005, Quarterly Review of Biophysics, pp. 1-47.

[0104] Yet another example of a targeting endonuclease that can be used is a transcription activator-like effector (TALE) nuclease. TALEs are transcription factors from the plant pathogen Xanthomonas that may be readily engineered to bind new DNA targets. TALEs or truncated versions thereof may be linked to the catalytic domain of endonucleases such as FokI to create targeting endonuclease called TALE nucleases or TALENs. See, e.g., Sanjana et al., 2012, Nature Protocols 7(1): 171-192; Bogdanove A J, Voytas D F., 2011, Science, 333(6051):1843-6; Bradley P, Bogdanove A J, Stoddard B L., 2013, Curr Opin Struct Biol., 23(l):93-9.IV. Immunotherapy

[0105] The methods include administration of an immunotherapy and / or treating subjects that are being treated or will be treated with an immunotherapy (i.e. have been prescribed an immunotherapy). Cancer immunotherapy (sometimes called immuno-oncology, abbreviated IO) is the use of the immune system to treat cancer. Immunotherapies can be categorized as active, passive or hybrid (active and passive). These approaches exploit the fact that cancer cells often have molecules on their surface that can be detected by the immune system, known as tumor-associated antigens (TAAs); they are often proteins or other macromolecules (e.g. carbohydrates). Active immunotherapy directs the immune system to attack tumor cells by27137193686targeting TAAs. Passive immunotherapies enhance existing anti-tumor responses and include the use of monoclonal antibodies, lymphocytes and cytokines. Immunotherapies are known in the art, and some are described below.A. Adoptive T-cell therapy

[0106] Adoptive T cell therapy is a form of passive immunization by the transfusion of T-cells (adoptive cell transfer). They are found in blood and tissue and usually activate when they find foreign pathogens or tumor antigens. Specifically they activate when the T-cell's surface receptors encounter cells that display parts of foreign proteins on their surface antigens. These can be either infected cells, or antigen presenting cells (APCs). They are found in normal tissue and in tumor tissue, where they are known as tumor infiltrating lymphocytes (TILs). They are activated by the presence of APCs such as dendritic cells that present tumor antigens. Although these cells can attack the tumor, the environment within the tumor is highly immunosuppressive, preventing immune-mediated tumor death.

[0107] Multiple ways of producing and obtaining tumor targeted T-cells have been developed. T-cells specific to a tumor antigen can be isolated from a tumor sample (TILs) or from blood. Subsequent activation and culturing is performed ex vivo, with the expansion and the reinfusion of the resulting cells. Activation can take place through gene therapy, or by exposing the T cells to tumor antigens. Additional details on the preparation, selection, use, combination with other therapies, an / or administration of cells for ACT treatment are described in the literature (Cook K et al., 2018, Elahi R et al., 2018; Sharma P. et al., 2017).

[0108] In some aspects, the adoptive cell therapy comprises dendritic cell therapy, which provokes anti-tumor responses by causing dendritic cells to present tumor antigens to lymphocytes, and then activates them, priming them to kill other cells that present the antigen. Dendritic cells are antigen presenting cells (APCs) in the mammalian immune system. In cancer treatment they aid cancer antigen targeting. One example of cellular cancer therapy based on dendritic cells is sipuleucel-T. One method of inducing dendritic cells to present tumor antigens is by vaccination with autologous tumor lysates or short peptides (small parts of protein that correspond to the protein antigens on cancer cells). These peptides are often given in combination with adjuvants (highly immunogenic substances) to increase the immune and anti-tumor responses. Other adjuvants include proteins or other chemicals that attract and / or activate dendritic cells, such as granulocyte macrophage colony-stimulating factor (GM-CSF).28137193686

[0109] Dendritic cells can also be activated in vivo by making tumor cells express GM-CSF. This can be achieved by either genetically engineering tumor cells to produce GM-CSF or by infecting tumor cells with an oncolytic virus that expresses GM-CSF. Another strategy is to remove dendritic cells from the blood of a patient and activate them outside the body. The dendritic cells are activated in the presence of tumor antigens, which may be a single tumorspecific peptide / protein or a tumor cell lysate (a solution of broken down tumor cells). These cells (with optional adjuvants) are infused and provoke an immune response.

[0110] Dendritic cell therapies may include the use of antibodies that bind to receptors on the surface of dendritic cells. Antigens can be added to the antibody and can induce the dendritic cells to mature and provide immunity to the tumor. Dendritic cell receptors such as TLR3, TLR7, TLR8 or CD40 have been used as antibody targets.[OHl] In some aspects, the adoptive cell therapy comprises CAR-T cell therapy. Chimeric antigen receptors (CARs, also known as chimeric immunoreceptors, chimeric T cell receptors or artificial T cell receptors) are engineered receptors that combine a new specificity with an immune cell to target cancer cells. Typically, these receptors graft the specificity of a monoclonal antibody onto a T cell. The receptors are called chimeric because they are fused of parts from different sources. CAR-T cell therapy refers to a treatment that uses such transformed cells for cancer therapy. Exemplary CAR-T therapies include Tisagenlecleucel (Kymriah) and Axicabtagene ciloleucel. In some aspects, the CAR-T therapy targets CD 19 or CD20.

[0112] In some aspects, the adoptive cell therapy comprises engineered TCR-T cell therapy, which is further described below.B. T Cell Receptors (TCRs) and TCR-T cell therapy

[0113] The T cell receptor or TCR is a molecule found on the surface of T lymphocytes (T cells) that is responsible for recognizing fragments of antigen as peptides bound to major histocompatibility complex (MHC) molecules. The TCR is composed of two different protein chains (that is, it is a heterodimer). In 95% of T cells in humans, the TCR consists of an alpha (a; also referred to herein as “a”) and beta (P - also referred to herein is “b”) chain, whereas in 5% of T cells the TCR consists of gamma and delta (y / 5) chains. This ratio changes during ontogeny and in diseased states as well as in different species.

[0114] When the TCR engages with antigenic peptide and MHC (peptide / MHC), the T lymphocyte is activated through signal transduction, that is, a series of biochemical events29137193686mediated by associated enzymes, co-receptors, specialized adaptor molecules, and activated or released transcription factors. The TCR is a disulfide-linked membrane-anchored heterodimeric protein normally consisting of the highly variable alpha (a) and beta (P) chains expressed as part of a complex with the invariant CD3 chain molecules. T cells expressing this receptor are referred to as a:P (or aP or ab) T cells, though a minority of T cells express an alternate receptor, formed by variable gamma (y - also referred to herein as “g”) and delta (8 -also referred to herein as “d”) chains, referred as y6 (or gd) T cells.

[0115] Each chain is composed of two extracellular domains: Variable (V) region and a Constant (C) region, both of Immunoglobulin superfamily (IgSF) domain forming antiparallel P-sheets. The constant region is proximal to the cell membrane, followed by a transmembrane region and a short cytoplasmic tail, while the Variable region binds to the peptide / MHC complex.

[0116] The variable domain of both the TCR a-chain and P-chain each have three hypervariable or complementarity determining regions (CDRs), whereas the variable region of the P-chain has an additional area of hypervariability (HV4) that does not normally contact antigen and, therefore, is not considered a CDR.

[0117] The residues are located in two regions of the TCR, at the interface of the a- and P-chains and in the P-chain framework region that is thought to be in proximity to the CD3 signaltransduction complex. CDR3 is the main CDR responsible for recognizing processed antigen, although CDR1 of the alpha chain has also been shown to interact with the N-terminal part of the antigenic peptide, whereas CDR1 of the P-chain interacts with the C-terminal part of the peptide. CDR2 is thought to recognize the MHC. CDR4 of the P-chain is not thought to participate in antigen recognition, but has been shown to interact with superantigens. The constant domain of the TCR domain consists of short connecting sequences in which a cysteine residue forms disulfide bonds, which forms a link between the two chains.

[0118] The TCR being a member of the IgSF protein means it may be compared to antibodies and BCR. In terms of similarity, TCR is like half an antibody with a heavy and a light chain, except the heavy chain is without its crystallisable fraction (Fc) (Note: ontogenically TCR alpha undergo VJ recombination, so it is like a light chain; TCR beta undergoes VDJ recombination, so it is like a heavy chain). So the TCR is ontologically like one of the antibody-binding fragments of the antibody. The two subunits of TCR are twisted together. Whereas the antibody uses its Fc region to bind to Fc Receptors on innate leukocytes, TCR is already docked onto the cell membrane. However, it is not able to mediate signal transduction itself due to its short cytoplasmic tail, so TCR still requires CD3 and zeta to carry 30137193686out the signal transduction in its place, just as antibodies requires binding to FcRs to initiate signal transduction. In this way the MHC-TCR-CD3 interaction for T cells is functionally similar to the Ag-Ig-FcR interaction for myeloid leukocytes, and Ag-Ig-CD79 interaction for B cells.

[0119] The cells of the disclosure may comprise an engineered TCR. The TCR may comprise proteins expressed from TCR-alpha and TCR-beta genes. The engineered TCR may comprise proteins expressed from TCR-gamma and TCR-delta genes. The engineered TCR may comprise proteins expressed from TCR-alpha and TCR-beta genes and the antigen recognition receptor comprises proteins expressed from the TCR-gamma and TCR-delta genes. The engineered TCR may comprise proteins expressed from TCR-gamma and TCR-delta genes and the antigen recognition receptor comprises proteins expressed from the TCR-alpha and TCR-beta genes.

[0120] Methods of identifying antigen-specific TCRs are known in the art. Methods may include, for example, 1) Synthesizing known or predicted HLA-restricted peptide epitopes derived from proteins of interest (e.g. tumor antigens, neoantigens from sequencing data, etc.); 2) presenting these via an antigen-presenting cell (for expansion) or tetramer (for direct sorting) to a pool of T cells from which TCR sequences are to be extracted (e.g. tumor infiltrating lymphocytes in the case of tumor-ag specific T cells); 3) selecting or screening for antigenspecific T cells (e.g. FACS sorting antigen-specific T cells based on tetramer binding); 4) cloning (via RT-PCR) and sequencing the TCR genes (i.e. alpha and beta chains or gamma and delta chains of the TCRs); cloning and sequencing may be done either on a population or single cell level; and 5) confirming and analyzing TCR specificity by, for example, testing the function of TCR clones by transducing peripheral blood T cells with these sequences and assessing their reactivity to target cells that express the cognate peptide-MHC complex. Reactivity is usually measured based on cytokine production (e.g. interferon gamma).

[0121] T cells can also be engineered to express TCRs. To generate T cells expressing a TCR of interested one can first thaw and activate PBMCs with CD3 / CD28 human activating beads. 48 hours after activation, one can perform T cell retroviral transduction with the TCR of interest.C. Chimeric Antigen Receptors (CARs) and CAR-T cell therapy

[0122] The immunotherapy may comprise CAR-T cell therapy. Chimeric antigen receptors (CARs, also known as chimeric immunoreceptors, chimeric T cell receptors or artificial T cell31137193686receptors) are engineered receptors that combine a new specificity with an immune cell to target cancer cells. Typically, these receptors graft the specificity of a monoclonal antibody onto a T cell. The receptors are called chimeric because they are fused of parts from different sources. CAR-T cell therapy refers to a treatment that uses such transformed cells for cancer therapy.

[0123] The basic principle of CAR-T cell design involves recombinant receptors that combine antigen-binding and T-cell activating functions. The general premise of CAR-T cells is to artificially generate T-cells targeted to markers found on cancer cells. Scientists can remove T-cells from a person, genetically alter them, and put them back into the patient for them to attack the cancer cells. Once the T cell has been engineered to become a CAR-T cell, it acts as a “living drug”. CAR-T cells create a link between an extracellular ligand recognition domain to an intracellular signaling molecule which in turn activates T cells. The extracellular ligand recognition domain is usually a single-chain variable fragment (scFv). An important aspect of the safety of CAR-T cell therapy is how to ensure that only cancerous tumor cells are targeted, and not normal cells. The specificity of CAR-T cells is determined by the choice of molecule that is targeted.

[0124] Exemplary CAR-T therapies include Tisagenlecleucel (Kymriah) and Axicabtagene ciloleucel (Yescarta).

[0125] Described below are domains / peptides that may be included in the CAR molecule.1. Signal peptide

[0126] Polypeptides of the present disclosure may comprise a signal peptide. A “signal peptide” refers to a peptide sequence that directs the transport and localization of the protein within a cell, e.g., to a certain cell organelle (such as the endoplasmic reticulum) and / or the cell surface. In some aspects, a signal peptide directs the nascent protein into the endoplasmic reticulum. This is essential if a receptor is to be glycosylated and anchored in the cell membrane. Generally, the signal peptide natively attached to the amino-terminal most component is used (e.g., in an scFv with orientation light chain - linker - heavy chain, the native signal of the light-chain is used).

[0127] In some aspects, the signal peptide is cleaved after passage of the endoplasmic reticulum (ER), i.e., is a cleavable signal peptide. In some aspects, a restriction site is at the carboxy end of the signal peptide to facilitate cleavage.321371936862. Antigen binding domain

[0128] Polypeptides of the present disclosure may comprise one or more antigen binding domains. An “antigen binding domain” describes a region of a polypeptide capable of binding to an antigen under appropriate conditions. In some aspects, an antigen binding domain is a single-chain variable fragment (scFv) based on one or more antibodies (e.g., CD20 antibodies). In some aspects, an antigen binding domain comprise a variable heavy (VH) region and a variable light (VL) region, with the VH and VL regions being on the same polypeptide. In some aspects, the antigen binding domain comprises a linker between the VH and VL regions. A linker may enable the antigen binding domain to form a desired structure for antigen binding.

[0129] The variable regions of the antigen-binding domains of the polypeptides of the disclosure can be modified by mutating amino acid residues within the VH and / or VL CDR 1, CDR 2 and / or CDR 3 regions to improve one or more binding properties (e.g., affinity) of the antibody. The term “CDR” refers to a complementarity-determining region that is based on a part of the variable chains in immunoglobulins (antibodies) and T cell receptors, generated by B cells and T cells respectively, where these molecules bind to their specific antigen. Since most sequence variation associated with immunoglobulins and T cell receptors is found in the CDRs, these regions are sometimes referred to as hypervariable regions. Mutations may be introduced by site-directed mutagenesis or PCR-mediated mutagenesis and the effect on antibody binding, or other functional property of interest, can be evaluated in appropriate in vitro or in vivo assays. Preferably conservative modifications are introduced and typically no more than one, two, three, four or five residues within a CDR region are altered. The mutations may be amino acid substitutions, additions or deletions.

[0130] Framework modifications can be made to the antibodies to decrease immunogenicity, for example, by “backmutating” one or more framework residues to the corresponding germline sequence.

[0131] It is also contemplated that the antigen binding domain may be multi-specific or multivalent by multimerizing the antigen binding domain with VH and VL region pairs that bind either the same antigen (multi -valent) or a different antigen (multi-specific).

[0132] The binding affinity of the antigen binding region, such as the variable regions (heavy chain and / or light chain variable region), or of the CDRs may be at least 10'5M, 10'6M, 10'7M, 10'8M, 10'9M, 10'10M, 10-11M, 10'12M, or 10'13M (or any range derivable therein). In some aspects, the KD of the antigen binding region, such as the variable regions (heavy chain33137193686and / or light chain variable region), or of the CDRs may be at least 10'5M, 10'6M, 10'7M, 10’8M, 10'9M, 1O'1OM, 10-11M, 10'12M, or 10'13M (or any derivable range therein).

[0133] Binding affinity, KA, or KD can be determined by methods known in the art such as by surface plasmon resonance (SRP)-based biosensors, by kinetic exclusion assay (KinExA), by optical scanner for microarray detection based on polarization-modulated oblique-incidence reflectivity difference (OI-RD), or by ELISA.

[0134] In some aspects, the polypeptide comprising the humanized binding region has equal, better, or at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 104, 106, 106, 108, 109, 110, 115, or 120% binding affinity and / or expression level in host cells (or any range derivable therein), compared to a polypeptide comprising a non-humanized binding region, such as a binding region from a mouse.In some aspects, the framework regions, such as FR1, FR2, FR3, and / or FR4 of a human framework can each or collectively have at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200 (or any derivable range therein) amino acid substitutions, contiguous amino acid additions, or contiguous amino acid deletions with respect to a mouse framework.

[0135] In some aspects, the framework regions, such as FR1, FR2, FR3, and / or FR4 of a mouse framework can each or collectively have at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182,34137193686183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200 (or any derivable range therein) amino acid substitutions, contiguous amino acid additions, or contiguous amino acid deletions with respect to a human framework.

[0136] The substitution may be at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 of FR1, FR2, FR3, or FR4 of a heavy or light chain variable region.

[0137] Non-limiting examples of tumor antigens that may be targeted by the CAR(s) of the present disclosure include at least the following: Differentiation antigens such as tyrosinase, TRP-1, TRP-2 and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pi 5; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor-suppressor genes such as p53, Ras, HER-2 / neu; unique tumor antigens resulting from chromosomal translocations; such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as the Epstein Barr virus antigens EBVA and the human papillomavirus (HPV) antigens E6 and E7. Other large, protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, pl85erbB2, pl80erbB-3, c-met, nm- 23H1, PSA, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-Catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha- fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1 , CO-029, FGF-5, G250, Ga733\EpCAM, HTgp- 175, M344, MA-50, MG7-Ag, M0V18, NB / 70K, NY-CO-1, RCASI, SDCCAG1 6, TA- 90\Mac-2 binding protein\cyclophilm C-associated protein, TAAL6, TAG72, TLP, TPS, GPC3, MUC16, MUC18, LMP1, EBMA-1, BARF-1, CS1, CD319, HER1, B7H6, LI CAM, IL6, and MET.

[0138] Tumor antigens also include, for example, a glioma-associated antigen, carcinoembryonic antigen (CEA), EGFRvlll, IL-IIRa, IL-13Ra, EGFR, FAP, B7H3, Kit, CA LX, CS-1, MUC1, BCMA, bcr-abl, HER2, b-human chorionic gonadotropin, alphafetoprotein (AFP), ALK, CD 19, cyclin Bl, lectin-reactive AFP, Fos-related antigen 1, ADRB3, thyroglobulin, EphA2, RAGE-1, RUI, RU2, SSX2, AKAP-4, LCK, OY-TESI, PAX5, SART3, CLL-1, fucosyl GM1 , GloboH, MN-CA IX, EPCAM, EVT6- AML, TGS5, human telomerase reverse transcriptase, ply sialic acid, PLAC1, RUI, RU2 (AS), intestinal carboxyl esterase, lewisY, sLe, LY6K, mut hsp70-2, M-CSF, MYCN, RhoC, TRP-2, CYPIBI, BORIS, prostase, prostate-specific antigen (PSA), PAX3, PAP, NY-ESO-1 , LAGE-la, LMP2, NCAM, p53, p5335137193686mutant, Ras mutant, gplOO, prostein, OR51 E2, PANX3, PSMA, PSCA, Her2 / neu, hTERT, HMWMAA, HAVCR1 , VEGFR2, PDGFR-beta, survivin and telomerase, legumain, HPV E6,E7, sperm protein 17, S SEA-4, tyrosinase, TARP, WT1, prostate-carcinoma tumor antigen-1 (PCTA-1), ML-IAP, MAGE, MAGE-A1.MAD-CT-1 , MAD-CT-2, MelanA / MART 1, XAGE1, ELF2M, ERG (TMPRSS2 ETS fusion gene), NA17, neutrophil elastase, sarcoma translocation breakpoints, NY-BR-1, ephnnB2, CD20, CD22, CD24, CD30, CD33, CD38, CD44v6, CD97, CD171 , CD 179a, androgen receptor, FAP, insulin growth factor (IGF)-I, IGFII, IGF-I receptor, GD2, o-acetyl-GD2, GD3, GM3, GPRC5D, GPR20, CX0RF61, folate receptor (FRa), folate receptor beta, R0R1, Flt3, TAG72, TN Ag, Tie 2, TEM1, TEM7R, CLDN6, TSHR, UPK2, mesothelin, and any combination thereof.

[0139] Further examples of tumor cell antigens to which a CAR may be directed include at least 5T4, 8H9, avP6 integrin, BCMA, B7-H3, B7-H6, CAIX, CA9, CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD123, CD 138, CD 171, CD 133, CEA, c-Met, CSPG4, EGFR, EGFR family including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, ERBB3, ERBB4, ErbB3 / 4, EPCAM, EphA2, EpCAM, folate receptor-a, FAP, FBP, fetal AchR, FRD, GD2, G250 / CAIX, GD3, Glypican-3 (GPC3), GUCY2C, HER1, HER2, ICAM-1, IL-13RD2, IL-llRa, Kras, Kras G12D, L1CAM, Lambda, Lewis- Y, Kappa, KDR, MAGE, MCSP, MET, Mesothelin, Mucl, Mucl6, MUC18, NCAM, NKG2D Ligands, NY-ESO-1, PRAME, PSC1, PSCA, PSMA, R0R1, SP17, Survivin, TAG72, TEMs, carcinoembryonic antigen, HMW-MAA, AFP, CA-125, ETA, Tyrosinase, MAGE, laminin receptor, HPV E6, E7, BING-4, Calcium-activated chloride channel 2, Cyclin-Bl, 9D7, EphA3, Telomerase, SAP-1, BAGE family, CAGE family, GAGE family, MAGE family, SAGE family, XAGE family, NY-ESO-l / LAGE-1, PAME, SSX-2, Melan-A / MART-1, GP100 / pmell7, TRP-1 / -2, P. polypeptide, MC1R, Prostate-specific antigen, P-catenin, BRCA1 / 2, CML66, Fibronectin, MART-2, TGF-PRII, TGF-P, WT-1, or VEGF receptors (e g., VEGFR2), for example. The CARs may be a first, second, third, or more generation CARs. The CARs may be bispecific for any two nonidentical antigens, or they may be specific for more than two nonidentical antigens.3. Peptide spacer

[0140] A peptide spacer, such as an extracellular spacer may link an antigen-binding domain to a transmembrane domain. In some aspects, a peptide spacer is flexible enough to allow the antigen-binding domain to orient in different directions to facilitate antigen binding.36137193686In one embodiment, the spacer comprises the hinge region from IgG. In some aspects, the spacer comprises or further comprises the CH2CH3 region of immunoglobulin and portions of CD3. In some aspects, the CH2CH3 region may have L235E / N297Q or L235D / N297Q modifications, or at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% amino acid sequence identity of the CH2CH3 region. In some aspects, the spacer is from IgG4. An extracellular spacer may comprise a hinge region.

[0141] As used herein, the term “hinge” refers to a flexible polypeptide connector region (also referred to herein as “hinge region”) providing structural flexibility and spacing to flanking polypeptide regions and can consist of natural or synthetic polypeptides. A “hinge” derived from an immunoglobulin (e.g., IgGl) is generally defined as stretching from Glu216 to Pro230 of human IgGl (Burton (1985) Molec. Immunol., 22: 161- 206). Hinge regions of other IgG isotypes may be aligned with the IgGl sequence by placing the first and last cysteine residues forming inter-heavy chain disulfide (S-S) bonds in the same positions. The hinge region may be of natural occurrence or non-natural occurrence, including but not limited to an altered hinge region as described in U.S. Pat. No. 5,677,425, incorporated by reference herein. The hinge region can include a complete hinge region derived from an antibody of a different class or subclass from that of the CHI domain. The term “hinge” > can also include regions derived from CD8 and other receptors that provide a similar function in providing flexibility and spacing to flanking regions.

[0142] The extracellular spacer can have a length of at least, at most, or exactly 4, 5, 6, 7, 8, 9, 10, 12, 15, 16, 17, 18, 19, 20, 20, 25, 30, 35, 40, 45, 50, 75, 100, 110, 119, 120, 130, 140, 150, 160, 170, 180, 190, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 260, 270, 280, 290, 300, 325, 350, or 400 amino acids (or any derivable range therein). In some aspects, the extracellular spacer consists of or comprises a hinge region from an immunoglobulin (e.g., IgG). Immunoglobulin hinge region amino acid sequences are known in the art; see, e.g., Tan et al. (1990) Proc. Natl. Acad. Sci. USA 87: 162; and Huck et al. (1986) Nucl. Acids Res.

[0143] The length of an extracellular spacer may have effects on the CAR’s signaling activity and / or the CAR-T cells’ expansion properties in response to antigen-stimulated CAR signaling. In some aspects, a shorter spacer such as less than 50, 45, 40, 30, 35, 30, 25, 20, 15, 14, 13, 12, 11, or 10 amino acids is used. In some aspects, a longer spacer, such as one that is at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 225, 226, 227, 228, 229,37137193686230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 260, 270, 280, or 290 amino acids (or any range derivable therein) may have the advantage of increased expansion in vivo or in vitro.

[0144] When the extracellular spacer comprises multiple parts, there may be anywhere from 0-50 amino acids in between the various parts. For example, there may be at least, at most, or exactly 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, or 50 amino acids (or any derivable range therein) between the hinge and the CH2 or CH3 region or between the CH2 and CH3 region when both are present. In some aspects, the extracellular spacer consists essentially of a hinge, CH2, and / or CH3 region, meaning that the hinge, CH2, and / or CH3 region is the only identifiable region present and all other domains or regions are excluded, but further amino acids not part of an identifiable region may be present.4. Transmembrane domain

[0145] Polypeptides of the present disclosure may comprise a transmembrane domain. In some aspects, a transmembrane domain is a hydrophobic alpha helix that spans the membrane. Different transmembrane domains may result in different receptor stability.

[0146] In some aspects, the transmembrane domain is interposed between the extracellular spacer and the cytoplasmic region. In some aspects, the transmembrane domain is interposed between the extracellular spacer and one or more costimulatory regions. In some aspects, a linker is between the transmembrane domain and the one or more costimulatory regions.

[0147] Any transmembrane domain that provides for insertion of a polypeptide into the cell membrane of a eukaryotic (e.g., mammalian) cell may be suitable for use. In some aspects, the transmembrane domain is derived from CD28, CD8, CD4, CD3-zeta, CD134, or CD7.5. Cytoplasmic region

[0148] After antigen recognition, receptors of the present disclosure may cluster and a signal transmitted to the cell through the cytoplasmic region. In some aspects, the costimulatory domains described herein are part of the cytoplasmic region. In some aspects, the cytoplasmic region comprises an intracellular signaling domain. An intracellular signaling domain may comprise a primary signaling domain and one or more costimulatory domains.

[0149] Cytoplasmic regions and / or costimulatiory regions suitable for use in the polypeptides of the disclosure include any desired signaling domain that provides a distinct and38137193686detectable signal (e.g., increased production of one or more cytokines by the cell; change in transcription of a target gene; change in activity of a protein; change in cell behavior, e.g., cell death; cellular proliferation; cellular differentiation; cell survival; modulation of cellular signaling responses; etc.) in response to activation by way of binding of the antigen to the antigen binding domain. In some aspects, the cytoplasmic region includes at least one (e.g., one, two, three, four, five, six, etc.) ITAM motif as described herein. In some aspects, the cytoplasmic region includes DAP10 / CD28 type signaling chains.

[0150] Cytoplasmic regions suitable for use in the polypeptides of the disclosure include immunoreceptor tyrosine-based activation motif (IT M)-containing intracellular signaling polypeptides. An ITAM motif is YX1X2(L / I), where XI and X2 are independently any amino acid. In some cases, the cytoplasmic region comprises 1, 2, 3, 4, or 5 ITAM motifs. In some cases, an ITAM motif is repeated twice in an endodomain, where the first and second instances of the ITAM motif are separated from one another by 6 to 8 amino acids, e.g., (YXlX2(L / I))(X3)n(YXlX2(L / I)), where n is an integer from 6 to 8, and each of the 6-8 X3 can be any amino acid.

[0151] A suitable cytoplasmic region may be an ITAM motif-containing portion that is derived from a polypeptide that contains an ITAM motif. For example, a suitable cytoplasmic region can be an ITAM motif-containing domain from any ITAM motif-containing protein. Thus, a suitable endodomain need not contain the entire sequence of the entire protein from which it is derived. Examples of suitable ITAM motif-containing polypeptides include, but are not limited to: DAP12, DAP10, FCER1G (Fc epsilon receptor I gamma chain); CD3D (CD3 delta); CD3E (CD3 epsilon); CD3G (CD3 gamma); CD3-zeta; and CD79A (antigen receptor complex-associated protein alpha chain).

[0152] Exemplary cytoplasmic regions are known in the art. The cytoplasmic regions shown below also provide examples of regions that may be incorporated in a CAR of the disclosure:

[0153] In some aspects, a suitable cytoplasmic region can comprise an ITAM motifcontaining portion of the full length DAP 12 amino acid sequence. In some aspects, the cytoplasmic region is derived from FCER1G (also known as FCRG; Fc epsilon receptor I gamma chain; Fc receptor gamma-chain; fc-epsilon Rl-gamma; fcRgamma; fceRI gamma; high affinity immunoglobulin epsilon receptor subunit gamma; immunoglobulin E receptor, high affinity, gamma chain; etc.). In some aspects, a suitable cytoplasmic region can comprise an ITAM motif-containing portion of the full length FCERI G amino acid sequence.39137193686

[0154] In some aspects, the cytoplasmic region is derived from T cell surface glycoprotein CD3 delta chain (also known as CD3D; CD3-DELTA; T3D; CD3 antigen, delta subunit; CD3 delta; CD3D; CD3d antigen, delta polypeptide (TiT3 complex); OKT3, delta chain; T cell receptor T3 delta chain; T cell surface glycoprotein CD3 delta chain; etc.). In some aspects, a suitable cytoplasmic region can comprise an ITAM motif-containing portion of the full length CD3 delta amino acid sequence. In some aspects, the cytoplasmic region is derived from T cell surface glycoprotein CD3 epsilon chain (also known as CD3e, CD3D; T cell surface antigen T3 / Leu-4 epsilon chain, T cell surface glycoprotein CD3 epsilon chain, AI504783, CD3, CD3-epsilon, T3e, etc.). In some aspects, a suitable cytoplasmic region can comprise an ITAM motif-containing portion of the full length CD3 epsilon amino acid sequence. In some aspects, the cytoplasmic region is derived from T cell surface glycoprotein CD3 gamma chain (also known as CD3G, CD3y, T cell receptor T3 gamma chain, CD3-GAMMA, T3G, gamma polypeptide (TiT3 complex), etc.). In some aspects, a suitable cytoplasmic region can comprise an ITAM motif-containing portion of the full length CD3 gamma amino acid sequence. In some aspects, the cytoplasmic region is derived from T cell surface glycoprotein CD3 zeta chain (also known as CD3Z, CD3(^, T cell receptor T3 zeta chain, CD247, CD3-ZETA, CD3H, CD3Q, T3Z, TCRZ, etc.). In some aspects, a suitable cytoplasmic region can comprise an ITAM motif-containing portion of the full length CD3 zeta amino acid sequence.

[0155] In some aspects, the cytoplasmic region is derived from CD79A (also known as B-cell antigen receptor complex-associated protein alpha chain; CD79a antigen (immunoglobulin-associated alpha); MB-1 membrane glycoprotein; ig-alpha; membranebound immunoglobulin-associated protein; surface IgM-associated protein; etc.). In some aspects, a suitable cytoplasmic region can comprise an ITAM motif-containing portion of the full length CD79A amino acid sequence.6. Costimulatory region

[0156] Non-limiting examples of suitable costimulatory regions, such as those included in the cytoplasmic region, include, but are not limited to, polypeptides from 4-1BB (CD 137), CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, andHVEM. It is specifically contemplated that any of these may be excluded from an embodiment.

[0157] A costimulatory region may have a length of at least, at most, or exactly 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, or 300 amino acids or any range derivable therein. In some aspects, the costimulatory region is derived from an intracellular portion of the40137193686transmembrane protein 4-1BB (also known as TNFRSF9; CD137; CDwl37; ILA; etc.). In some aspects, the costimulatory region is derived from an intracellular portion of the transmembrane protein CD28 (also known as Tp44). In some aspects, the costimulatory region is derived from an intracellular portion of the transmembrane protein ICOS (also known as AILIM, CD278, and CVID1). In some aspects, the costimulatory region is derived from an intracellular portion of the transmembrane protein OX-40 (also known as TNFRSF4, RP5-902P8.3, ACT35, CD134, 0X40, TXGP1L). In some aspects, the costimulatory region is derived from an intracellular portion of the transmembrane protein BTLA (also known as BTLA1 and CD272). In some aspects, the costimulatory region is derived from an intracellular portion of the transmembrane protein CD27 (also known as S 152, T14, TNFRSF7, and Tp55). In some aspects, the costimulatory region is derived from an intracellular portion of the transmembrane protein CD30 (also known as TNFRSF8, D1S166E, and Ki-1). In some aspects, the costimulatory region is derived from an intracellular portion of the transmembrane protein GITR (also known as TNFRSF18, RP5-902P8.2, AITR, CD357, and GITR-D). In some aspects, the costimulatory region derived from an intracellular portion of the transmembrane protein HVEM (also known as TNFRSF14, RP3-395M20.6, ATAR, CD270, HVEA, HVEM, LIGHTR, and TR2).7. Detection peptides

[0158] In some aspects, the polypeptides described herein may further comprise a detection peptide. Suitable detection peptides include hemagglutinin (HA; e.g., YPYDVPDYA (SEQ ID NO:1); FLAG (e.g, DYKDDDDK (SEQ ID NO:2); c-myc (e.g, EQKLISEEDL; SEQ ID NO:3), and the like. Other suitable detection peptides are known in the art.8. Peptide linkers

[0159] In some aspects, the polypeptides of the disclosure include peptide linkers (sometimes referred to as a linker). A peptide linker may be used to separate any of the peptide domain / regions described herein. As an example, a linker may be between the signal peptide and the antigen binding domain, between the VH and VL of the antigen binding domain, between the antigen binding domain and the peptide spacer, between the peptide spacer and the transmembrane domain, flanking the costimulatory region or on the N- or C- region of the costimulatory region, and / or between the transmembrane domain and the endodomain. The peptide linker may have any of a variety of amino acid sequences. Domains and regions can41137193686be joined by a peptide linker that is generally of a flexible nature, although other chemical linkages are not excluded. A linker can be a peptide of between about 6 and about 40 amino acids in length, or between about 6 and about 25 amino acids in length. These linkers can be produced by using synthetic, linker-encoding oligonucleotides to couple the proteins.

[0160] Peptide linkers with a degree of flexibility can be used. The peptide linkers may have virtually any amino acid sequence, bearing in mind that suitable peptide linkers will have a sequence that results in a generally flexible peptide. The use of small amino acids, such as glycine and alanine, are of use in creating a flexible peptide. The creation of such sequences is routine to those of skill in the art.

[0161] Suitable linkers can be readily selected and can be of any suitable length, such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and may be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0162] Suitable linkers can be readily selected and can be of any of a suitable of different lengths, such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and may be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0163] Example flexible linkers include glycine polymers (G)n, glycine- serine polymers (including, for example, (GS)n, (GSGGS)n (SEQ ID NO:4), (G4S)n and (GGGS)n (SEQ ID NO:5), where n is an integer of at least one. In some aspects, n is at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (or any derivable range therein). Glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers can be used; both Gly and Ser are relatively unstructured, and therefore can serve as a neutral tether between components. Glycine polymers can be used; glycine accesses significantly more phi-psi space than even alanine, and is much less restricted than residues with longer side chains. Exemplary spacers can comprise amino acid sequences including, but not limited to, GGSG (SEQ ID NO:6), GGSGG (SEQ ID NO:7), GSGSG (SEQ ID NO:8), GSGGG (SEQ ID NO:9), GGGSG (SEQ ID NO: 10), or GSSSG (SEQ ID NO: 11).42137193686D. Checkpoint Inhibitors and Combination Treatment

[0164] Aspects of the disclosure may include administration of immune checkpoint inhibitors, which are further described below.1. PD-1, PDL1, and PDL2 inhibitors

[0165] PD-1 can act in the tumor microenvironment where T cells encounter an infection or tumor. Activated T cells upregulate PD-1 and continue to express it in the peripheral tissues. Cytokines such as IFN-gamma induce the expression of PDL1 on epithelial cells and tumor cells. PDL2 is expressed on macrophages and dendritic cells. The main role of PD-1 is to limit the activity of effector T cells in the periphery and prevent excessive damage to the tissues during an immune response. Inhibitors of the disclosure may block one or more functions of PD-1 and / or PDL1 activity.

[0166] Alternative names for “PD-1” include CD279 and SLEB2. Alternative names for “PDL1” include B7-H1, B7-4, CD274, and B7-H. Alternative names for “PDL2” include B7-DC, Btdc, and CD273. In some aspects, PD-1, PDL1, and PDL2 are human PD-1, PDL1 and PDL2.

[0167] In some aspects, the PD-1 inhibitor is a molecule that inhibits the binding of PD-1 to its ligand binding partners. In a specific aspect, the PD-1 ligand binding partners are PDL1 and / or PDL2. In another aspect, a PDL1 inhibitor is a molecule that inhibits the binding of PDL1 to its binding partners. In a specific aspect, PDL1 binding partners are PD-1 and / or B7-1. In another aspect, the PDL2 inhibitor is a molecule that inhibits the binding of PDL2 to its binding partners. In a specific aspect, a PDL2 binding partner is PD-1. The inhibitor may be an antibody, an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide. Exemplary antibodies are described in U.S. Patent Nos. 8,735,553, 8,354,509, and 8,008,449, all incorporated herein by reference. Other PD-1 inhibitors for use in the methods and compositions provided herein are known in the art such as described in U. S. Patent Application Nos. US2014 / 0294898, US2014 / 022021, and US2011 / 0008369, all incorporated herein by reference.

[0168] In some aspects, the PD-1 inhibitor is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some aspects, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and pidilizumab. In some aspects, the PD-1 inhibitor is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g., an43137193686Fc region of an immunoglobulin sequence). In some aspects, the PDL1 inhibitor comprises AMP- 224. Nivolumab, also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, is an anti-PD-1 antibody described in W02006 / 121168. Pembrolizumab, also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA®, and SCH-900475, is an anti-PD-1 antibody described in W02009 / 114335. Pidilizumab, also known as CT-011, hBAT, or hBAT-1, is an anti-PD-1 antibody described in W02009 / 101611. AMP-224, also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor described in W02010 / 027827 and WO2011 / 066342. Additional PD-1 inhibitors include MEDI0680, also known as AMP-514, and REGN2810.

[0169] In some aspects, the immune checkpoint inhibitor is a PDL1 inhibitor such as Durvalumab, also known as MEDI4736, atezolizumab, also known as MPDL3280A, avelumab, also known as MSB00010118C, MDX-1105, BMS-936559, or combinations thereof. In certain aspects, the immune checkpoint inhibitor is a PDL2 inhibitor such as rHIgM12B7.

[0170] In some aspects, the inhibitor comprises the heavy and light chain CDRs or VRs of nivolumab, pembrolizumab, or pidilizumab. Accordingly, in one aspect, the inhibitor comprises the CDR1, CDR2, and CDR3 domains of the VH region of nivolumab, pembrolizumab, or pidilizumab, and the CDR1, CDR2 and CDR3 domains of the VL region of nivolumab, pembrolizumab, or pidilizumab. In another aspect, the antibody competes for binding with and / or binds to the same epitope on PD-1, PDL1, or PDL2 as the above-mentioned antibodies. In another aspect, the antibody has at least about 70, 75, 80, 85, 90, 95, 97, or 99% (or any derivable range therein) variable region amino acid sequence identity with the above-mentioned antibodies.2. CTLA-4, B7-1, and B7-2

[0171] Another immune checkpoint that can be targeted in the methods provided herein is the cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence of human CTLA-4 has the Genbank accession number LI 5006. CTLA-4 is found on the surface of T cells and acts as an “off’ switch when bound to B7-1 (CD80) or B7-2 (CD86) on the surface of antigen-presenting cells. CTLA4 is a member of the immunoglobulin superfamily that is expressed on the surface of Helper T cells and transmits an inhibitory signal to T cells. CTLA4 is similar to the T-cell co-stimulatory protein, CD28, and both molecules bind to B7-1 and B7-2 on antigen-presenting cells. CTLA-4 transmits an44137193686inhibitory signal to T cells, whereas CD28 transmits a stimulatory signal. Intracellular CTLA-4 is also found in regulatory T cells and may be important to their function. T cell activation through the T cell receptor and CD28 leads to increased expression of CTLA-4, an inhibitory receptor for B7 molecules. Inhibitors of the disclosure may block one or more functions of CTLA-4, B7-1, and / or B7-2 activity. In some aspects, the inhibitor blocks the CTLA-4 and B7-1 interaction. In some aspects, the inhibitor blocks the CTLA-4 and B7-2 interaction.

[0172] In some aspects, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.

[0173] Anti-human-CTLA-4 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art recognized anti-CTLA-4 antibodies can be used. For example, the anti-CTLA-4 antibodies disclosed in: US 8,119,129, WO 01 / 14424, WO 98 / 42752; WO 00 / 37504 (CP675,206, also known as tremelimumab; formerly ticilimumab), U.S. Patent No. 6,207,156; Hurwitz et al., 1998; can be used in the methods disclosed herein. The teachings of each of the aforementioned publications are hereby incorporated by reference. Antibodies that compete with any of these art-recognized antibodies for binding to CTLA-4 also can be used. For example, a humanized CTLA-4 antibody is described in International Patent Application No. W02001 / 014424, W02000 / 037504, and U.S. Patent No. 8,017,114; all incorporated herein by reference.

[0174] A further anti-CTLA-4 antibody useful as a checkpoint inhibitor in the methods and compositions of the disclosure is ipilimumab (also known as 10D1, MDX- 010, MDX- 101, and Yervoy®) or antigen binding fragments and variants thereof (see, e.g., WOO 1 / 14424).

[0175] In some aspects, the inhibitor comprises the heavy and light chain CDRs or VRs of tremelimumab or ipilimumab. Accordingly, in one aspect, the inhibitor comprises the CDR1, CDR2, and CDR3 domains of the VH region of tremelimumab or ipilimumab, and the CDR1, CDR2 and CDR3 domains of the VL region of tremelimumab or ipilimumab. In another aspect, the antibody competes for binding with and / or binds to the same epitope on PD-1, B7-1, or B7-2 as the above- mentioned antibodies. In another aspect, the antibody has at least about 70, 75, 80, 85, 90, 95, 97, or 99% (or any derivable range therein) variable region amino acid sequence identity with the above-mentioned antibodies.45137193686E. LAG3 and TIGIT

[0176] In some embodiments, the immunotherapy comprises an inhibitor of lymphocyte activation gene 3 (LAG-3) and / or an inhibitor of T cell immunoreceptor with Ig and ITIM domains (TIGIT). Without being bound by theory, LAG-3 and TIGIT are inhibitory immune checkpoint receptors that can be expressed by activated T cells and that can reduce T cell activation, cytokine production, and / or cytotoxic effector function within the tumor microenvironment. In certain embodiments, blockade of LAG-3 and / or TIGIT increases antitumor immunity by increasing activation, proliferation, and / or effector function of endogenous tumor-reactive T cells and / or adoptively transferred T cells (including, for example, CAR-T cells, TCR-T cells, and / or tumor infiltrating lymphocytes (TILs)).LAG-3 (also known as CD223) is an inhibitory receptor that can be expressed on activated CD4+ and / or CD8+ T cells and, in some contexts, on other immune cell types. In some embodiments, LAG-3 binds one or more ligands including major histocompatibility complex class II (MHC-II) molecules. In some embodiments, a LAG-3 inhibitor reduces, blocks, or otherwise interferes with binding of LAG-3 to a LAG-3 ligand and thereby increases T cell activation and / or T cell effector function. In some embodiments, a LAG-3 inhibitor comprises an antibody or antigenbinding fragment that specifically binds LAG-3. In some embodiments, the LAG-3 inhibitor comprises a monoclonal antibody, a human antibody, a humanized antibody, a chimeric antibody, a single-domain antibody, a Fab, an F(ab')2, a scFv, or a multispecific antibody that binds LAG-3 and one or more additional targets. In some embodiments, the LAG-3 inhibitor is administered alone or in combination with an additional checkpoint inhibitor, such as an inhibitor of PD-1 and / or PD-L1 and / or CTLA-4. In some embodiments, the LAG-3 inhibitor is administered concurrently with, before, and / or after administration of the T cell modulator described herein. In some embodiments, the LAG-3 inhibitor comprises relatlimab. TIGIT is an inhibitory receptor that can be expressed on activated T cells, including CD8+ T cells, and can also be expressed on natural killer (NK) cells. In some embodiments, TIGIT binds one or more ligands including CD155 (also known as PVR) and / or CD112 (also known as Nectin-2), which may be expressed by tumor cells and / or antigen-presenting cells. In some embodiments, a TIGIT inhibitor reduces, blocks, or otherwise interferes with binding of TIGIT to a TIGIT ligand and thereby increases T cell activation and / or cytotoxic effector function and / or increases NK cell activity. In some embodiments, the TIGIT inhibitor comprises an antibody or antigen-binding fragment that specifically binds TIGIT. In some embodiments, the TIGIT inhibitor comprises a monoclonal antibody, a human antibody, a humanized antibody, a46137193686chimeric antibody, a single-domain antibody, a Fab, an F(ab')2, a scFv, or a multispecific antibody that binds TIGIT and one or more additional targets. In some embodiments, the TIGIT inhibitor is administered alone or in combination with an inhibitor of PD-1 and / or PD-L1 and / or CTLA-4 and / or LAG-3. In some embodiments, the TIGIT inhibitor is administered concurrently with, before, and / or after administration of the T cell modulator described herein. In some embodiments, the subject is administered a combination therapy comprising (i) a T cell modulator as described herein (for example, Telmisartan and / or AZD4573), and (ii) a checkpoint inhibitor selected from a LAG-3 inhibitor, a TIGIT inhibitor, or a combination thereof. In some embodiments, the combination therapy further comprises an additional checkpoint inhibitor, optionally an inhibitor of PD-1 and / or PD-L1. In some embodiments, the checkpoint inhibitor(s) are administered systemically. In some embodiments, the checkpoint inhibitor(s) are administered intravenously. In some embodiments, the checkpoint inhibitor(s) are administered according to a dosing regimen selected by a treating clinician. In some embodiments, the checkpoint inhibitor comprises a nucleic acid encoding a LAG-3 inhibitor and / or a nucleic acid encoding a TIGIT inhibitor, wherein the nucleic acid is delivered to the subject using a delivery system selected from a viral vector, a lipid nanoparticle, or a non-viral delivery system. In some embodiments, the checkpoint inhibitor comprises an engineered immune cell that expresses a LAG-3 inhibitor and / or a TIGIT inhibitor, optionally as a secreted antibody or antibody fragment.In some embodiments, administration of the T cell modulator described herein in combination with LAG-3 blockade and / or TIGIT blockade results in one or more of increased tumor infiltration by T cells, increased persistence of transferred T cells, increased cytokine production by T cells, increased tumor cell killing, increased duration of response, reduced tumor burden, or improved survival.

[0177] The first approved therapy that targets LAG-3 is Relatlimab, a monoclonal antibody against LAG-3. Relatlimab is commercially available in a fixed-dose combination product with Nivolumab (an anti-PD-1 antibody) under the brand name Opdualag. Opdualag is FDA-approved for treatment of unresectable or metastatic melanoma. In this combination, dual checkpoint blockade (PD-1 plus LAG-3) is intended to relieve inhibitory signaling on tumor-reactive T cells through complementary pathways, thereby enhancing antitumor immune responses relative to PD-1 blockade alone in at least some patients.47137193686F. Dendritic cell therapy

[0178] The immunotherapy may comprise dendritic cell therapy. Dendritic cell therapy provokes anti-tumor responses by causing dendritic cells to present tumor antigens to lymphocytes, which activates them, priming them to kill other cells that present the antigen. Dendritic cells are antigen presenting cells (APCs) in the mammalian immune system. In cancer treatment they aid cancer antigen targeting. One example of cellular cancer therapy based on dendritic cells is sipuleucel-T.

[0179] One method of inducing dendritic cells to present tumor antigens is by vaccination with autologous tumor lysates or short peptides (small parts of protein that correspond to the protein antigens on cancer cells). These peptides are often given in combination with adjuvants (highly immunogenic substances) to increase the immune and anti-tumor responses. Other adjuvants include proteins or other chemicals that attract and / or activate dendritic cells, such as granulocyte macrophage colony-stimulating factor (GM-CSF).

[0180] Dendritic cells can also be activated in vivo by making tumor cells express GM-CSF. This can be achieved by either genetically engineering tumor cells to produce GM-CSF or by infecting tumor cells with an oncolytic virus that expresses GM-CSF.

[0181] Another strategy is to remove dendritic cells from the blood of a patient and activate them outside the body. The dendritic cells are activated in the presence of tumor antigens, which may be a single tumor-specific peptide / protein or a tumor cell lysate (a solution of broken down tumor cells). These cells (with optional adjuvants) are infused and provoke an immune response.

[0182] Dendritic cell therapies include the use of antibodies that bind to receptors on the surface of dendritic cells. Antigens can be added to the antibody and can induce the dendritic cells to mature and provide immunity to the tumor. Dendritic cell receptors such as TLR3, TLR7, TLR8 or CD40 have been used as antibody targets.G. Cytokine therapy

[0183] Cytokines are proteins produced by many types of cells present within a tumor. They can modulate immune responses. The tumor often employs them to allow it to grow and reduce the immune response. These immune-modulating effects allow them to be used as drugs to provoke an immune response. Two commonly used cytokines are interferons and interleukins.48137193686

[0184] Interferons are produced by the immune system. They are usually involved in antiviral response, but also have use for cancer. They fall in three groups: type I (IFNa and IFNP), type II (IFNy) and type III (IFNI).

[0185] Interleukins have an array of immune system effects. IL-2 is an exemplary interleukin cytokine therapy.V. Additional Therapies

[0186] The methods of the disclosure may include administration of an additional therapy. The subject may be one that is being treated with an additional therapy. The additional therapy may be an additional immunotherapy or an additional therapy described herein.A. Chemotherapies

[0187] In some aspects, the additional therapy comprises a chemotherapy. Suitable classes of chemotherapeutic agents include (a) Alkylating Agents, such as nitrogen mustards (e.g., mechlorethamine, cylophosphamide, ifosfamide, melphalan, chlorambucil), ethylenimines and methylmelamines (e.g., hexamethylmelamine, thiotepa), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomustine, chlorozoticin, streptozocin) and triazines (e.g., dicarbazine), (b) Antimetabolites, such as folic acid analogs (e.g., methotrexate), pyrimidine analogs (e.g., 5-fluorouracil, floxuridine, cytarabine, azauridine) and purine analogs and related materials (e.g., 6-mercaptopurine, 6-thioguanine, pentostatin), (c) Natural Products, such as vinca alkaloids (e.g., vinblastine, vincristine), epipodophylotoxins (e.g., etoposide, teniposide), antibiotics (e.g., dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin and mitoxanthrone), enzymes (e.g., L-asparaginase), and biological response modifiers (e.g., Interferon-a), and (d) Miscellaneous Agents, such as platinum coordination complexes (e.g., cisplatin, carboplatin), substituted ureas (e.g., hydroxyurea), methylhydiazine derivatives (e.g., procarbazine), and adreocortical suppressants (e.g., taxol and mitotane). In some aspects, cisplatin is a particularly suitable chemotherapeutic agent.

[0188] Cisplatin has been widely used to treat cancers such as, for example, metastatic testicular or ovarian carcinoma, advanced bladder cancer, head or neck cancer, cervical cancer, lung cancer or other tumors. Cisplatin is not absorbed orally and must therefore be delivered via other routes such as, for example, intravenous, subcutaneous, intratumoral or intraperitoneal injection. Cisplatin can be used alone or in combination with other agents, with efficacious doses used in clinical applications including about 15 mg / m2 to about 20 mg / m249137193686for 5 days every three weeks for a total of three courses being contemplated in certain aspects. In some aspects, the amount of cisplatin delivered to the cell and / or subject in conjunction with the construct comprising an Egr-1 promoter operably linked to a polynucleotide encoding the therapeutic polypeptide is less than the amount that would be delivered when using cisplatin alone.

[0189] Other suitable chemotherapeutic agents include antimicrotubule agents, e.g., Paclitaxel (“Taxol”) and doxorubicin hydrochloride (“doxorubicin”). The combination of an Egr-1 promoter / TNFa construct delivered via an adenoviral vector and doxorubicin was determined to be effective in overcoming resistance to chemotherapy and / or TNF-a, which suggests that combination treatment with the construct and doxorubicin overcomes resistance to both doxorubicin and TNF-a.

[0190] Doxorubicin is absorbed poorly and is preferably administered intravenously. In certain aspects, appropriate intravenous doses for an adult include about 60 mg / m2 to about 75 mg / m2 at about 21 -day intervals or about 25 mg / m2 to about 30 mg / m2 on each of 2 or 3 successive days repeated at about 3 week to about 4 week intervals or about 20 mg / m2 once a week. The lowest dose should be used in elderly patients, when there is prior bone-marrow depression caused by prior chemotherapy or neoplastic marrow invasion, or when the drug is combined with other myelopoietic suppressant drugs.

[0191] Nitrogen mustards are another suitable chemotherapeutic agent useful in the methods of the disclosure. A nitrogen mustard may include, but is not limited to, mechlorethamine (HN2), cyclophosphamide and / or ifosfamide, melphalan (L-sarcolysin), and chlorambucil. Cyclophosphamide (CYTOXAN®) is available from Mead Johnson and NEOSTAR® is available from Adria), is another suitable chemotherapeutic agent. Suitable oral doses for adults include, for example, about 1 mg / kg / day to about 5 mg / kg / day, intravenous doses include, for example, initially about 40 mg / kg to about 50 mg / kg in divided doses over a period of about 2 days to about 5 days or about 10 mg / kg to about 15 mg / kg about every 7 days to about 10 days or about 3 mg / kg to about 5 mg / kg twice a week or about 1.5 mg / kg / day to about 3 mg / kg / day. Because of adverse gastrointestinal effects, the intravenous route is preferred. The drug also sometimes is administered intramuscularly, by infiltration or into body cavities.

[0192] Additional suitable chemotherapeutic agents include pyrimidine analogs, such as cytarabine (cytosine arabinoside), 5 -fluorouracil (fluouracil; 5-FU) and floxuridine (fluorodeoxyuridine; FudR). 5-FU may be administered to a subject in a dosage of anywhere between about 7.5 to about 1000 mg / m2. Further, 5-FU dosing schedules may be for a variety of time 50137193686periods, for example up to six weeks, or as determined by one of ordinary skill in the art to which this disclosure pertains.

[0193] Gemcitabine diphosphate (GEMZAR®, Eli Lilly & Co., “gemcitabine”), another suitable chemotherapeutic agent, is recommended for treatment of advanced and metastatic pancreatic cancer, and will therefore be useful in the present disclosure for these cancers as well.

[0194] The amount of the chemotherapeutic agent delivered to the patient may be variable. In one suitable aspect, the chemotherapeutic agent may be administered in an amount effective to cause arrest or regression of the cancer in a host, when the chemotherapy is administered with the construct. In other aspects, the chemotherapeutic agent may be administered in an amount that is anywhere between 2 to 10,000 fold less than the chemotherapeutic effective dose of the chemotherapeutic agent. For example, the chemotherapeutic agent may be administered in an amount that is about 20 fold less, about 500 fold less or even about 5000 fold less than the chemotherapeutic effective dose of the chemotherapeutic agent. The chemotherapeutics of the disclosure can be tested in vivo for the desired therapeutic activity in combination with the construct, as well as for determination of effective dosages. For example, such compounds can be tested in suitable animal model systems prior to testing in humans, including, but not limited to, rats, mice, chicken, cows, monkeys, rabbits, etc. In vitro testing may also be used to determine suitable combinations and dosages, as described in the examples.B. Radiotherapy

[0195] In some aspects, the additional therapy or prior therapy comprises radiation, such as ionizing radiation. As used herein, “ionizing radiation” means radiation comprising particles or photons that have sufficient energy or can produce sufficient energy via nuclear interactions to produce ionization (gain or loss of electrons). An exemplary and preferred ionizing radiation is an x-radiation. Means for delivering x-radiation to a target tissue or cell are well known in the art.C. Surgery

[0196] In some aspects, the additional therapy comprises surgery. Approximately 60% of persons with cancer will undergo surgery of some type, which includes preventative, diagnostic or staging, curative, and palliative surgery. Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and / or destroyed and may be used in51137193686conjunction with other therapies, such as the treatment of the present aspects, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy, and / or alternative therapies. Tumor resection refers to physical removal of at least part of a tumor. In addition to tumor resection, treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and microscopically-controlled surgery (Mohs’ surgery).

[0197] Upon excision of part or all of cancerous cells, tissue, or tumor, a cavity may be formed in the body. Treatment may be accomplished by perfusion, direct injection, or local application of the area with an additional anti-cancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months (or any range derivable therein). These treatments may be of varying dosages as well.VI. Treatment of Disease

[0198] Methods may be employed with respect to individuals who have tested positive for such disorders or who are deemed to be at risk for developing such a condition or related condition. In some aspects, the compositions and methods described herein are used to treat an inflammatory or autoimmune component of a disorder listed herein and / or known in the art.

[0199] Certain aspects of the disclosure relate to the treatment of cancer and / or use of cancer antigens. The cancer to be treated or antigen may be an antigen associated with any cancer known in the art or, for example, epithelial cancer, (e.g., breast, gastrointestinal, lung), prostate cancer, bladder cancer, lung (e.g., small cell lung) cancer, colon cancer, ovarian cancer, brain cancer, gastric cancer, renal cell carcinoma, pancreatic cancer, liver cancer, esophageal cancer, head and neck cancer, or a colorectal cancer. In some aspects, the cancer to be treated or antigen is from one of the following cancers: adenocortical carcinoma, agnogenic myeloid metaplasia, AIDS-related cancers (e.g., AIDS-related lymphoma), anal cancer, appendix cancer, astrocytoma (e.g., cerebellar and cerebral), basal cell carcinoma, bile duct cancer (e.g., extrahepatic), bladder cancer, bone cancer, (osteosarcoma and malignant fibrous histiocytoma), brain tumor (e.g., glioma, brain stem glioma, cerebellar or cerebral astrocytoma (e.g., pilocytic astrocytoma, diffuse astrocytoma, anaplastic (malignant) astrocytoma), malignant glioma, ependymoma, oligodenglioma, meningioma, meningiosarcoma, craniopharyngioma, haemangioblastomas, medulloblastoma, supratentorial primitive neuroectodermal tumors, visual pathway and hypothalamic glioma, and glioblastoma), breast cancer, bronchial adenomas / carcinoids, carcinoid tumor (e.g., gastrointestinal carcinoid52137193686tumor), carcinoma of unknown primary, central nervous system lymphoma, cervical cancer, colon cancer, colorectal cancer, chronic myeloproliferative disorders, endometrial cancer (e.g., uterine cancer), ependymoma, esophageal cancer, Ewing’s family of tumors, eye cancer (e.g., intraocular melanoma and retinoblastoma), gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor, (e.g., extracranial, extragonadal, ovarian), gestational trophoblastic tumor, head and neck cancer, hepatocellular (liver) cancer (e.g., hepatic carcinoma and heptoma), hypopharyngeal cancer, islet cell carcinoma (endocrine pancreas), laryngeal cancer, laryngeal cancer, leukemia, lip and oral cavity cancer, oral cancer, liver cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), lymphoid neoplasm (e.g., lymphoma), medulloblastoma, ovarian cancer, mesothelioma, metastatic squamous neck cancer, mouth cancer, multiple endocrine neoplasia syndrome, myelodysplastic syndromes, myelodysplastic / myeloproliferative diseases, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, neuroendocrine cancer, oropharyngeal cancer, ovarian cancer (e.g., ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor), pancreatic cancer, parathyroid cancer, penile cancer, cancer of the peritoneal, pharyngeal cancer, pheochromocytoma, pineoblastoma and supratentorial primitive neuroectodermal tumors, pituitary tumor, pleuropulmonary blastoma, lymphoma, primary central nervous system lymphoma (microglioma), pulmonary lymphangiomyomatosis, rectal cancer, renal cancer, renal pelvis and ureter cancer (transitional cell cancer), rhabdomyosarcoma, salivary gland cancer, skin cancer (e.g., non-melanoma (e.g., squamous cell carcinoma), melanoma, and Merkel cell carcinoma), small intestine cancer, squamous cell cancer, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, tuberous sclerosis, urethral cancer, vaginal cancer, vulvar cancer, Wilms’ tumor, and posttransplant lymphoproliferative disorder (PTLD), abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), or Meigs’ syndrome. Any of these cancers or conditions may be excluded in an embodiment.

[0200] Methods may be employed with respect to individuals who have tested positive for such disorders or who are deemed to be at risk for developing such a condition or related condition. The compositions and methods described herein may be used to treat an inflammatory or autoimmune component of a disorder listed herein and / or known in the art.

[0201] The autoimmune disease to be treated may be comprise or exclude diabetes, graft rejection, GVHC, arthritis (rheumatoid arthritis such as acute arthritis, chronic rheumatoid arthritis, gout or gouty arthritis, acute gouty arthritis, acute immunological arthritis, chronic 53137193686inflammatory arthritis, degenerative arthritis, type II collagen-induced arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, Still’s disease, vertebral arthritis, and juvenile-onset rheumatoid arthritis, osteoarthritis, arthritis chronica progrediente, arthritis deformans, polyarthritis chronica primaria, reactive arthritis, and ankylosing spondylitis), inflammatory hyperproliferative skin diseases, psoriasis such as plaque psoriasis, gutatte psoriasis, pustular psoriasis, and psoriasis of the nails, atopy including atopic diseases such as hay fever and Job’s syndrome, dermatitis including contact dermatitis, chronic contact dermatitis, exfoliative dermatitis, allergic dermatitis, allergic contact dermatitis, dermatitis herpetiformis, nummular dermatitis, seborrheic dermatitis, non-specific dermatitis, primary irritant contact dermatitis, and atopic dermatitis, x-linked hyper IgM syndrome, allergic intraocular inflammatory diseases, urticaria such as chronic allergic urticaria and chronic idiopathic urticaria, including chronic autoimmune urticaria, myositis, polymyositis / dermatomyositis, juvenile dermatomyositis, toxic epidermal necrolysis, scleroderma (including systemic scleroderma), sclerosis such as systemic sclerosis, multiple sclerosis (MS) such as spino-optical MS, primary progressive MS (PPMS), and relapsing remitting MS (RRMS), progressive systemic sclerosis, atherosclerosis, arteriosclerosis, sclerosis disseminata, ataxic sclerosis, neuromyelitis optica (NMO), inflammatory bowel disease (IBD) (for example, Crohn’s disease, autoimmune-mediated gastrointestinal diseases, colitis such as ulcerative colitis, colitis ulcerosa, microscopic colitis, collagenous colitis, colitis polyposa, necrotizing enterocolitis, and transmural colitis, and autoimmune inflammatory bowel disease), bowel inflammation, pyoderma gangrenosum, erythema nodosum, primary sclerosing cholangitis, respiratory distress syndrome, including adult or acute respiratory distress syndrome (ARDS), meningitis, inflammation of all or part of the uvea, iritis, choroiditis, an autoimmune hematological disorder, rheumatoid spondylitis, rheumatoid synovitis, hereditary angioedema, cranial nerve damage as in meningitis, herpes gestationis, pemphigoid gestationis, pruritis scroti, autoimmune premature ovarian failure, sudden hearing loss due to an autoimmune condition, IgE-mediated diseases such as anaphylaxis and allergic and atopic rhinitis, encephalitis such as Rasmussen’s encephalitis and limbic and / or brainstem encephalitis, uveitis, such as anterior uveitis, acute anterior uveitis, granulomatous uveitis, nongranulomatous uveitis, phacoantigenic uveitis, posterior uveitis, or autoimmune uveitis, glomerulonephritis (GN) with and without nephrotic syndrome such as chronic or acute glomerulonephritis such as primary GN, immune-mediated GN, membranous GN (membranous nephropathy), idiopathic membranous GN or idiopathic membranous nephropathy, membrano- or membranous proliferative GN (MPGN), including Type I and 54137193686Type II, and rapidly progressive GN, proliferative nephritis, autoimmune polyglandular endocrine failure, balanitis including balanitis circumscripta plasmacellularis, balanoposthitis, erythema annulare centrifugum, erythema dyschromicum perstans, eythema multiform, granuloma annulare, lichen nitidus, lichen sclerosus et atrophicus, lichen simplex chronicus, lichen spinulosus, lichen planus, lamellar ichthyosis, epidermolytic hyperkeratosis, premalignant keratosis, pyoderma gangrenosum, allergic conditions and responses, allergic reaction, eczema including allergic or atopic eczema, asteatotic eczema, dyshidrotic eczema, and vesicular palmoplantar eczema, asthma such as asthma bronchiale, bronchial asthma, and auto-immune asthma, conditions involving infiltration of T cells and chronic inflammatory responses, immune reactions against foreign antigens such as fetal A-B-0 blood groups during pregnancy, chronic pulmonary inflammatory disease, autoimmune myocarditis, leukocyte adhesion deficiency, lupus, including lupus nephritis, lupus cerebritis, pediatric lupus, non-renal lupus, extra-renal lupus, discoid lupus and discoid lupus erythematosus, alopecia lupus, systemic lupus erythematosus (SLE) such as cutaneous SLE or subacute cutaneous SLE, neonatal lupus syndrome (NLE), and lupus erythematosus disseminatus, juvenile onset (Type I) diabetes mellitus, including pediatric insulin-dependent diabetes mellitus (IDDM), and adult onset diabetes mellitus (Type II diabetes) and autoimmune diabetes. Also contemplated are immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes, sarcoidosis, granulomatosis including lymphomatoid granulomatosis, Wegener’s granulomatosis, agranulocytosis, vasculitides, including vasculitis, large-vessel vasculitis (including polymyalgia rheumatica and gianT cell (Takayasu’s) arteritis), mediumvessel vasculitis (including Kawasaki’s disease and polyarteritis nodosa / periarteritis nodosa), microscopic polyarteritis, immunovasculitis, CNS vasculitis, cutaneous vasculitis, hypersensitivity vasculitis, necrotizing vasculitis such as systemic necrotizing vasculitis, and ANCA-associated vasculitis, such as Churg-Strauss vasculitis or syndrome (CSS) and ANCA-associated small-vessel vasculitis, temporal arteritis, aplastic anemia, autoimmune aplastic anemia, Coombs positive anemia, Diamond Blackfan anemia, hemolytic anemia or immune hemolytic anemia including autoimmune hemolytic anemia (AIHA), Addison’s disease, autoimmune neutropenia, pancytopenia, leukopenia, diseases involving leukocyte diapedesis, CNS inflammatory disorders, Alzheimer’s disease, Parkinson’s disease, multiple organ injury syndrome such as those secondary to septicemia, trauma or hemorrhage, antigen-antibody complex-mediated diseases, anti -glomerular basement membrane disease, anti-phospholipid antibody syndrome, allergic neuritis, Behcet’s disease / syndrome, Castleman’s syndrome, Goodpasture’s syndrome, Reynaud’s syndrome, Sjogren’s syndrome, Stevens-Johnson 55137193686syndrome, pemphigoid such as pemphigoid bullous and skin pemphigoid, pemphigus (including pemphigus vulgaris, pemphigus foliaceus, pemphigus mucus-membrane pemphigoid, and pemphigus erythematosus), autoimmune polyendocrinopathies, Reiter’s disease or syndrome, thermal injury, preeclampsia, an immune complex disorder such as immune complex nephritis, antibody-mediated nephritis, polyneuropathies, chronic neuropathy such as IgM polyneuropathies or IgM-mediated neuropathy, autoimmune or immune-mediated thrombocytopenia such as idiopathic thrombocytopenic purpura (ITP) including chronic or acute ITP, scleritis such as idiopathic cerato-scleritis, episcleritis, autoimmune disease of the testis and ovary including autoimmune orchitis and oophoritis, primary hypothyroidism, hypoparathyroidism, autoimmune endocrine diseases including thyroiditis such as autoimmune thyroiditis, Hashimoto’s disease, chronic thyroiditis (Hashimoto’s thyroiditis), or subacute thyroiditis, autoimmune thyroid disease, idiopathic hypothyroidism, Grave’s disease, polyglandular syndromes such as autoimmune polyglandular syndromes (or polyglandular endocrinopathy syndromes), paraneoplastic syndromes, including neurologic paraneoplastic syndromes such as Lambert-Eaton myasthenic syndrome or Eaton-Lambert syndrome, stiff-man or stiff-person syndrome, encephalomyelitis such as allergic encephalomyelitis or encephalomyelitis allergica and experimental allergic encephalomyelitis (EAE), experimental autoimmune encephalomyelitis, myasthenia gravis such as thymoma-associated myasthenia gravis, cerebellar degeneration, neuromyotonia, opsoclonus or opsoclonus myoclonus syndrome (OMS), and sensory neuropathy, multifocal motor neuropathy, Sheehan’s syndrome, autoimmune hepatitis, chronic hepatitis, lupoid hepatitis, gianT cell hepatitis, chronic active hepatitis or autoimmune chronic active hepatitis, lymphoid interstitial pneumonitis (LIP), bronchiolitis obliterans (non-transplant) vs NSIP, Guillain-Barre syndrome, Berger’s disease (IgA nephropathy), idiopathic IgA nephropathy, linear IgA dermatosis, acute febrile neutrophilic dermatosis, subcorneal pustular dermatosis, transient acantholytic dermatosis, cirrhosis such as primary biliary cirrhosis and pneumonocirrhosis, autoimmune enteropathy syndrome, Celiac or Coeliac disease, celiac sprue (gluten enteropathy), refractory sprue, idiopathic sprue, cryoglobulinemia, amylotrophic lateral sclerosis (ALS; Lou Gehrig’s disease), coronary artery disease, autoimmune ear disease such as autoimmune inner ear disease (AIED), autoimmune hearing loss, polychondritis such as refractory or relapsed or relapsing polychondritis, pulmonary alveolar proteinosis, Cogan’s syndrome / nonsyphilitic interstitial keratitis, Bell’s palsy, Sweet’s disease / syndrome, rosacea autoimmune, zoster-associated pain, amyloidosis, a non-cancerous lymphocytosis, a primary lymphocytosis, which includes monoclonal B cell lymphocytosis (e.g., benign monoclonal 56137193686gammopathy and monoclonal gammopathy of undetermined significance, MGUS), peripheral neuropathy, paraneoplastic syndrome, channelopathies such as epilepsy, migraine, arrhythmia, muscular disorders, deafness, blindness, periodic paralysis, and channelopathies of the CNS, autism, inflammatory myopathy, focal or segmental or focal segmental glomerulosclerosis (FSGS), endocrine opthalmopathy, uveoretinitis, chorioretinitis, autoimmune hepatological disorder, fibromyalgia, multiple endocrine failure, Schmidt’s syndrome, adrenalitis, gastric atrophy, presenile dementia, demyelinating diseases such as autoimmune demyelinating diseases and chronic inflammatory demyelinating polyneuropathy, Dressier’s syndrome, alopecia greata, alopecia totalis, CREST syndrome (calcinosis, Raynaud’s phenomenon, esophageal dysmotility, sclerodactyl), and telangiectasia), male and female autoimmune infertility, e.g., due to anti-spermatozoan antibodies, mixed connective tissue disease, Chagas’ disease, rheumatic fever, recurrent abortion, farmer’s lung, erythema multiforme, postcard! otomy syndrome, Cushing’s syndrome, bird-fancier’s lung, allergic granulomatous angiitis, benign lymphocytic angiitis, Alport’s syndrome, alveolitis such as allergic alveolitis and fibrosing alveolitis, interstitial lung disease, transfusion reaction, leprosy, malaria, parasitic diseases such as leishmaniasis, kypanosomiasis, schistosomiasis, ascariasis, aspergillosis, Sampler’s syndrome, Caplan’s syndrome, dengue, endocarditis, endomyocardial fibrosis, diffuse interstitial pulmonary fibrosis, interstitial lung fibrosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endophthalmitis, erythema elevatum et diutinum, erythroblastosis fetalis, eosinophilic faciitis, Shulman’s syndrome, Felty’s syndrome, flariasis, cyclitis such as chronic cyclitis, heterochronic cyclitis, iridocyclitis (acute or chronic), or Fuch’s cyclitis, Henoch-Schonlein purpura, human immunodeficiency virus (HIV) infection, SCID, acquired immune deficiency syndrome (AIDS), echovirus infection, sepsis, endotoxemia, pancreatitis, thyroxicosis, parvovirus infection, rubella virus infection, postvaccination syndromes, congenital rubella infection, Epstein-Barr virus infection, mumps, Evan’s syndrome, autoimmune gonadal failure, Sydenham’s chorea, post-streptococcal nephritis, thromboangitis ubiterans, thyrotoxicosis, tabes dorsalis, chorioiditis, gianT cell polymyalgia, chronic hypersensitivity pneumonitis, keratoconjunctivitis sicca, epidemic keratoconjunctivitis, idiopathic nephritic syndrome, minimal change nephropathy, benign familial and ischemia-reperfusion injury, transplant organ reperfusion, retinal autoimmunity, joint inflammation, bronchitis, chronic obstructive airway / pulmonary disease, silicosis, aphthae, aphthous stomatitis, arteriosclerotic disorders, aspemiogenese, autoimmune hemolysis, Boeck’s disease, cryoglobulinemia, Dupuytren’s contracture, endophthalmia phacoanaphylactica, enteritis allergica, erythema nodosum leprosum, idiopathic facial 57137193686paralysis, chronic fatigue syndrome, febris rheumatica, Hamman-Rich’s disease, sensoneural hearing loss, haemoglobinuria paroxysmatica, hypogonadism, ileitis regionalis, leucopenia, mononucleosis infectiosa, traverse myelitis, primary idiopathic myxedema, nephrosis, ophthalmia symphatica, orchitis granulomatosa, pancreatitis, polyradiculitis acuta, pyoderma gangrenosum, Quervain’s thyreoiditis, acquired spenic atrophy, non-malignant thymoma, vitiligo, toxic-shock syndrome, food poisoning, conditions involving infiltration of T cells, leukocyte-adhesion deficiency, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes, diseases involving leukocyte diapedesis, multiple organ injury syndrome, antigen-antibody complex-mediated diseases, antiglomerular basement membrane disease, allergic neuritis, autoimmune polyendocrinopathies, oophoritis, primary myxedema, autoimmune atrophic gastritis, sympathetic ophthalmia, rheumatic diseases, mixed connective tissue disease, nephrotic syndrome, insulitis, polyendocrine failure, autoimmune polyglandular syndrome type I, adultonset idiopathic hypoparathyroidism (AOIH), cardiomyopathy such as dilated cardiomyopathy, epidermolisis bullosa acquisita (EBA), hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosing cholangitis, purulent or nonpurulent sinusitis, acute or chronic sinusitis, ethmoid, frontal, maxillary, or sphenoid sinusitis, an eosinophil-related disorder such as eosinophilia, pulmonary infiltration eosinophilia, eosinophilia-myalgia syndrome, Loftier’ s syndrome, chronic eosinophilic pneumonia, tropical pulmonary eosinophilia, bronchopneumonic aspergillosis, aspergilloma, or granulomas containing eosinophils, anaphylaxis, seronegative spondyloarthritides, polyendocrine autoimmune disease, sclerosing cholangitis, sclera, episclera, chronic mucocutaneous candidiasis, Bruton’s syndrome, transient hypogammaglobulinemia of infancy, Wiskott-Aldrich syndrome, ataxia telangiectasia syndrome, angiectasis, autoimmune disorders associated with collagen disease, rheumatism, neurological disease, lymphadenitis, reduction in blood pressure response, vascular dysfunction, tissue injury, cardiovascular ischemia, hyperalgesia, renal ischemia, cerebral ischemia, and disease accompanying vascularization, allergic hypersensitivity disorders, glomerulonephritides, reperfusion injury, ischemic re-perfusion disorder, reperfusion injury of myocardial or other tissues, lymphomatous tracheobronchitis, inflammatory dermatoses, dermatoses with acute inflammatory components, multiple organ failure, bullous diseases, renal cortical necrosis, acute purulent meningitis or other central nervous system inflammatory disorders, ocular and orbital inflammatory disorders, granulocyte transfusion-associated syndromes, cytokine-induced toxicity, narcolepsy, acute serious inflammation, chronic intractable inflammation, pyelitis, endarterial hyperplasia, peptic 58137193686ulcer, valvulitis, graft versus host disease, contact hypersensitivity, asthmatic airway hyperreaction, and endometriosis.

[0202] Embodiment one is a method for making T cells and / or for increasing the cytotoxicity of T cells, comprising contacting the T cell ex vivo with a T cell modulator, wherein the T cell modulator comprises Telmisartan or AZD4573 and wherein the T cells comprise engineered CAR-T cells or TCR-T cells.

[0203] Embodiment two is a method for making T cells comprising contacting the T cell ex vivo with a T cell modulator, wherein the T cell modulator comprises Telmisartan, Calcimycin, Calmidazolium, Thapsigargin, a CDK9 inhibitor, Riviciclib, a BET inhibitor, a CDK8 inhibitor, AZD4573, NSC-95397, PMA, or combinations thereof.

[0204] Embodiment three is the method of Embodiment one or two, wherein the method increases the cytotoxicity of the T cells and / or increases the antitumor activity of the cells

[0205] Embodiment four is the method of any one of Embodiments two to three, wherein the CDK9 inhibitor comprises PHA-767491 or the CDK8 inhibitor comprises CDK8-IN-4.

[0206] Embodiment five is the method of any one of Embodiments one to four, wherein the concentration of the T cell modulator in contact with the cell is 1 nM - 1 mM.

[0207] Embodiment six is the method of any one of Embodiments one to five, wherein contacting the T cell ex vivo with the T cell modulator comprises culturing the cell in cell medium comprising the T cell modulator.

[0208] Embodiment seven is the method of Embodiment six, wherein the concentration of the T cell modulator in the cell medium is 1 nM - 1 mM.

[0209] Embodiment eight is the method of Embodiment seven, wherein the T cell modulator comprises Telmisartan at a concentration of 0.1 nM - 100 pM.

[0210] Embodiment nine is the method of Embodiment seven, wherein the T cell modulator comprises AZD4573 at a concentration of 0.1 nM 100 pM.

[0211] Embodiment ten is the method of any one of Embodiments one to nine, wherein the T cell is derived from a CD3+ T cell.

[0212] Embodiment eleven is the method of any one of Embodiments one to ten, wherein the T cell is derived from a CD8+ T cell.

[0213] Embodiment twelve is the method of any one of Embodiments one to eleven, wherein the T cell is derived from a naive or memory T cell.

[0214] Embodiment thirteen is the method of any one of Embodiments one to twelve, wherein the cell is contacted with the T cell modulator for a period of time of at least zero point one hours.59137193686

[0215] Embodiment fourteen is the method of any one of Embodiments one to thirteen, wherein the cell has been isolated from a patient by leukapheresis.

[0216] Embodiment fifteen is the method of any one of Embodiments one to fourteen, wherein the T cell comprises an engineered T cell..

[0217] Embodiment sixteen is the method of Embodiment fifteen, wherein the T cell comprises a heterologous nucleic acid encoding a T cell receptor (TCR) or chimeric antigen receptor (CAR).

[0218] Embodiment seventeen is the method of Embodiment sixteen, wherein the cell has been transduced with a viral vector comprising a nucleic acid encoding a TCR or CAR.

[0219] Embodiment eighteen is the method of Embodiment seventeen, wherein the viral vector comprises a lentiviral vector, AAV vector, or retroviral vector.

[0220] Embodiment nineteen is the method of any one of Embodiments one to sixteen, wherein a nucleic acid encoding the TCR or CAR has been transferred into the cell by a non-viral method.

[0221] Embodiment twenty is the method of any one of Embodiments one to nineteen, wherein the T cell comprises T cells isolated from a subject.

[0222] Embodiment twenty-one is the method of Embodiment twenty, wherein the T cells comprise tumor infiltrating lymphocytes (TILs)

[0223] Embodiment twenty -two is the method of any one of Embodiments one to twenty-one, wherein the method further comprises cryopreservation of the cell before or after contacting the cell with the T cell modulator.

[0224] Embodiment twenty-three is a T cell made by the method of any one of Embodiments one to twenty-two.

[0225] Embodiment twenty -four is a population of T cells made by the method of any one of Embodiments one to twenty -two or derived from the cell of Embodiment twenty -three.

[0226] Embodiment twenty-five is the population of cells of Embodiment twenty-four, wherein the population comprises CD4+ T cells, CD8+ T cells, or a mixture of CD4+ and CD8+ T cells.

[0227] Embodiment twenty-six is a method for treating a subj ect comprising administering the cell or cells of any one of Embodiments twenty -three to twenty-five.

[0228] Embodiment twenty-seven is the method of Embodiment twenty-six, wherein IxlO6- IxlO10cells are administered.

[0229] Embodiment twenty-eight is the method of Embodiment twenty-seven, wherein the cells are determined to be positive for expression of the TCR or CAR.60137193686

[0230] Embodiment twenty -nine is the method of any one of Embodiments twenty-six to twenty-eight, wherein the cells are autologous cells.

[0231] Embodiment thirty is the method of any one of Embodiments twenty-six to twenty-nine, wherein the method further comprises administration of an additional therapy.

[0232] Embodiment thirty-one is the method of Embodiment thirty, wherein the additional therapy comprises a chemotherapy.

[0233] Embodiment thirty -two is the method of Embodiment thirty or thirty-one, wherein the additional therapy comprises lymphodepletion.

[0234] Embodiment thirty -three is the method of any one of Embodiments thirty to thirty-two, wherein the additional therapy comprises fludarabine and / or cyclophosphamide.

[0235] Embodiment thirty-four is the method of any one of Embodiments thirty to thirty-three, wherein the additional therapy is given prior to administration of the cells.

[0236] Embodiment thirty-five is the method of Embodiment thirty-four, wherein the additional therapy is given to the subject starting at five days prior to administration of the cells.

[0237] Embodiment thirty-six is the method of any one of Embodiments twenty-six to thirty-five, wherein the method is for treating cancer and wherein the subject has cancer.

[0238] Embodiment thirty-seven is the method of Embodiment thirty-six, wherein the cancer is selected from lung cancer, prostate cancer, ovarian cancer, testicular cancer, brain cancer, neuroblastoma, sarcoma, osteosarcoma, skin cancer, melanoma, colon cancer, rectal cancer, gastric cancer, esophageal cancer, tracheal cancer, head & neck cancer, pancreatic cancer, liver cancer, breast cancer, ovarian cancer, glioblastoma, glioma, a B-cell malignancy, melanoma, leukemia, sarcomas of bone or soft tissue, cervical cancer, and vulvar cancer.

[0239] Embodiment thirty-eight is the method of Embodiment thirty-seven, wherein the cancer comprises melanoma.

[0240] Embodiment thirty-nine is the method of Embodiment thirty-eight, wherein the melanoma is further classified as superficial spreading, nodular, lentigo maligna, acral lentiginous, amelanotic, nevoid, spitzoid, desmoplastic, cutaneous, eye, choroidal, onjunctival, iris, and / or mucosal.

[0241] Embodiment forty is the method of any one of Embodiments twenty-six to thirty-nine, wherein the additional therapy comprises an immunotherapy.

[0242] Embodiment forty-one is the method of Embodiment forty, wherein the T cell modulator and immunotherapy are administered at the same time.61137193686

[0243] Embodiment forty-two is the method of Embodiment forty, wherein the T cell modulator is administered prior to the immunotherapy.

[0244] Embodiment forty-three is the method of Embodiment forty, wherein the T cell modulator is administered after the immunotherapy.

[0245] Embodiment forty-four is the method of any one of Embodiments forty to forty-three, wherein the immunotherapy comprises an immune checkpoint inhibitor (ICI), an immune agonist antibody, or nucleic acids encoding for an immune checkpoint inhibitor or an immune agonist antibody.

[0246] Embodiment forty -five is the method of Embodiment forty -four, wherein the ICI or immune agonist antibody comprises an anti-CD40 agonistic antibody, anti-PDl blocking antibody, anti-PDLl blocking antibody, anti-CTLA4 blocking antibody, anti-LAG3 blocking antibody, or combinations thereof.

[0247] Embodiment forty-six is the method of Embodiment forty-five, wherein the ICI comprises one or more of Ipilimumab, Tremelimumab, Nivolumab, Pembrolizumab, Atezolizumab, Avelumab, Durvalumab, Spartalizumab, Cemiplimab, and Opdualag.

[0248] Embodiment forty-seven is the method of any one of Embodiments twenty-six to forty-six, wherein the method is for treating an autoimmune disorder.

[0249] Embodiment forty-eight is a method for treating cancer in a subject, the method comprising administering a T cell modulator to the subject, wherein the subject is being treated with one or more immunotherapies and wherein the T cell modulator comprises Telmisartan or AZD4573.

[0250] Embodiment forty-nine is a method for treating cancer in a subject comprising administering a T cell modulator and one or more immunotherapies to the subject, wherein the T cell modulator comprises Telmisartan.

[0251] Embodiment fifty is a method for treating cancer in a subject, the method comprising administering a T cell modulator to the subject, wherein the subject is being treated with one or more immunotherapies and wherein the T cell modulator comprises Telmisartan, Calcimycin, Calmidazolium, Thapsigargin, a CDK9 inhibitor, Riviciclib, a BET inhibitor, a CDK8 inhibitor, AZD4573, NSC-95397, PMA, or combinations thereof.

[0252] Embodiment fifty-one is a method for treating cancer in a subject comprising administering a T cell modulator and one or more immunotherapies to the subject, wherein the T cell modulator comprises Telmisartan, Calcimycin, Calmidazolium, Thapsigargin, a CDK9 inhibitor, Riviciclib, a BET inhibitor, a CDK8 inhibitor, AZD4573, NSC-95397, PMA, or combinations thereof.62137193686

[0253] Embodiment fifty -two is the method of Embodiment fifty or fifty-one, wherein the CDK9 inhibitor comprises PHA-767491 or the CDK8 inhibitor comprises CDK8-IN-4.

[0254] Embodiment fifty-three is the method of any one of Embodiments forty-eight to fifty -two, wherein the T cell modulator and immunotherapy are administered at the same time.

[0255] Embodiment fifty-four is the method of any one of Embodiments forty-eight to fifty-three, wherein the T cell modulator is administered prior to the immunotherapy.

[0256] Embodiment fifty-five is the method of any one of Embodiments forty-eight to fifty-three, wherein the T cell modulator is administered after the immunotherapy.

[0257] Embodiment fifty-six is the method of any one of Embodiments forty-eight to fifty-five, wherein the immunotherapy comprises adoptive cell therapy and optionally wherein the adoptive cell therapy comprises TCR-T and / or CAR-T cell therapy.

[0258] Embodiment fifty-seven is the method of any one of Embodiments forty-eight to fifty-six, wherein the immunotherapy comprises an immune checkpoint inhibitor (ICI), an immune agonist antibody, or nucleic acids encoding for an immune checkpoint inhibitor or an immune agonist antibody.

[0259] Embodiment fifty-eight is the method of Embodiment fifty-seven, wherein the ICI or immune agonist antibody comprises an anti-CD40 agonistic antibody, anti-PDl blocking antibody, anti-PDLl blocking antibody, anti-CTLA4 blocking antibody, anti-LAG3 blocking antibody, or combinations thereof.

[0260] Embodiment fifty -nine is the method of Embodiment fifty-eight, wherein the ICI comprises one or more of Ipilimumab, Tremelimumab, Nivolumab, Pembrolizumab, Atezolizumab, Avelumab, Durvalumab, Spartalizumab, Cemiplimab, and Opdualag.

[0261] Embodiment sixty is the method of any one of Embodiments forty-eight to fifty-nine, wherein the immunotherapy comprises pro-inflammatory molecules or nucleic acids encoding for pro-inflammatory molecules.

[0262] Embodiment sixty-one is the method of Embodiment sixty, wherein the pro-inflammatory molecule comprises IL-2, IL-12, IL-15, or combinations thereof.

[0263] Embodiment sixty-two is the method of any one of Embodiments forty-eight to sixty-one, wherein the method comprises administration of an additional therapy.

[0264] Embodiment sixty-three is the method of any one of Embodiments forty-eight to sixty-one, wherein the subject is being treated with or has been prescribed an additional therapy.63137193686

[0265] Embodiment sixty-four is the method of Embodiment sixty-two or sixty-three, wherein the additional therapy comprises an immunotherapy, a chemotherapy, radiotherapy, or surgery.

[0266] Embodiment sixty-five is the method of any one of Embodiments forty-eight to sixty-four, wherein the cancer is selected from lung cancer, prostate cancer, ovarian cancer, testicular cancer, brain cancer, neuroblastoma, sarcoma, osteosarcoma, skin cancer, melanoma, colon cancer, rectal cancer, gastric cancer, esophageal cancer, tracheal cancer, head & neck cancer, pancreatic cancer, liver cancer, breast cancer, ovarian cancer, glioblastoma, glioma, a B-cell malignancy, melanoma, leukemia, sarcomas of bone or soft tissue, cervical cancer, and vulvar cancer.

[0267] Embodiment sixty-six is the method of Embodiment sixty-five, wherein the cancer comprises melanoma.

[0268] Embodiment sixty-seven is the method of Embodiment sixty-five, wherein the melanoma is further classified as superficial spreading, nodular, lentigo maligna, acral lentiginous, amelanotic, nevoid, spitzoid, desmoplastic, cutaneous, eye, choroidal, onjunctival, iris, and / or mucosal.

[0269] Embodiment sixty-eight is a method for treating an autoimmune disorder in a subject or for suppressing an immune response in a subject, the method comprising administering a T cell modulator to the subject, wherein the T cell modulator comprises Telmisartan or AZD4573.

[0270] Embodiment sixty-nine is a method for treating an autoimmune disorder in a subj ect or for suppressing an immune response in a subject, the method comprising administering a T cell modulator to the subject, wherein the T cell modulator comprises Telmisartan, Calcimycin, Calmidazolium, Thapsigargin, a CDK9 inhibitor, Riviciclib, a BET inhibitor, a CDK8 inhibitor, AZD4573, NSC-95397, PMA, or combinations thereof.

[0271] Embodiment seventy is the method of Embodiment sixty-nine, wherein the CDK9 inhibitor comprises PHA-767491 or the CDK8 inhibitor comprises CDK8-IN-4.

[0272] Embodiment seventy-one is the method of any one of Embodiments sixty-eight to seventy, wherein the autoimmune or inflammatory condition comprises inflammatory bowel disease, idiopathic pulmonary fibrosis, multiple sclerosis, type one diabetes, Crohn’s disease, psoriasis, acute inflammation, chronic inflammation, neuroinflammation, arthritis, rheumatoid arthritis, fibrosis, infection, allergy, inflammatory therapy-related adverse events, and -related inflammatory illness.64137193686

[0273] Embodiment seventy -two is the method of any one of Embodiments sixty-eight to seventy-one, wherein the subject is being treated with an immunotherapy or the method further comprises administration of an immunotherapy.

[0274] Embodiment seventy-three is the method of Embodiment seventy-two, wherein the method is for suppressing an immune response in a subject and wherein the immune response comprises a toxic immune response associated with administration of an immunotherapy.

[0275] Embodiment seventy-four is the method of any one of Embodiments sixty-eight to seventy-three, wherein the immune response comprises cytokine release syndrome.

[0276] Embodiment seventy-five is the method of any one of Embodiments forty-eight to seventy-four, wherein the T cell modulator is administered systemically.

[0277] Embodiment seventy-six is the method of any one of Embodiments forty-eight to seventy-five, wherein the T cell modulator is administered by intravenous injection.

[0278] Embodiment seventy-seven is the method of any one of Embodiments forty-eight to seventy-five, wherein the T cell modulator is administered intratum orally.

[0279] Embodiment seventy-eight is the method of any one of Embodiments twenty-six to seventy-seven, wherein the subject is a human subject.

[0280] Embodiment seventy-nine is a method comprising contacting a composition comprising a MHC molecule and a TCR with T cell modulator, wherein the T cell modulator comprises Telmisartan or AZD4573.

[0281] Embodiment eighty is a method comprising contacting a composition comprising a MHC molecule and a TCR with T cell modulator, wherein the T cell modulator comprises Telmisartan, Calcimycin, Calmidazolium, Thapsigargin, a CDK9 inhibitor, Riviciclib, a BET inhibitor, a CDK8 inhibitor, AZD4573, NSC-95397, PMA, or combinations thereof.

[0282] Embodiment eighty-one is the method of Embodiment seventy-nine or eighty, wherein the method is for increasing the binding of the TCR and the MHC molecule.

[0283] Embodiment eighty-two is the method of any one of Embodiments eighty to eighty-one, wherein the CDK9 inhibitor comprises PHA-767491 or the CDK8 inhibitor comprises CDK8-IN-4.

[0284] Embodiment eighty -three is the method of any one of Embodiments seventy -nine to eighty-two, wherein the MHC molecule and / or TCR is on a cell.

[0285] Embodiment eighty-four is the method of Embodiment eighty-three, wherein the cell comprises a stem cell or an immune cell.

[0286] Embodiment eighty-five is the method of Embodiment eighty-four, wherein the immune cell comprises a T cell.65137193686

[0287] Embodiment eighty-six is the method of any one of Embodiments seventy-nine to eighty-five, wherein the MHC molecule comprises a MHC tetramer.

[0288] Embodiment eighty-seven is a method for performing a tetramer assay comprising contacting a composition comprising a MHC tetramer and T cells with a T cell modulator, wherein the T cell modulator comprises telmisartan or AZD4573.

[0289] Embodiment eighty-eight is a method for performing a tetramer assay comprising contacting a composition comprising a MHC tetramer and T cells with a T cell modulator, wherein the T cell modulator comprises Telmisartan, Calcimycin, Calmidazolium, Thapsigargin, a CDK9 inhibitor, Riviciclib, a BET inhibitor, a CDK8 inhibitor, AZD4573, NSC-95397, PMA, or combinations thereof.

[0290] Embodiment eighty-nine is the method of Embodiment eighty-seven or eightyeight, wherein the method further comprises isolating T cells that are specifically bound to the MHC tetramer.VII. Examples

[0291] The following examples are included to demonstrate preferred aspects of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific aspects which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.Example 1: USE OF TELMISARTAN AND AZD4573 TO IMPROVE T CELL CYTOTOXICITY FOR CANCER IMMUNOTHERAPY

[0292] The inventors have established a T cell-based screening platform capable of identifying novel hits that pharmacologically rewire specific T cell responses. The strength of this high-throughput drug screen relies on several factors including the use of a unique panel of patient-derived neoantigen-specific TCRs targeting the same mutation but with different cytotoxic profiles (TCR66 with no cytotoxicity, TCR 67 cytotoxic and TCR 68 with intermediate cytotoxicity). In addition, the inventors have used an autologous melanoma cell line derived from the same patient, M490, which have been genetically modified to express GFP and luciferase, allowing a two-readout system that increased the sensitivity of the screen.66137193686Using this screen, they have identified key known T cell regulators of T cell activation as well as drugs that inhibit TCR signaling.

[0293] The screen is also able to identify novel drugs that have not been previously associated with T cell cytotoxicity. The inventors have also found an FDA approved drug, Telmisartan, which increased T cell cytotoxicity in all three TCRs. They have validated that Telmisartan increases T cell cytotoxicity in an antigen-specific manner, leading to increased cytotoxicity of all three TCRs upon co-culture with the autologous cell line, M490, but not when co-cultured with a mismatched cell line derived from a different patient or when using an irrelevant TCR (NY-ESO1 TCR) that targets an antigen not expressed in the M490 target cell line. They have also shown that telmisartan does not lead to T cell killing by TCRs 66, 67 and 68 when co-cultured with an HLA-A2 melanoma cell line that express the WT version of the gene that is targeted by these TCRs.

[0294] In addition, the inventors have expanded the list of TCRs with increased cytotoxicity when combined with Telmisartan. To date they have a total of 11 TCRs targeting different antigens and restricted to different HLA classes that benefit from Telmisartan. In summary the list of TCRs is: 3 TCRs (66,67,68) that target a mutation in ATP11 A restricted to HLA-A2. 5 TCRs (69,109,110,111,112) that target a mutation in NAT10 restricted to HLA-A2. 1 TCR (106) that target a mutation in HP1BP3 restricted to HLA-B15:01. 1 TCR (107) that target a mutation in PRPSAP2 restricted to HLA-C3 :04. 1 TCR (108) that target a mutation in UVSSA restricted to HLA-B57:01.

[0295] In summary, these results demonstrate that Telmisartan effect on T cell cytotoxicity is antigen-specific and it is not limited to a single neoantigen or HLA class. Also, Telmisartan does not induce T cell cytotoxicity when co-cultured with cells bearing WT gene versions of the antigen these TCRs recognize.

[0296] Furthermore, Telmisartan increased T cell activation as measured by 4-1BB expression levels in T cells engineered to express either TCR66, TCR67 or TCR68 and co-cultured with the autologous M490 melanoma cells for 24 hours. T cells expressing the NY-ESO-1 TCR were used as a negative control and we did not observe any 4- IBB increase with the addition of Telmisartan. Also, the effect of Telmisartan is dose dependent as observed in co-cultures of TCR-engineered T cells with the M490 cell line and doses of 0.1 pM, 0.25pM, 0,5pM, 2.5pM and 5pM of Telmisartan. Of note, T cell killing was observed in doses as low as lOOnM. Furthermore, Telmisartan allows the reduction of effector to target (E:T) ratios without compromising T cell cytotoxicity and bypasses the need of pre-treating M490 cells with IFNy to increase HLA-expression, suggesting that Telmisartan is able to increase the 67137193686killing of melanoma target cells with few HLA-antigen molecules on the surface. Importantly, Telmisartan maintains its effect on T cells through multiple repetitive antigen challenges. Telmisartan allowed T cells engineered to express TCR66 and TCR68 to maintain its cytotoxic activity through three antigen challenges, while T cells engineered to express TCR67, maintained its cytotoxic activity through two antigen challenges.

[0297] Importantly, Telmisartan improves adoptive cell therapy efficacy in vivo. The inventors have observed that the use of Telmisartan in combination with T cells engineered to express TCR68, significantly decreased M490 tumor growth control in NSG mice. They observed that this effect was dose dependent, with 500mg / kg Telmisartan dose being the most effective dose tested. In addition, Telmisartan increased tumor T cell infiltration. Furthermore, the inventors used the colorectal mouse model MC38, to study the effect of Telmisartan and checkpoint blockade in an immunocompetent mouse. They observed that the antitumor activity of the combination of anti-PDl together with Telmisartan was increased compared to anti-PD-1 or Telmisartan alone. Of note, Telmisartan does not have any effect on the MC38 tumor growth in vitro.

[0298] Interestingly, Telmisartan does not improve functional avidity when using a concentration gradient of plate-bound peptide-MHC and measuring 4- IBB expression levels as a T cell activation readout.

[0299] The inventors are actively working to determine Telmisartan mechanism of action. Telmisartan is an Angiotensin II Receptor Blocker (ARB) that is commonly used to treat hypertension and has not been previously associated with T cell cytotoxicity. However, its main target, angiotensin receptor 1 (AGTR1) is not expressed in T cells. In addition, Telmisartan is the only antihypertensive drug in our screen that has an effect on T cell cytotoxicity suggesting an independent mechanism of angiotensin receptor blockade. Of note, blockade of PPARy agonistic effect of Telmisartan did not prevent the increase in T cell cytotoxicity. The inventors also observed few transcriptomic changes upon Telmisartan treatment, suggesting a direct drug-protein effect which they are currently investigating. Finally, we performed cell surface proteomics to identify changes in protein expression driven by Telmisartan. Interestingly, they found that Telmisartan increased the expression of protein related to the immunological synapse such as ICAM2, TMIGD2 and MSN, which could improve the interaction of the TCR with the peptide / MHC and lead to increased cytotoxicity.

[0300] As shown in FIG. 25, AZD4573 also showed similar effects of sensitizing melanoma cells to T cell killing.68137193686

[0301] Overall, these experiments depict the potential of Telmisartan and AZD4572 as an immunotherapy drug which could be used to increase the potency of TCR-engineered T cells used in combination with the adoptively transferred T cells. Additionally, these compounds could be used to increase the antitumor activity of immune checkpoint blockade therapy and other immunotherapies that rely on the activation of endogenous tumor specific T cells.* * *

[0302] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred aspects, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims. The references cited in the disclosure, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.69137193686

Claims

WHAT IS CLAIMED IS:

1. A method for treating cancer in a subj ect, the method comprising administering a T cell modulator to the subject, wherein the subject is being treated with one or more immunotherapies and wherein the T cell modulator comprises Telmisartan, Calcimycin, Calmidazolium, Thapsigargin, a CDK9 inhibitor, Riviciclib, a BET inhibitor, a CDK8 inhibitor, AZD4573, NSC-95397, and / or PMA.

2. The method of claim 1, wherein the T cell modulator comprises Telmisartan.

3. The method of claim 2, wherein the immunotherapies comprise TCR-T or CAR-T cell therapy.

4. A method for making T cells comprising contacting the T cell ex vivo with a T cell modulator, wherein the T cell modulator comprises Telmisartan, Calcimycin, Calmidazolium, Thapsigargin, a CDK9 inhibitor, Riviciclib, a BET inhibitor, a CDK8 inhibitor, AZD4573, NSC-95397, PMA, or combinations thereof.

5. The method of any one of claims 1-4, wherein the CDK9 inhibitor comprises PHA-767491 or the CDK8 inhibitor comprises CDK8-IN-4.

6. The method of any one of claims 4-5, wherein contacting the T cell ex vivo with the T cell modulator comprises culturing the cell in cell medium comprising the T cell modulator.

7. The method of any one of claims 1-6, wherein the T cell is derived from a CD3+ T cell, CD8+ T cell, naive T cell, or a memory T cell.

8. The method of any one of claims 1-7, wherein the cell has been isolated from a patient by leukapheresis.

9. The method of any one of claims 1-8, wherein the T cell comprises an engineered T cell.

10. The method of claim 9, wherein the T cell comprises a heterologous nucleic acid encoding a T cell receptor (TCR) or chimeric antigen receptor (CAR) or wherein the T cells comprise tumor infiltrating lymphocytes.

11. A T cell made by the method of any one of claims 4-10.

12. A population of T cells made by the method of any one of claims 1-10 or derived from the cell of claim 11.

13. A method for treating a subject comprising administering the cell or cells of claims 11 or 12.

14. The method of claim 13, wherein the cells are autologous cells.7013719368615. The method of claim 13 or 14, wherein the method further comprises administration of an additional therapy and wherein the additional therapy comprises lymphodepletion, fludarabine, and / or cyclosphosphamide.

16. The method of any one of claims 13-15, wherein the method is for treating cancer in the subject and wherein the cancer comprises lung cancer, prostate cancer, ovarian cancer, testicular cancer, brain cancer, neuroblastoma, sarcoma, osteosarcoma, skin cancer, melanoma, colon cancer, rectal cancer, gastric cancer, esophageal cancer, tracheal cancer, head & neck cancer, pancreatic cancer, liver cancer, breast cancer, ovarian cancer, glioblastoma, glioma, a B-cell malignancy, melanoma, leukemia, sarcomas of bone or soft tissue, cervical cancer, or vulvar cancer.

17. The method of any one of claims 13-16, wherein the immunotherapies comprise or wherein the subject is being treated with an additional therapy and wherein the additional therapy comprises an immunotherapy, and wherein the immunotherapy comprises an immune checkpoint inhibitor (ICI), an immune agonist antibody, or nucleic acids encoding for an immune checkpoint inhibitor or an immune agonist antibody; optionally wherein the ICI or immune agonist antibody comprises an anti-CD40 agonistic antibody, anti-PDl blocking antibody, anti-PDLl blocking antibody, anti-CTLA4 blocking antibody, anti-LAG3 blocking antibody, or combinations thereof; optionally wherein the ICI comprises one or more of Ipilimumab, Tremelimumab, Nivolumab, Pembrolizumab, Atezolizumab, Avelumab, Durvalumab, Spartalizumab, Cemiplimab, and Opdualag.

18. A method for treating cancer in a subject comprising administering a T cell modulator and one or more immunotherapies to the subject, wherein the T cell modulator comprises Telmisartan, Calcimycin, Calmidazolium, Thapsigargin, a CDK9 inhibitor, Riviciclib, a BET inhibitor, a CDK8 inhibitor, AZD4573, NSC-95397, PMA, or combinations thereof.

19. A method for treating an autoimmune disorder in a subject or for suppressing an immune response in a subject, the method comprising administering a T cell modulator to the subject, wherein the T cell modulator comprises Telmisartan, Calcimycin, Calmidazolium, Thapsigargin, a CDK9 inhibitor, Riviciclib, a BET inhibitor, a CDK8 inhibitor, AZD4573, NSC-95397, PMA, or combinations thereof.

20. A method comprising contacting a composition comprising a MHC molecule and a TCR with T cell modulator, wherein the T cell modulator comprises Telmisartan, Calcimycin, Calmidazolium, Thapsigargin, a CDK9 inhibitor, Riviciclib, a BET inhibitor, a CDK8 inhibitor, AZD4573, NSC-95397, PMA, or combinations thereof.7113719368621. A method for performing a tetramer assay comprising contacting a composition comprising a MHC tetramer and T cells with a T cell modulator, wherein the T cell modulator comprises Telmisartan, Calcimycin, Calmidazolium, Thapsigargin, a CDK9 inhibitor, Riviciclib, a BET inhibitor, a CDK8 inhibitor, AZD4573, NSC-95397, PMA, or combinations thereof.72137193686

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