Off-the-shelf cell therapy based on engineered INKT cells from stem cells

Abstract

To provide a stem cell-engineered INKT cell-based off-the-shelf cellular therapy.SOLUTION: Embodiments of the disclosure include compositions and methods related to engineered invariant natural killer T (iNKT) cells for off-the-shelf use for clinical therapy. In particular embodiments, the iNKT cells are produced from hematopoietic stem progenitorcells and also are suitable for allogeneic cellular therapy because they are HLA negative. In specific embodiments, the cells are cultured in a particular in vitro three-dimensional artificial thymic organoid system and the cells have imaging and suicide targeting capabilities. Embodiments of the disclosure concern at least the fields of immunology, cell biology, molecular biology, and medicine, including at least cancer medicine.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 683,750, filed June 12, 2018, the entire contents of which are incorporated herein by reference.

[0002] Embodiments of the present disclosure relate to the fields of at least immunology, cell biology, molecular biology, and medicine, including at least cancer medicine. [Background technology]

[0003] Cancer affects tens of millions of people worldwide and is a major threat to public health in the United States and California. It is the second leading cause of death in California, resulting in over 56,000 deaths annually, and it also has a devastating economic impact on the state. Despite existing treatments, cancer patients still suffer from the ineffectiveness of these treatments, treatment toxicity, and the risk of recurrence. Therefore, novel cancer treatments are desperately needed. Over the past decade, immunotherapy has become a new generation of cancer medicine. In particular, cell-based therapies have shown great promise. A prominent example is adoptive T-cell therapy via chimeric antigen receptor (CAR) engineering, which targets certain hematologic cancers with impressive efficacy.

[0004] However, the majority of current treatment protocols consist of autologous adoptive cell transfer, in which immune cells harvested from a patient are manufactured and used to treat that single patient. Such approaches are expensive, labor-intensive to manufacture, and difficult to deliver widely to all patients in need. Therefore, there is a great demand for allogeneic immune cell products that can be manufactured on a large scale and easily distributed to treat multiple patients.

[0005] Despite existing treatments, cancer patients still suffer from the ineffectiveness of these treatments, the toxicity of the treatments, and the risk of recurrence. Therefore, there is a dire need for new treatments for diseases such as cancer and autoimmune diseases. The present disclosure not only provides a solution to a long-standing need for a treatment, but also provides a treatment that can be delivered or distributed more widely. Summary of the Invention [Means for solving the problem]

[0006] Embodiments are provided to address the need for new therapies, and more particularly, the need for cell therapies that are not hindered by the challenges posed by personalized therapies using autologous cells. The ability to manufacture therapeutic cell populations or cell populations that can be used to create "off-the-shelf" therapeutic cell populations increases the availability and usefulness of new cell therapies.

[0007] Embodiments relate to engineered invariant natural killer T (iNKT) cells or populations of engineered iNKT cells. In at least some cases, the engineered iNKT cells comprise an engineered chimeric antigen receptor (CAR; CAR-iNKT cells) and / or an engineered T cell receptor (TCR-iNKT cells). Any of the embodiments discussed with respect to cells can be applied to populations of such cells. In certain embodiments, the engineered iNKT cells comprise a nucleic acid comprising one, two, and / or three of the following: i) all or a portion of an invariant alpha T cell receptor coding sequence; ii) all or a portion of an invariant beta T cell receptor coding sequence; or iii) a suicide gene. In further embodiments, there are engineered iNKT cells comprising a nucleic acid having a sequence encoding i) all or a portion of an invariant alpha T cell receptor; ii) all or a portion of an invariant beta T cell receptor; and / or iii) a suicide gene product.

[0008] Further aspects relate to engineered iNKT cells having increased levels of NK-activating receptors, decreased levels of NK inhibitory receptors, and / or increased levels of cytotoxic molecules. In some embodiments, the NK-activating receptor comprises NKG2D and / or DNAM-1. In some embodiments, the cytotoxic molecule comprises perforin and / or granzyme B. In some embodiments, the inhibitory receptor comprises KIR. The increase or decrease can be relative to the level of the same marker in unengineered iNKTs isolated from healthy individuals. Further aspects relate to a population of engineered iNKT cells having increased levels of NK-activating receptors, decreased levels of NK inhibitory receptors, and / or increased levels of cytotoxic molecules. In some embodiments, at least 70%, exactly 70% or more, at least 71%, exactly 71% or more, at least 72%, exactly 72% or more, at least 73%, exactly 73% or more, at least 74%, exactly 74% or more, at least 75%, exactly 75% or more, at least 76%, exactly 76% or more, at least 77%, exactly 77% or more, at least 78%, exactly 78% or more, at least 79%, exactly 79% or more, at least 80%, exactly ... 0%, exactly 80% or more than 80%, at least 81%, exactly 81% or more than 81%, at least 82%, exactly 82% or more than 82%, at least 83%, exactly 83% or more than 83%, at least 84%, exactly 84% or more than 84%, at least 85%, exactly 85% or more than 85%, at least 86%, exactly 86% or more than 86%, at least 87%, exactly 87% or more than 87%, at least 88%, exactly 88% or more than 88%, at least 89%, exactly 89% or more than 89%, at least 90%, exactly 90% or more than 90%, at least 91%,Exactly 91%, or more than 91%, at least 92%, exactly 92% or more than 92%, at least 93%, exactly 93% or more than 93%, at least 94%, exactly 94% or more than 94%, at least 95%, exactly 95% or more than 95%, at least 96%, exactly 96% or more than 96%, at least 97%, exactly 97% or more than 97%, at least 98%, exactly 98% or more than 98%, or at least 99%, exactly 99% or more than 99% of the cells express high levels of NKG2D. In some embodiments, at least 80%, exactly 80% or more, at least 81%, exactly 81% or more, at least 82%, exactly 82% or more, at least 83%, exactly 83% or more, at least 84%, exactly 84% or more, at least 85%, exactly 85% or more, at least 86%, exactly 86% or more, at least 87%, exactly 87% or more, at least 88%, exactly 88% or more, at least 89%, exactly 89% or more , at least 90%, exactly 90% or more, at least 91%, exactly 91% or more, at least 92%, exactly 92% or more, at least 93%, exactly 93% or more, at least 94%, exactly 94% or more, at least 95%, exactly 95% or more, at least 96%, exactly 96% or more, at least 97%, exactly 97% or more, at least 98%, exactly 98% or more, or at least 99%, exactly 99% or more, of the cells express high levels of DNAM-1. In some embodiments, up to 1%, exactly 1% or less, up to 2%, exactly 2% or less, up to 3%, exactly 3% or less, up to 4%,Exactly 4% or less than 4%, maximum 5%, exactly 5% or less than 5%, maximum 6%, exactly 6% or less than 6%, maximum 7%, exactly 7% or less than 7%, maximum 8%, exactly 8% or less than 8%, maximum 9%, exactly 9% or less than 9%, maximum 10%, exactly 10% or less than 10%, maximum 11%, exactly 11% or less than 11%, maximum 12%, exactly 12% or less than 12%, maximum 13%, exactly 13% or less than 13%, maximum 14%, exactly 14% or less than 14%, maximum 15%, exactly 15% or less than 15%, maximum 16%, exactly 16% or less than 16%, maximum 17%, exactly 17% or less than 17%, maximum 18%, exactly At or below 18%, up to 19%, exactly 19% or less, up to 20%, exactly 20% or less, up to 21%, exactly 21% or less, up to 22%, exactly 22% or less, up to 23%, exactly 23% or less, up to 24%, exactly 24% or less, up to 25%, exactly 25% or less, up to 26%, exactly 26% or less, up to 27%, exactly 27% or less, up to 28%, exactly 28% or less, up to 29%, exactly 29% or less, or up to 30%, exactly 30% or less, of the cells express high levels of KIR. In some embodiments, at least 65%, exactly 65% ​​or more, at least 66%, exactly 66% or more, at least 67%, exactly 67% or more, at least 68%, exactly 68% or more, at least 69%, exactly 69% or more, at least 70%, exactly 70% or more, at least 71%, exactly 71% or more, at least 72%, exactly 72% or more, at least 73%, exactly 73% or more, at least 74%, exactly 74% or more, at least 75%, exactly 75% or more, at least 76%, exactly 76% or more, at least 77%,exactly 77% or more, at least 78%, exactly 78% or more, at least 79%, exactly 79% or more, at least 80%, exactly 80% or more, at least 81%, exactly 81% or more, at least 82%, exactly 82% or more, at least 83%, exactly 83% or more, at least 84%, exactly 84% or more, at least 85%, exactly 85% or more, at least 86%, exactly 86% or more, at least 87%, exactly 87% or more, at least 88%, exactly 88% or more, at least At least 89%, exactly 89% or more, at least 90%, exactly 90% or more, at least 91%, exactly 91% or more, at least 92%, exactly 92% or more, at least 93%, exactly 93% or more, at least 94%, exactly 94% or more, at least 95%, exactly 95% or more, at least 96%, exactly 96% or more, at least 97%, exactly 97% or more, at least 98%, exactly 98% or more, or at least 99%, exactly 99% or more, of the cells express high levels of perforin. In some embodiments, at least 50%, exactly 50% or more, at least 51%, exactly 51% or more, at least 52%, exactly 52% or more, at least 53%, exactly 53% or more, at least 54%, exactly 54% or more, at least 55%, exactly 55% or more, at least 56%, exactly 56% or more, at least 57%, exactly 57% or more, at least 58%, exactly 58% or more, at least 59%, exactly 59% or more,At least 60%, exactly 60% or more than 60%, at least 61%, exactly 61% or more than 61%, at least 62%, exactly 62% or more than 62%, at least 63%, exactly 63% or more than 63%, at least 64%, exactly 64% or more than 64%, at least 65%, exactly 65% ​​or more than 65%, at least 66%, exactly 66% or more than 66%, at least 67%, exactly 67% or more than 67%, at least 68%, exactly 68% or more than 68%, at least 69%, exactly 69% or more than 69%, at least 70%, exactly 70% or more than 70%, at least 71%, exactly 71% or more than 71%, at least 72%, exactly 72% or more than 72%, at least 73%, exactly 73% or more than 73%, at least 74%, exactly 74% or more than 74%, at least 75%, exactly 75% or more than 75%, at least 76%, exactly 76% or more than 76%, at least at least 77%, exactly 77% or more than 77%, at least 78%, exactly 78% or more than 78%, at least 79%, exactly 79% or more than 79%, at least 80%, exactly 80% or more than 80%, at least 81%, exactly 81% or more than 81%, at least 82%, exactly 82% or more than 82%, at least 83%, exactly 83% or more than 83%, at least 84%, exactly 84% or more than 84%, at least 85%, exactly 85% or more than 85% greater than 5%, at least 86%, exactly 86% or more than 86%, at least 87%, exactly 87% or more than 87%, at least 88%, exactly 88% or more than 88%, at least 89%, exactly 89% or more than 89%, at least 90%, exactly 90% or more than 90%, at least 91%, exactly 91% or more than 91%, at least 92%, exactly 92% or more than 92%, at least 93%, exactly 93% or more than 93%, at least 94%,exactly 94% or more, at least 95%, exactly 95% or more, at least 96%, exactly 96% or more, at least 97%, exactly 97% or more, at least 98%, exactly 98% or more, Or at least 99%, exactly 99%, or more than 99% of the cells express high levels of granzyme B. Further embodiments of the present disclosure relate to engineered iNKT cells or populations of cells that contain high levels of the NK activators NKG2D and DNAM-1, low or undetectable levels of the NK inhibitory receptor KIR, and high levels of the cytotoxic molecules perforin and granzyme B. Further embodiments of the present disclosure relate to engineered populations of iNKT cells, more than 90% of which contain high levels of the NK activators NKG2D and DNAM-1, low or undetectable levels of the NK inhibitory receptor KIR, and high levels of the cytotoxic molecules perforin and granzyme B.

[0009] In some embodiments, the engineered iNKT cells comprise a nucleic acid under the control of a heterologous promoter, meaning that the promoter is not the same as the genomic promoter that controls transcription of the nucleic acid. It is contemplated that the engineered iNKT cells comprise an exogenous nucleic acid comprising one or more coding sequences, some or all of which are under the control of a heterologous promoter in many embodiments described herein.

[0010] It is particularly noted that any embodiment discussed with respect to a particular cell or cell population embodiment can be used with respect to any other cell or cell population embodiment. Furthermore, any embodiment used with respect to a particular method can be implemented with respect to any other method described herein. Furthermore, aspects of different methods described herein can be combined to realize other methods, as well as to create or describe the use of any cell or cell population. Specifically, it is contemplated that aspects of one or more embodiments can be combined with aspects of one or more other embodiments described herein. Furthermore, any method described herein can be said to describe one or more uses of the cells or cell populations described herein. For example, the use of engineered iNKT cells or iNKT cell populations can be described from any method described herein.

[0011] In certain embodiments, there are engineered invariant natural killer T (iNKT) cells that express at least one invariant natural killer T cell receptor (iNKT TCR) and an exogenous suicide gene product, where the at least one iNKT TCR is expressed from an exogenous nucleic acid and / or from an endogenous invariant TCR gene under the transcriptional control of a recombinantly modified promoter region. iNKT TCR refers to a "TCR that recognizes lipid antigens presented by CD1d molecules." iNKT TCRs can include alpha-TCRs, beta-TCRs, or both. In some cases, the utilized TCR may belong to the broader group of "invariant TCRs," such as MAIT cell TCRs, GEM cell TCRs, or gamma / delta TCRs, which arise from the engineering of HSCs to MAIT cells, GEM cells, or gamma / delta T cells, respectively.

[0012] In certain embodiments, there is an engineered iNKT cell population. In certain embodiments, there is an engineered iNKT cell population, comprising engineered iNKT clonal cells that contain an altered genomic invariant T cell receptor sequence or an exogenous nucleic acid encoding an invariant T cell receptor (TCR) and lack expression of one or more HLA-I or HLA-II genes. "Altered genomic invariant T cell receptor sequence" refers to a sequence that has been altered by recombinant DNA technology. The term "clonal" cells refers to iNKT cells that have been engineered to express a clonal transgenic iNKT TCR. In some embodiments, the clonal cells are derived from the same progenitor cell. In some embodiments, it is contemplated that there is a population of mixed clonal cells, which refers to a population comprising clonal cells derived from a set of progenitor cells, the set being 10, at least 10 or up to 10, 20, at least 20 or up to 20, 30, at least 30 or up to 30, 40, at least 40 or up to 40, 50, at least 50 or up to 50, 60, at least 60 or up to 60, 70, at least 70 or up to 70, 80, at least 80 or up to 80, 90, at least 90 or up to 90, 100, at least 100 or up to 100, 200, at least 200, The population may be 200 or up to 200, 300, at least 300 or up to 300, 400, at least 400 or up to 400, 500, at least 500 or up to 500, 600, at least 600 or up to 600, 700, at least 700 or up to 700, 800, at least 800 or up to 800, 900, at least 900 or up to 900, 1000, at least 1000 or up to 1000 or more (or any range derivable therein) progenitor cells, meaning that the cells in the population are progeny of an originally transfected / infected set of progenitor cells. In the case of cells containing exogenous nucleic acid or altered genomic DNA sequences, the clonal cells may arise from an ancestral cell into which the exogenous nucleic acid was introduced.Some embodiments relate to a population of clonal cells, which refers to a population containing progeny cells derived from the same ancestral cell. It is intended that some populations of cells may contain a mixture of different clonal cells, meaning that the population originated from different ancestral cells that contain exogenous nucleic acid but may differ in a distinguishable way, such as the integration site of the exogenous nucleic acid. A nucleic acid sequence that is introduced into a cell (either alone or as part of a longer nucleic acid sequence) and becomes integrated, such that progeny cells contain the integrated nucleic acid sequence, is considered an exogenous nucleic acid. An introduced nucleic acid sequence that is maintained extrachromosomally is also considered an exogenous nucleic acid.

[0013] In embodiments utilizing a portion of an iNKT alpha T cell receptor or a portion of an iNKT beta T cell receptor, it is contemplated that the embodiments require a functional portion of an iNKT alpha T cell receptor or a functional portion of an iNKT beta T cell receptor such that cells expressing both are functional iNKT cells based on, at least, an assay assessing their ability to recognize lipid antigens presented by CD1d molecules.

[0014] In some embodiments, the nucleic acid is an iNKT TCR-alpha or iNKT 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, 1100, 1111, 1120, 1121, 1122, 1123, 1124, 1125, 1126, 1127, 1128, 1129, 1130, 1131, 1132, 1133, 1134, 1135, 1136, 1137, 1138, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, 1151, 1152, 1153, 1154, 1155, 1156, 1157, 1158, 1159, 1160, 1161, 11 5 pieces, 106 pieces, 107 pieces, 108 pieces, 109 pieces, 110 pieces, 111 pieces, 112 pieces, 113 pieces, 114 pieces, 115 pieces, 116 pieces, 11 7 pieces, 118 pieces, 119 pieces, 120 pieces, 121 pieces, 122 pieces, 123 pieces, 124 pieces, 125 pieces, 126 pieces, 127 pieces, 128 pieces, 12 9 pieces, 130 pieces, 131 pieces, 132 pieces, 133 pieces, 134 pieces, 135 pieces, 136 pieces, 137 pieces, 138 pieces, 139 pieces, 140 pieces, 14 1 piece, 142 pieces, 143 pieces, 144 pieces, 145 pieces, 146 pieces, 147 pieces, 148 pieces, 149 pieces, 150 pieces, 151 pieces, 152 pieces, 153 pieces, 154 pieces, 155 pieces, 156 pieces, 157 pieces, 158 pieces, 159 pieces, 160 pieces, 161 pieces, 162 pieces, 163 pieces, 164 pieces, 165 pieces pieces, 166 pieces, 167 pieces, 168 pieces, 169 pieces, 170 pieces, 171 pieces, 172 pieces, 173 pieces, 174 pieces, 175 pieces, 176 pieces, 177 pieces, 178 pieces, 179 pieces, 180 pieces, 181 pieces, 182 pieces, 183 pieces, 184 pieces, 185 pieces, 186 pieces, 187 pieces, 188 pieces, 189 pieces, 190 pieces, 191 pieces, 192 pieces, 193 pieces, 194 pieces, 195 pieces, 196 pieces, 197 pieces, 198 pieces, 199 pieces, 200 pieces, 201 pieces , 202 pieces, 203 pieces, 204 pieces, 205 pieces, 206 pieces, 207 pieces, 208 pieces, 209 pieces, 210 pieces, 211 pieces, 212 pieces, 213 pieces , 214 pieces, 215 pieces, 216 pieces, 217 pieces, 218 pieces, 219 pieces, 220 pieces, 221 pieces, 222 pieces, 223 pieces, 224 pieces, 225 pieces , 226 pieces, 227 pieces, 228 pieces, 229 pieces, 230 pieces, 231 pieces, 232 pieces, 233 pieces, 234 pieces, 235 pieces, 236 pieces, 237 pieces , 238 pieces, 239 pieces, 240 pieces, 241 pieces, 242 pieces, 243 pieces, 244 pieces, 245 pieces, 246 pieces, 247 pieces, 248 pieces, 249 pieces,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 pieces, 451 pieces, 452 pieces, 453 pieces, 454 pieces, 455 pieces, 456 pieces, 457 pieces, 458 pieces, 459 pieces, 460 pieces, 461 pieces, 4 62 pieces, 463 pieces, 464 pieces, 465 pieces, 466 pieces, 467 pieces, 468 pieces, 469 pieces, 470 pieces, 471 pieces, 472 pieces, 473 pieces, 474 pieces, 475 pieces, 476 pieces, 477 pieces, 478 pieces, 479 pieces, 480 pieces, 481 pieces, 482 pieces, 483 pieces, 484 pieces, 485 pieces, 486 pieces, 487 pieces, 488 pieces, 489 pieces, 490 pieces, 491 pieces, 492 pieces, 493 pieces, 494 pieces, 495 pieces, 496 pieces, 497 pieces, 498 pieces, 49 For a sequence encoding 9, 500 (or any range derivable therein) amino acids or consecutive amino acid residues, 60%, at least 60% or up to 60%, 61%, at least 61% or up to 61%, 62%, at least 62% or up to 62%, 63%, at least 63% or up to 63%, 64%, at least 64% or up to 64%, 65%, at least 65% or up to 65%, 66%, at least 66% or up to 66%, 67%, at least 67% or up to 67%, 68%, at least 68% or is at most 68%, 69%, at least 69% or up to 69%, 70%, at least 70% or up to 70%, 71%, at least 71% or up to 71%, 72%, at least 72% or up to 72%, 73%, at least 73% or up to 73%, 74%, at least 74% or up to 74%, 75%, at least 75% or up to 75%, 76%, at least 76% or up to 76%, 77%, at least 77% or up to 77%, 78%, at least 78% or up to 78%, 79%, at least 79% or up to 79% %, 80%, at least 80% or up to 80%, 81%, at least 81% or up to 81%, 82%, at least 82% or up to 82%, 83%, at least 83% or up to 83%, 84%, at least 84% or up to 84%, 85%, at least 85% or up to 85%, 86%, at least 86% or up to 86%, 87%, at least 87% or up to 87%, 88%, at least 88% or up to 88%, 89%, at least 89% or up to 89%, 90%, at least 90% or up to 90%, 91%,91%, or up to 91%, 92%, at least 92% or up to 92%, 93%, at least 93% or up to 93%, 94%, at least 94% or up to 94%, 95%, at least 95% or up to 95%, 96%, at least 96% or up to 96%, 97%, at least 97% or up to 97%, 98%, at least 98% or up to 98%, 99%, at least 99% or up to 99%, 100%, at least 100% or up to 100% (or any range derivable therein) identical.

[0015] In certain embodiments, the suicide gene is enzyme-based, meaning that the gene product of the suicide gene is an enzyme and the suicide function depends on enzymatic activity. One or more suicide genes can be utilized in a single cell or a clonal population. In some embodiments, the suicide gene is selected from the group consisting of herpes simplex virus thymidine kinase (HSV-TK), purine nucleoside phosphorylase (PNP), cytosine deaminase (CD), carboxypeptidase G2, cytochrome P450, linamarase, beta- The suicide gene product encodes -lactamase, nitroreductase (NTR), carboxypeptidase A, or inducible caspase 9. Methods in the art for using suicide genes can be used, such as those in U.S. Patent No. 8,628,767, U.S. Patent Application Publication No. 20140369979, U.S. Patent No. 20140242033, and U.S. Patent No. 20040014191, all of which are incorporated by reference in their entireties. In further embodiments, the TK gene is a viral TK gene, i.e., a TK gene derived from a virus. In certain embodiments, the TK gene is a herpes simplex virus TK gene. In some embodiments, the suicide gene product is activated by a substrate. Thymidine kinase is a suicide gene product activated by ganciclovir, penciclovir, or derivatives thereof. In certain embodiments, the substrate that activates the suicide gene product is labeled for detection. In some examples, the substrate can be labeled for imaging. In some embodiments, the suicide gene products may be encoded by the same or different nucleic acid molecules encoding one or both of TCR-alpha or TCR-beta. In certain embodiments, the suicide gene is sr39TK or inducible caspase 9. In alternative embodiments, the cells do not express an exogenous suicide gene. In some embodiments, the engineered iNKT cells specifically bind alpha-galactosylceramide (α-GC).

[0016] In additional embodiments, the cells lack or have reduced surface expression of at least one HLA-I or HLA-II molecule. In some embodiments, the lack of surface expression of HLA-I and / or HLA-II molecules is achieved by disrupting the genes encoding individual HLA-I / II molecules, or by disrupting the gene encoding B2M (beta 2 microglobulin), a component common to all HLA-I complex molecules, or by disrupting the gene encoding CIITA (class II major histocompatibility complex transactivator), a critical transcription factor that controls the expression of all HLA-II genes. In certain embodiments, the cells lack one or more of the following: or lack surface expression of multiple HLA-I and / or HLA-II molecules, or express such molecules at reduced levels by 50% (or at least 50%), 60% (or at least 60%), 70% (or at least 70%), 80% (or at least 80%), 90% (or at least 90%), 100% (or at least 100%) (or any range derivable therein). In some embodiments, the iNKT cells have been manipulated by gene editing such that HLA-I and / or HLA-II molecules are not expressed in the cells. Or HLA-II is not expressed. In some embodiments, gene editing involves CRISPR-Cas9. Instead of Cas9, CasX or CasY may be involved. Zinc finger nucleases (ZFNs) and TALENs, as well as Cpf1, are other gene editing techniques, all of which can be used. In other embodiments, the iNKT cells contain one or more different siRNA or miRNA molecules targeted to reduce the expression of HLA-I / II molecules, B2M, and / or CIITA.

[0017] In some embodiments, the iNKT cells comprise a recombinant vector or a nucleic acid sequence derived from a recombinant vector introduced into the cells. In certain embodiments, the recombinant vector is or was a viral vector. In further embodiments, the viral vector is or was a lentivirus, retrovirus, adeno-associated virus (AAV), herpesvirus, or adenovirus. It is understood that the nucleic acid of certain viral vectors is integrated into the host genome sequence.

[0018] In some embodiments, the cells have not been exposed to a medium containing animal serum. In further embodiments, the cells are or have been frozen. In some embodiments, the cells are pre-frozen, and the pre-frozen cells are stable at room temperature for at least 1 hour. In some embodiments, the cells are pre-frozen, and the pre-frozen cells are stable at room temperature for at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 10 hours, 15 hours, 20 hours, 24 hours, 30 hours, or 48 hours (or any range derivable therein). In certain embodiments, the cells or population of cells present in solution comprise dextrose, one or more electrolytes, albumin, dextran, and / or DMSO. In further embodiments, the cells are present in a sterile, nonpyogenic, and isotonic solution.

[0019] In certain embodiments, the iNKT cells are activated or are activated. In certain embodiments, the iNKT cells are activated with alpha-galactosylceramide (α-GC).

[0020] In embodiments involving large numbers of cells, the cell population may be about 10 2 pieces, at least about 10 2 pieces or up to about 10 2 pieces, about 10 3 pieces, at least about 10 3 pieces or up to about 10 3 pieces, about 10 4 pieces, at least about 104 pieces or up to about 10 4 pieces, about 10 5 pieces, at least about 10 5 pieces or up to about 10 5 pieces, about 10 6 pieces, at least about 10 6 pieces or up to about 10 6 pieces, about 10 7 pieces, at least about 10 7 pieces or up to about 10 7 pieces, about 10 8 pieces, at least about 10 8 pieces or up to about 10 8 pieces, about 10 9 pieces, at least about 10 9 pieces or up to about 10 9 pieces, about 10 10 pieces, at least about 10 10 pieces or up to about 10 10 pieces, about 10 11 pieces, at least about 10 11 pieces or up to about 10 11 pieces, about 10 12 pieces, at least about 10 12 pieces or up to about 10 12 pieces, about 10 13 pieces, at least about 10 13 pieces or up to about 10 13 pieces, about 10 14 pieces, at least about 10 14 pieces or up to about 10 14 pieces, about 10 15 pieces, at least about 10 15 pieces or up to about 10 15 or more (or any range derivable therein), which in some embodiments are engineered iNKT cells. In some cases, the cell population comprises at least about 10 engineered iNKT cells. 6 ~10 12 In some embodiments, it is contemplated that these numbers of populations of cells are generated from a single batch of cells and are not the result of pooling batches of cells generated separately.

[0021] In certain embodiments, an iNKT cell population comprising clonal iNKT cells comprising an iNKT T-cell receptor (TCR) and one or more exogenous nucleic acids encoding a suicide thymidine kinase gene product, wherein the clonal iNKT cells have been engineered not to express functional beta 2-microglobulin (B2M) and / or class II major histocompatibility complex or transactivator (CIITA), and the cell population comprises at least about 10 total cells. 6 ~10 12 and the engineered iNKT cells are at least about 10 2 ~10 6 There is a population of iNKT cells, including 100 cells. In certain instances, the cells are frozen in solution.

[0022] Some embodiments relate to methods of preparing iNKT cells or populations of cells, particularly populations in which some or all of the cells are clonal. In certain embodiments, the cell population comprises cells in which at least or up to 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% (or any range derivable therein) of the cells are clonal, i.e., the percentage of cells that are derived from the same ancestral cell as other cells in the population. In other embodiments, the cell population is 1, at least 1 or up to 1, 2, at least 2 or up to 2, 3, at least 3 or up to 3, 4, at least 4 or up to 4, 5, at least 5 or up to 5, 6, at least 6 or up to 6, 7, at least 7 or up to 7, 8, at least 8 or up to 8, 9, at least 9 or up to 9, 10, at least 10 or up to 10, 11, at least 11 or up to 11, 12, at least 12 or up to 12, 13, at least 13 or up to 13, 14, at least 14 or up to 14, 15, at least 15 or up to 15, 16, at least 16 or up to 16, 17, at least 17 or up to 17, 18, at least 18 or up to 18 species, 19 species, at least 19 species or up to 19 species, 20 species, at least 20 species or up to 20 species, 21 species, at least 21 species or up to 21 species, 22 species, at least 22 species or up to 22 species, 23 species, at least 23 species or up to 23 species, 24 species, at least 24 species or up to 24 species, 25 species, at least 25 species or up to 25 species, 26 species, at least 26 species or up to 26 species, 7 species, at least 7 species or up to 7 species, 28 species, at least 28 species or up to 28 species, 29 species, at least 29 species or up to 29 species, 30 species, at least 30 species or up to 30 species, 31 species, at least 31 species or up to 31 species, 32 species, at least 32 species or up to 32 species, 33 species, at least 33 species or up to 33 species, 34 species, at least 34 species or up to 34 species, 35 species, at least 35 species or up to 35 species, 36 species,at least 36 species or up to 36 species, 37 species, at least 37 species or up to 37 species, 38 species, at least 38 species or up to 38 species, 39 species, at least 39 species or up to 39 species, 40 species, at least 40 species or up to 40 species, 41 species, at least 41 species or up to 41 species, 42 species, at least 42 species or up to 42 species, 43 species, at least 43 species or up to 43 species, 44 species, at least 44 species or up to 44 species, 45 species, at least 45 species or up to 45 species, 46 species, at least 46 species or up to 46 species, 47 species, at least 47 species or up to 47 species, 48 ​​species, at least 48 species or up to 48 species, 49 species, at least 49 species or up to 49 species, 50 species, at least 50 species or up to 50 species, 51 species, at least 51 species or up to 51 species, 52 species, at least 52 species or up to 52 species, 53 species, at least 53 species or up to 53 species, 54 species, at least 54 species or up to 54 species, 55 species, at least 55 species or up to 55 species, 56 species, at least 56 species or up to 56 species, 57 species, at least 57 species or up to 57 species, 58 species, at least 58 species or up to 58 species, 59 species, at least 59 species or up to 59 species, 60 species, at least 60 species or up to 60 species, 61 species, at least 61 species or up to 61 species, 62 species, at least 62 species or up to 62 species, 63 species, at least 63 species or up to 63 species, 64 species, at least 64 species or up to 64 species, 65 species, at least 65 species or up to 65 species, 66 species, at least 66 species or up to 66 species, 67 species, at least 67 species or up to 67 species, 68 species, at least 68 species or up to 68 species, 69 species, at least 69 species or up to 69 species species, 70 species, at least 70 species or up to 70 species, 71 species, at least 71 species or up to 71 species, 72 species, at least 72 species or up to 72 species, 73 species, at least 73 species or up to 73 species, 74 species, at least 74 species or up to 74 species, 75 species, at least 75 species or up to 75 species, 76 species, at least 76 species or up to 76 species, 77 species, at least 77 species or up to 77 species, 78 species, at least 78 species or up to 78 species, 79 species, at least 79 species or up to 79 species, 80 species, at least 80 species or up to 80 species, 81 species,At least 81 species or up to 81 species, 82 species, at least 82 species or up to 82 species, 83 species, at least 83 species or up to 83 species, 84 species, at least 84 species or up to 84 species, 85 species, at least 85 species or up to 85 species, 86 species, at least 86 species or up to 86 species, 87 species, at least 87 species or up to 87 species, 88 species, at least 88 species or up to 88 species, 89 species, at least 89 species or up to 89 species, 90 species, at least 90 species or up to 90 species, 91 species, at least 91 species or up to 91 species, 92 species, 93, at least 93 or up to 93, 94, at least 94 or up to 94, 95, at least 95 or up to 95, 96, at least 96 or up to 96, 97, at least 97 or up to 97, 98, at least 98 or up to 98, 99, at least 99 or up to 99, 100, at least 100 or up to 100 (or any range derivable therein) different parent cells.

[0023] Methods for preparing, generating, producing, and using engineered iNKT cells and iNKT cell populations are provided. The method, in embodiments, comprises one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or more of the following steps: obtaining hematopoietic cells; obtaining hematopoietic progenitor cells; obtaining progenitor cells capable of becoming one or more hematopoietic cells; obtaining progenitor cells capable of becoming iNKT cells; selecting cells from a mixed cell population using one or more cell surface markers; selecting CD34+ cells from the population of cells; isolating CD34+ cells from the population of cells; separating CD34+ cells and CD34− cells from each other; selecting cells based on a cell surface marker other than or in addition to CD34; introducing into the cells one or more nucleic acids encoding an iNKT T cell receptor (TCR); infecting the cells with a viral vector encoding an iNKT T cell receptor (TCR); transfecting the cell with one or more nucleic acids encoding a T cell receptor (TCR); transfecting the cell with an expression construct encoding an iNKT T cell receptor (TCR); integrating an exogenous nucleic acid encoding an iNKT T cell receptor (TCR) into the genome of the cell; introducing nucleic acids encoding one or more suicide gene products into the cell; infecting the cell with a viral vector encoding the suicide gene products; transfecting the cell with nucleic acids encoding one or more suicide gene products; transfecting the cell with an expression construct encoding the suicide gene products; integrating an exogenous nucleic acid encoding a suicide gene product into the genome of the cell; introducing into the cell one or more nucleic acids encoding one or more polypeptides and / or nucleic acid molecules for gene editing; infecting the cell with a viral vector encoding one or more polypeptides and / or nucleic acid molecules for gene editing;transfecting cells with one or more nucleic acids encoding one or more polypeptides and / or nucleic acid molecules for gene editing; transfecting cells with an expression construct encoding one or more polypeptides and / or nucleic acid molecules for gene editing; incorporating exogenous nucleic acids encoding one or more polypeptides and / or nucleic acid molecules for gene editing; editing the genome of the cells; editing the promoter region of the cells; editing the promoter and / or enhancer region for the iNKT TCR gene; eliminating expression of one or more genes; eliminating expression of one or more HLA-I / II genes in isolated human CD34+ cells; transfecting cells with one or more nucleic acids for gene editing; culturing the isolated or selected cells; expanding the isolated or selected cells; culturing cells selected for one or more cell surface markers; iNKT Culturing isolated CD34+ cells that express a TCR; expanding the isolated CD34+ cells; culturing the cells under conditions that produce or expand iNKT cells; culturing the cells in an artificial thymic organoid (ATO) system to produce iNKT cells; culturing the cells in serum-free medium; culturing the cells in an ATO system, the ATO system comprising 3D cell aggregates containing a selected population of stromal cells that express a Notch ligand and serum-free medium. It is specifically contemplated that one or more steps can be eliminated in certain embodiments.

[0024] In some embodiments, there is a method of preparing a population of clonal iNKT cells, comprising: a) selecting CD34+ cells from human peripheral blood cells (PBMCs); b) introducing one or more nucleic acids encoding a human T cell receptor (TCR); c) eliminating surface expression of one or more HLA-I / II genes in the isolated human CD34+ cells; and d) culturing the isolated CD34+ cells that express the iNKT TCR in an artificial thymic organoid (ATO) system to produce iNKT cells, wherein the ATO system comprises 3D cell aggregates comprising a selected population of stromal cells that express a Notch ligand and serum-free medium.

[0025] The cells that can be used to generate engineered iNKT cells are hematopoietic progenitor stem cells. The cells can be derived from peripheral blood mononuclear cells (PBMCs), bone marrow cells, fetal liver cells, embryonic stem cells, umbilical cord blood cells, induced pluripotent stem cells (iPS cells), or a combination thereof.

[0026] In some embodiments, the method involves isolating CD34- cells or separating CD34- and CD34+ cells. In some embodiments, the CD34- cells can be used to generate iNKT cells, while further manipulation of the CD34+ cells is involved. Thus, in some embodiments, the CD34- cells can be subsequently used and banked for this purpose.

[0027] Certain methods involve culturing selected CD34+ cells in a medium before introducing one or more nucleic acids into the cells. The step of culturing the cells can include incubating the selected CD34+ cells in a medium containing one or more growth factors. In some embodiments, the one or more growth factors include c-kit ligand, flt-3 ligand, and / or human thrombopoietin (TPO). In further embodiments, the medium includes c-kit ligand, flt-3 ligand, and TPO. In some embodiments, the concentration of the one or more growth factors is between about 5 ng / ml and about 500 ng / ml for each growth factor individually or for the sum of all of these particular growth factors. The concentration of a single growth factor or combination of growth factors in the medium can be about, at least about, or up to about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 500, 510, 515, 520, 525 400, 410, 420, 425, 430, 440, 441, 450, 460, 470, 475, 480, 490, 500 ng / ml or μg / ml (or any derivable range), or may be higher.

[0028] In some embodiments, the nucleic acid may comprise a nucleic acid sequence encoding an α-TCR and / or a β-TCR as discussed herein. In certain embodiments, a single nucleic acid encodes both an α-TCR and a β-TCR. In additional embodiments, the nucleic acid further comprises a nucleic acid sequence encoding a suicide gene product. In some embodiments, the nucleic acid molecules introduced into the selected CD34+ cells encode the α-TCR, the β-TCR, and the suicide gene product. In other embodiments, the method also involves introducing into the selected CD34+ cells a nucleic acid encoding a suicide gene product, where a nucleic acid molecule different from the nucleic acid encoding at least one of the TCR genes encodes the suicide gene product.

[0029] As discussed, in some embodiments, iNKT cells do not express HLA-I and / or HLA-II molecules on the cell surface, which can be achieved by disrupting the expression of genes encoding beta2-microglobulin (B2M), transactivator (CIITA), or HLA-I and HLA-II molecules. In certain embodiments, the method involves eliminating the surface expression of one or more HLA-I / II molecules in isolated human CD34+ cells. In certain embodiments, elimination of expression can be achieved by gene editing the genomic DNA of the cells. Some methods include introducing a CRISPR and one or more guide RNAs (gRNAs) corresponding to B2M or CIITA into the cells. In certain embodiments, the CRISPR or one or more gRNAs are transfected into the cells by electroporation or lipid-mediated transfection. Thus, the method can involve introducing a CRISPR and one or more gRNAs into the cells by transfecting the cells with nucleic acids encoding the CRISPR and one or more gRNAs. In some embodiments, different gene editing techniques can be used.

[0030] Similarly, in some embodiments, one or more nucleic acids encoding TCR receptors are introduced into cells. This can be done by transfecting or infecting cells with a recombinant vector, which may or may not be a viral vector as discussed herein. In some embodiments, the exogenous nucleic acid can be integrated into the genome of the cell.

[0031] In some embodiments, the cells are cultured in a cell-free medium. In certain embodiments, the serum-free medium further comprises exogenously added ascorbic acid. In certain embodiments, the method involves adding ascorbic acid to the medium. In further embodiments, the serum-free medium further comprises one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or all sixteen (or a derivable range therein) of the following exogenously added components: FLT3 ligand (FLT3L), interleukin-7 (IL-7), stem cell factor (SCF), thrombopoietin (TPO), stem cell factor (SCF), IL-2, IL-4, IL-6, IL-15, IL-21, TNF-alpha, TGF-beta, interferon-gamma, interferon-lambda, TSLP, thymopentin, pleotrophin, or midkine. Follow up In additional embodiments, the serum-free medium comprises one or more vitamins. In some cases, the serum-free medium comprises one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve of the following vitamins (or any range derivable therein): biotin, DL-alpha tocopherol acetate, DL-alpha tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, folic acid, nicotinamide, pyridoxine, riboflavin, thiamine, inositol, vitamin B12, or a salt thereof. In certain embodiments, the medium comprises at least biotin, DL-alpha tocopherol acetate, DL-alpha tocopherol, vitamin A, or a combination or salt thereof. In additional embodiments, the serum-free medium comprises one or more proteins. In some embodiments, the serum-free medium comprises one, two, three, four, five, six, or more of the following proteins (or any range derivable therein): albumin or bovine serum albumin (BSA), a fraction of BSA, catalase, insulin, transferrin, superoxide dismutase, or a combination thereof. In other embodiments, the serum-free medium comprises one, two, three, four, five, seven, eight, nine, ten, or eleven of the following compounds: corticosterone, D-galactose, ethanolamine, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, or triodo-L-thyronine, or a combination thereof. In further embodiments, the serum-free medium comprises B-27® supplement, Xeno-Free B-27® supplement, GS21™ supplement, or a combination thereof. In additional embodiments, the serum-free medium comprises or further comprises amino acids, simple sugars, and / or inorganic ions.In some embodiments, the serum-free medium comprises one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or thirteen of the following amino acids: arginine, cysteine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine, or a combination thereof. In other embodiments, the serum-free medium comprises one, two, three, four, five, or six of the following inorganic ions: sodium, potassium, calcium, magnesium, nitrogen, or phosphorus, or a combination or salt thereof. In additional embodiments, the serum-free medium comprises one, two, three, four, five, six, or seven of the following elements: molybdenum, vanadium, iron, zinc, selenium, copper, or manganese, or a combination thereof.

[0032] In some methods, cells are cultured in an artificial thymic organoid (ATO) system. The ATO system involves three-dimensional (3D) cell aggregates, which are aggregates of cells. In certain embodiments, the 3D cell aggregates contain a selected population of stromal cells that express a Notch ligand. In some embodiments, the 3D cell aggregates are created by mixing CD34+ transduced cells and a selected population of stromal cells on a physical matrix or scaffold. In further embodiments, the method includes centrifuging the CD34+ transduced cells and stromal cells to form a cell pellet that is placed on the physical matrix or scaffold. In certain embodiments, the stromal cells express a Notch ligand that is intact, partial, or modified DLL1, DLL4, JAG1, JAG2, or a combination thereof. In further embodiments, the Notch ligand is a human Notch ligand. In other embodiments, the Notch ligand is human DLL1.

[0033] In further aspects, the ratio of stromal cells to CD34+ cells is about, at least about, or at most about 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50 (or any range derivable therein). In specific embodiments, the ratio of stromal cells to CD34+ cells is about 1:5 to 1:20. In certain embodiments, the stromal cells are a murine stromal cell line, a human stromal cell line, a selected population of primary stromal cells, a selected population of stromal cells differentiated in vitro from pluripotent stem cells, or a combination thereof. In certain embodiments, the stromal cells are a selected population of stromal cells differentiated in vitro from hematopoietic stem or progenitor cells. Co-culture of CD34+ cells with stromal cells can be carried out for about, at least about, or up to about 1, 2, 3, 4, 5, 6, 7 days and / 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 weeks, or longer (or any range derivable therein). In some embodiments, the stromal cells are irradiated prior to co-culture.

[0034] The disclosed methods allow for the collection of at least 1 x 10 cells that may express a marker or have high or low levels of a particular marker. 2 pieces, 1×10 3 pieces, 1×10 4 pieces, 1×10 5 pieces, 1×10 6 pieces, 1×10 7 pieces, 1×10 8 pieces, 1×10 9 pieces, 1×10 10 pieces, 1×10 11 pieces, 1×10 12 pieces, 1×10 13 pieces, 1×10 14 pieces, 1×10 15 pieces, 1×10 16 pieces, 1×10 17 pieces, 1×10 18pieces, 1×10 19 pieces, 1×10 20 pieces, or 1 x 10 21 A population of cells containing (or any derivable range therein) can be produced. The cell population number can be achieved without cell sorting based on marker expression, or without cell sorting based on NK marker expression, or without cell sorting based on T cell marker expression. In some embodiments, the cell population size can be achieved without cell sorting based on antigen binding to a heterologous targeting element such as a CAR, TCR, BiTE, or other heterologous tumor targeting agent. Additionally, the population of cells achieved is at least 1 x 10 generated within a certain period of time, such as at least, at most, or exactly 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 days or 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 weeks (or any range derivable therein). 2 pieces, 1×10 3 pieces, 1×10 4 pieces, 1×10 5 pieces, 1×10 6 pieces, 1×10 7 pieces, 1×10 8 pieces, 1×10 9 pieces, 1×10 10 pieces, 1×10 11 pieces, 1×10 12 pieces, 1×10 13 pieces, 1×10 14 pieces, 1×10 15 pieces, 1×10 16 pieces, 1×10 17 pieces, 1×10 18 pieces, 1×10 19 pieces, 1×10 20 pieces, or 1 x 10 21The expression level may include cells (or any range derivable therein). A high or low level of marker expression, such as an NK activator, inhibitor, or cytotoxic molecule, may relate to high expression as determined by FACS analysis. In some embodiments, a high level relates to non-NK cells or non-iNKT cells, or cells that are not T cells. In some embodiments, a high or low level is determined by FACS analysis.

[0035] In some embodiments, the feeder cells used in the method comprise CD34- cells. These CD34- cells may be derived from the same population of cells selected for CD34+ cells. In additional embodiments, the cells may be activated. In certain embodiments, the method includes activating iNKT cells. In certain embodiments, iNKT cells are activated and expanded with alpha-galactosylceramide (α-GC). To activate and expand the cells, the cells may be incubated or cultured with α-GC. In some embodiments, the feeder cells are pulsed with α-GC.

[0036] In some methods, iNKT cells are selected that lack surface expression of one or more HLA-I or HLA-II molecules. In some embodiments, selecting iNKT cells that lack surface expression of HLA-I and / or HLA-II molecules protects these cells from depletion by recipient immune cells.

[0037] The cells can be used immediately or stored for later use. In certain embodiments, the cells used to generate iNKT cells are frozen, while in some embodiments, the generated iNKT cells can be frozen. In some aspects, the cells are present in a solution comprising dextrose, one or more electrolytes, albumin, dextran, and DMSO. In other embodiments, the cells are present in a sterile, non-pyrogenic, and isotonic solution. In some embodiments, the engineered iNKT cells are derived from hematopoietic stem cells. In some embodiments, the engineered iNKT cells are derived from CD34+ cells mobilized with G-CSF. In some embodiments, the cells are derived from cells from a human patient without cancer. In some embodiments, the cells do not express an endogenous TCR.

[0038] The number of cells produced by a production cycle is about, at least about, or at most about 10 2 pieces, 10 3 pieces, 10 4 pieces, 10 5 pieces, 10 6 pieces, 10 7 pieces, 10 8 pieces, 10 9 pieces, 10 10 pieces, 10 11 pieces, 10 12 pieces, 10 13 pieces, 10 14 pieces, 10 15 The cell population may be at least about 10 (or any range derivable therein) of cells or more, which in some embodiments are engineered iNKT cells. In some cases, the cell population comprises at least about 10 engineered iNKT cells. 6 ~10 12 In some embodiments, it is intended that these numbers of populations of cells are produced from a single batch of cells and are not the result of pooling separately produced batches of cells, i.e., derived from a single production cycle.

[0039] In some embodiments, the cell population is frozen and then thawed. The cell population can be used to generate or can include engineered iNKT cells.

[0040] The engineered iNKT cells can be used to treat patients.In some embodiments, the method comprises introducing one or more additional nucleic acids into a cell population that may or may not have been previously frozen and thawed.This use provides one of the advantages of producing ready-made iNKT cells.In certain embodiments, the one or more additional nucleic acids encode one or more therapeutic gene products. Examples of therapeutic gene products include at least the following: 1. antigen recognition molecules, such as CAR (chimeric antigen receptor) and / or TCR (T cell receptor); 2. costimulatory molecules, such as CD28, 4-1BB, 4-1BBL, CD40, CD40L, ICOS; and / or 3. cytokines, such as IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-7, IL-9, IL-15, IL-12, IL-17, IL-21, IL-23, IFN-γ, TNF-α, TGF-β, G-CSF, GM-CSF; 4. transcription factors, such as T-bet, GATA-3, RORγt, FOXP3, and Bcl-6. Therapeutic antibodies are included as chimeric antigen receptors, single-chain antibodies, monobodies, humanized antibodies, bispecific antibodies, single-chain FV antibodies, or combinations thereof.

[0041] In some embodiments, a method for preparing a cell population comprising engineered invariant natural killer (iNKT) T cells includes the steps of: a) selecting CD34+ cells from human peripheral blood cells (PBMCs); b) culturing the CD34+ cells in a medium containing growth factors including c-kit ligand, flt-3 ligand, and human thrombopoietin (TPO); c) transducing the selected CD34+ cells with a lentiviral vector comprising nucleic acid sequences encoding α-TCR, β-TCR, thymidine kinase, and a suicide gene, such as sr39TK; and d) transducing the selected CD34+ cells with Cas9 and a vector. a) introducing gRNA against B2M and / or CTIIA to disrupt expression of B2M and / or CTIIA; b) culturing the transduced cells with an irradiated stromal cell line expressing an exogenous Notch ligand for 2-12 weeks (e.g., 2-10 weeks or 6-12 weeks) to expand iNKT cells in 3D aggregate cell culture; c) selecting iNKT cells lacking surface expression of HLA-I and / or HLA-II molecules; and d) culturing the selected iNKT cells with irradiated feeder cells loaded with α-GC.

[0042] In some embodiments, the method includes the steps of: a) selecting CD34+ cells from human peripheral blood cells (PBMCs); b) culturing the CD34+ cells in a medium containing growth factors including c-kit ligand, flt-3 ligand, and human thrombopoietin (TPO); c) transducing the selected CD34+ cells with a lentiviral vector comprising nucleic acid sequences encoding α-TCR, β-TCR, thymidine kinase, and a reporter gene product; and d) transducing the selected CD34+ cells with a lentiviral vector comprising Cas9 and beta-2 microglobulin (B). e) introducing gRNA against B2M and / or CTIIA to eliminate expression of B2M or CTIIA; f) culturing the transduced cells with an irradiated stromal cell line expressing an exogenous Notch ligand for 2 to 10 weeks to expand the iNKT cells in 3D aggregate cell culture; f) selecting iNKT cells lacking expression of B2M and / or CTIIA; and g) culturing the selected iNKT cells with irradiated feeder cells.

[0043] Also provided are methods of treating patients with iNKT cells or cell populations. In certain embodiments, the patient has cancer. In some embodiments, the patient has a disease or condition involving inflammation, in some embodiments excluding cancer. In certain embodiments, the patient has an autoimmune disease or condition. In certain aspects, the cells or cell population are allogeneic to the patient. In additional embodiments, the patient does not show signs of rejection or depletion of the cells or cell population. Some treatment methods further include administering to the patient a stimulatory molecule that activates iNKT cells (e.g., α-GC, alone or loaded onto APCs), or a compound that initiates a suicide gene product.

[0044] In some embodiments, the cancer treated with engineered iNKT cells comprises leukemia. In some embodiments, the cancer treated with engineered iNKT cells comprises chronic myeloid leukemia cells. In some embodiments, the cancer treated with engineered iNKT cells comprises blood cancer. In some embodiments, the cancer treated with engineered iNKT cells comprises multiple myeloma. In some embodiments, the cancer treated with engineered iNKT cells comprises prostate cancer. In some embodiments, the cancer treated with engineered iNKT cells comprises lung cancer.

[0045] Treatment of a cancer patient with iNKT cells can result in the killing of tumor cells in the cancer patient after administration of the cells or cell population to the patient. Treatment for an inflammatory disease or condition can result in a reduction in inflammation. In other embodiments, patients with an autoimmune disease or condition may experience an improvement in the symptoms of the disease or condition or experience other therapeutic benefits from iNKT cells. Treatment combining iNKT cells with standard therapeutic regimens or other immunotherapy regimens can be used.

[0046] The foregoing has outlined, rather broadly, the features and technical advantages of the present disclosure in order that the detailed description that follows may be better understood. Additional features and advantages will be described below which form the subject of the claims herein. Those skilled in the art will appreciate that the conception and specific embodiments disclosed may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present design. Those skilled in the art will also appreciate that such equivalent constructions do not depart from the spirit and scope of the appended claims. Both as to organization and method of operation The novel features believed characteristic of the design disclosed herein, together with further objects and advantages, will be better understood from a consideration of the following description taken in conjunction with the accompanying drawings, in which: It is to be expressly understood, however, that each of the figures is presented for the purpose of illustration and description only and is not intended as a definition of the limits of the present disclosure. In certain embodiments, for example, the following items are provided: (Item 1) An engineered invariant natural killer T (iNKT) cell expressing at least one invariant natural killer (iNKT) T cell receptor (TCR) wherein one or both of: (1) expresses an exogenous suicide gene product; and (2) the genome of the cell has been modified to eliminate surface expression of at least one HLA-I or HLA-II molecule, and wherein the at least one iNKT TCR is expressed from an exogenous nucleic acid and / or from an endogenous invariant TCR gene under the transcriptional control of a recombinantly modified promoter region. (Item 2) 2. The engineered iNKT cell of item 1, wherein the genome of the cell has been modified to eliminate surface expression of at least one HLA-I or HLA-II molecule. (Item 3) 3. The engineered iNKT cell of item 1 or 2, wherein the invariant TCR gene product is an alpha TCR gene product. (Item 4) 4. The engineered iNKT cell of any of items 1 to 3, wherein the invariant TCR gene product is a beta TCR gene product. (Item 5) 5. The engineered iNKT cell of any of items 1 to 4, wherein both an alpha TCR gene product and a beta TCR gene product are expressed. (Item 6) 6. The engineered iNKT cell of any of items 1 to 5, wherein at least one invariant TCR gene product is expressed from an exogenous nucleic acid. (Item 7) 7. The engineered iNKT cell of any of items 1 to 6, wherein the exogenous suicide gene product and / or the exogenous nucleic acid has one or more codons optimized for expression in the cell. (Item 8) 8. The engineered iNKT cell of any of items 1 to 7, wherein the suicide gene product is herpes simplex virus thymidine kinase (HSV-TK), purine nucleoside phosphorylase (PNP), cytosine deaminase (CD), carboxypeptidase G2, cytochrome P450, linamarase, beta-lactamase, nitroreductase (NTR), carboxypeptidase A, or inducible caspase 9. (Item 9) 9. The engineered iNKT cell of any of items 1 to 8, wherein the suicide gene is enzyme-based. (Item 10) 10. The engineered iNKT cell of item 9, wherein the suicide gene encodes thymidine kinase (TK) or inducible caspase 9. (Item 11) 11. The engineered iNKT cell of item 10, wherein the TK gene is a viral TK gene. (Item 12) 12. The engineered iNKT cell of item 11, wherein the TK gene is a herpes simplex virus TK gene. (Item 13) 13. The engineered iNKT cell of any of items 1 to 12, wherein the suicide gene product is activated by a substrate. (Item 14) 14. The engineered iNKT cells of item 13, wherein the substrate is ganciclovir, penciclovir, or a derivative thereof. (Item 15) 15. The engineered iNKT cell of any of items 1 to 14, comprising an exogenous nucleic acid encoding a polypeptide having a substrate that can be labeled for imaging. (Item 16) 16. The engineered iNKT cell of item 15, wherein the suicide gene product is a polypeptide having a substrate that can be labeled for imaging. (Item 17) 17. The engineered iNKT cell of any of items 1 to 16, wherein the suicide gene is sr39TK or inducible caspase 9. (Item 18) 18. The engineered iNKT cell of any one of items 1 to 17, wherein the iNKT TCR specifically binds alpha-galactosylceramide (α-GC). (Item 19) 19. The engineered iNKT cells of any of items 1 to 18, which do not express surface HLA-I and surface HLA-II molecules by disrupting expression of genes encoding beta2-microglobulin (B2M), major histocompatibility complex II transactivator (CIITA), and / or individual HLA-I and HLA-II molecules. (Item 20) 20. The engineered iNKT cells of item 19, wherein the iNKT cells are engineered by gene editing such that the HLA-I or HLA-II is not expressed on the surface of the cells. (Item 21) 21. The engineered iNKT cell of item 20, wherein the gene editing involves CRISPR-Cas9. (Item 22) 22. The engineered iNKT cell of any of items 1 to 21, comprising a nucleic acid derived from a recombinant vector introduced into the iNKT cell. (Item 23) 23. The engineered iNKT cell of item 22, wherein the nucleic acid is integrated into the genome of the cell. (Item 24) 24. The engineered iNKT cell of item 22 or 23, wherein the recombinant vector is a viral vector. (Item 25) 25. The engineered iNKT cell of item 24, wherein the viral vector is a lentivirus, retrovirus, adeno-associated virus (AAV), herpesvirus, or adenovirus. (Item 26) 26. The engineered iNKT cells of any of items 1 to 25, which have not been exposed to a medium containing animal serum. (Item 27) 27. The engineered iNKT cells of any of items 1 to 26, which are frozen. (Item 28) 27. The engineered iNKT cells of any one of paragraphs 1 to 26, which have been previously frozen and are stable at room temperature for at least 1 hour. (Item 29) 29. The engineered iNKT cells of any of items 1 to 28, wherein the engineered iNKT cells are present in a solution comprising one or more of dextrose, one or more electrolytes, albumin, dextran, and DMSO. (Item 30) 30. The engineered iNKT cells of any of items 1 to 29, wherein the engineered iNKT cells are present in a sterile, non-pyrogenic, and isotonic solution. (Item 31) 31. The engineered iNKT cells of any of items 1 to 30, which are derived from hematopoietic stem cells. (Item 32) 32. The engineered iNKT cells of item 31, which are derived from G-CSF-mobilized CD34+ cells. (Item 33) 33. The engineered iNKT cells of any of items 1 to 32, derived from cells from a human patient without cancer. (Item 34) 34. The engineered iNKT cell of any of items 1 to 33, which does not express an endogenous TCR. (Item 35) 35. A cell population comprising engineered invariant natural killer T (iNKT) cells according to any of items 1 to 34. (Item 36) 36. The cell population of item 35, wherein the iNKT cells comprise an exogenous nucleic acid encoding a suicide gene. (Item 37) 37. The cell population of item 36, wherein the suicide gene is enzyme-based. (Item 38) 38. The cell population of item 37, wherein the suicide gene encodes thymidine kinase (TK) or inducible caspase 9. (Item 39) 39. The cell population of item 38, wherein the TK gene is a viral TK gene. (Item 40) 40. The cell population of item 39, wherein the TK gene is a herpes simplex virus TK gene. (Item 41) 41. The cell population of any of items 36 to 40, wherein the suicide gene product is activated by a substrate. (Item 42) 42. The cell population of item 41, wherein the substrate is ganciclovir, penciclovir, or a derivative thereof. (Item 43) 43. The cell population of any of items 35 to 42, wherein the cells comprise an exogenous nucleic acid encoding a polypeptide having a substrate that can be labeled for imaging. (Item 44) 44. The cell population of item 43, wherein the suicide gene product is a polypeptide having a substrate that can be labeled for imaging. (Item 45) 45. The cell population of any of items 39 to 44, wherein the suicide gene is sr39TK. (Item 46) 46. ​​The cell population of any of items 35 to 45, wherein the iNKT cells do not express surface HLA-I and surface HLA-II molecules by disrupting expression of beta2-microglobulin (B2M), major histocompatibility complex II transactivator (CIITA), and / or genes encoding individual HLA-I and HLA-II molecules. (Item 47) 47. The cell population of item 46, wherein the iNKT cells are engineered by gene editing such that the HLA-I or HLA-II is not expressed on the surface of the cells. (Item 48) The cell population according to Item 47, wherein the gene editing is mediated by CRISPR-Cas9. 49. The cell population of any of items 35 to 48, wherein the iNKT cells comprise a nucleic acid sequence derived from a recombinant vector introduced into the cells. (Item 50) 50. The cell population of item 49, wherein the recombinant vector is a viral vector. (Item 51) 51. The cell population of item 50, wherein the viral vector is a lentivirus, retrovirus, adeno-associated virus (AAV), herpesvirus, or adenovirus. (Item 52) 52. The cell population of any of items 35 to 51, wherein the cells have not been exposed to a medium containing animal serum. (Item 53) 53. The cell population of any of items 35 to 52, wherein the cells are frozen. (Item 54) 54. The cell population of any of items 35 to 53, wherein the cells are present in a solution comprising dextrose, one or more electrolytes, albumin, dextran, and DMSO. (Item 55) 55. The cell population of any of items 35 to 54, wherein the cells are present in a sterile, apyrogenic, and isotonic solution. (Item 56) 57. The cell population of any one of items 35 to 56, wherein the iNKT TCR specifically binds to alpha-galactosylceramide (α-GC). (Item 57) 57. The cell population of any of items 35 to 56, wherein the iNKT cells are activated. (Item 58) 58. The cell population of item 57, wherein the iNKT cells are activated and expanded with alpha-galactosylceramide (α-GC). (Item 59) engineered iNKT cells at least approximately 10 2 ~10 6 59. The cell population according to any of items 35 to 58, comprising: (Item 60) engineered iNKT cells at least approximately 10 6 ~10 12 60. The cell population according to any of items 35 to 59, comprising: (Item 61) 61. The cell population of any of items 35 to 60, wherein greater than 70% of the cells are engineered iNKT cells. (Item 62) 62. The cell population of item 61, wherein greater than 80% of the cells are engineered iNKT cells. (Item 63) 63. The cell population of paragraph 62, wherein greater than 90% of the cells are engineered iNKT cells. (Item 64) 64. The cell population of paragraph 63, wherein greater than 95% of the cells are engineered iNKT cells. (Item 65) 65. The cell population of paragraph 64, wherein greater than 99% of the cells are engineered iNKT cells. (Item 66) 1. A cell population comprising engineered invariant natural killer T (iNKT) cells comprising an iNKT T cell receptor (T cell receptor) and one or more exogenous nucleic acids encoding suicide thymidine kinase, wherein the iNKT cells have been engineered not to express one or more surface HLA-I and / or surface HLA-II molecules, and wherein the cell population comprises at least about 10 6 ~10 12 A cell population that is engineered iNKT cells. (Item 67) 67. The cell population of item 66, wherein the cells are frozen. (Item 68) 68. The cell population of any one of paragraphs 35 to 67, wherein the cells comprise one or more of high levels of the NK activators NKG2D and DNAM-1, low or undetectable levels of the NK inhibitory receptor KIR, and high levels of the cytotoxic molecules perforin and granzyme B. (Item 69) 69. The cell population of any one of items 35 to 68, wherein greater than 90% of the population comprises one or more of high levels of the NK activators NKG2D and DNAM-1, low or undetectable levels of the NK inhibitory receptor KIR, and high levels of the cytotoxic molecules perforin and granzyme B. (Item 70) 70. The cell population of item 69, wherein greater than 90% of the population comprises high levels of the NK activators NKG2D and DNAM-1, low or undetectable levels of the NK inhibitory receptor KIR, and high levels of the cytotoxic molecules perforin and granzyme B. (Item 71) 1. A method for preparing a population of engineered invariant natural killer T (iNKT) cells, comprising: a) selecting CD34+ cells from a plurality of hematopoietic stem or progenitor cells; b) introducing one or more nucleic acids encoding at least one human invariant natural killer (iNKT) T cell receptor (TCR); c) eliminating the surface expression of one or more HLA-I and / or HLA-II molecules on the isolated human CD34+ cells; d) culturing the isolated CD34+ cells expressing the iNKT TCR to produce iNKT cells; A method comprising: (Item 72) 72. The method of claim 71, wherein the step of culturing the isolated CD34+ cells that express iNKT TCR comprises culturing the CD34+ cells in an artificial thymic organoid (ATO) system to produce iNKT cells, wherein the ATO system comprises 3D cell aggregates comprising a selected population of stromal cells that express a Notch ligand and serum-free medium. (Item 73) 73. The method of paragraph 71 or 72, wherein the CD34+ cells are derived from a population comprising differentiated hematopoietic cells. (Item 74) 74. The method of claim 73, wherein the differentiated hematopoietic cells are peripheral blood mononuclear cells (PBMCs). (Item 75) 73. The method of item 71 or 72, wherein the stem or progenitor cells comprise umbilical cord blood cells, fetal liver cells, embryonic stem cells, induced pluripotent stem cells, or bone marrow cells. (Item 76) 76. The method of any of items 71 to 75, further comprising the step of isolating CD34 − cells. (Item 77) 77. The method of any of items 71 to 76, further comprising culturing the selected CD34+ cells in culture medium before introducing the one or more nucleic acids into the cells. (Item 78) 78. The method of claim 77, wherein the culturing step comprises incubating the selected CD34+ cells with a medium containing one or more growth factors. (Item 79) 79. The method of claim 78, wherein the one or more growth factors comprise c-kit ligand, flt-3 ligand, and / or human thrombopoietin (TPO). (Item 80) 80. The method of claim 79, wherein the concentration of the one or more growth factors is between about 5 ng / ml and about 500 ng / ml. (Item 81) 81. The method of any of items 71 to 80, wherein one nucleic acid comprises a nucleic acid sequence encoding an α-TCR. (Item 82) 82. The method of any one of items 71 to 81, wherein one nucleic acid comprises a nucleic acid sequence encoding a β-TCR. (Item 83) 83. The method of any of items 71 to 82, wherein one nucleic acid comprises a nucleic acid encoding both an α-TCR and a β-TCR. (Item 84) 84. The method of any of items 71 to 83, wherein one nucleic acid comprises a nucleic acid sequence encoding an α-TCR and the second nucleic acid comprises a nucleic acid sequence encoding a β-TCR. (Item 85) 72. The method of item 71, further comprising the step of introducing a nucleic acid encoding a suicide gene into the selected CD34+ cells. (Item 86) 86. The method of item 85, wherein one nucleic acid encodes both the α-TCR and the β-TCR. (Item 87) 87. The method of item 86, wherein one nucleic acid encodes the α-TCR, the β-TCR, and the suicide gene. (Item 88) 88. The method of any of items 85 to 87, wherein the suicide gene is enzyme-based. (Item 89) 89. The method of item 88, wherein the suicide gene encodes thymidine kinase (TK) or inducible caspase 9. (Item 90) 90. The method of item 89, wherein the TK gene is a viral TK gene. (Item 91) Item 92. The method of Item 89, wherein the TK gene is a herpes simplex virus TK gene. 92. The method of any of items 85 to 91, wherein the suicide gene product is activated by a substrate. (Item 93) 93. The method of claim 92, wherein the substrate is ganciclovir, penciclovir, or a derivative thereof. (Item 94) 94. The method of any of items 71 to 93, wherein the cells comprise an exogenous nucleic acid encoding a polypeptide having a substance that can be labeled for imaging. (Item 95) 95. The method of claim 94, wherein the suicide gene product is a polypeptide having a substrate that can be labeled for imaging. (Item 96) 96. The method of any of items 91 to 95, wherein the suicide gene is sr39TK. (Item 97) 97. The method of any of items 71 to 96, wherein the iNKT cells do not express surface HLA-I molecules and / or surface HLA-II molecules upon disruption of expression of genes encoding beta2-microglobulin (B2M), major histocompatibility complex II transactivator (CIITA), and / or individual HLA-I and HLA-II molecules. (Item 98) 98. The method of claim 97, wherein the step of eliminating expression of cell surface HLA-I and / or cell surface HLA-II molecules in the isolated human CD34+ cells comprises introducing CRISPR and B2M, CIITA, and / or one or more guide RNAs (gRNAs) corresponding to individual HLA-I and HLA-II molecules into the cells. (Item 99) 99. The method of claim 98, wherein CRISPR or the one or more gRNAs are transfected into the cells by electroporation or lipid-mediated transfection. (Item 100) 99. The method of any of items 71 to 99, wherein the nucleic acid encoding the TCR receptor is introduced into the cell using a recombinant vector. (Item 101) Item 101. The method of item 100, wherein the recombinant vector is a viral vector. (Item 102) 102. The method of claim 101, wherein the viral vector is a lentivirus, retrovirus, adeno-associated virus (AAV), herpesvirus, or adenovirus. (Item 103) 103. The method of claim 102, wherein the viral vector is a lentivirus. (Item 104) 104. The method according to any of items 71 to 103, wherein the serum-free medium further comprises exogenously added ascorbic acid. (Item 105) Item 106. The method according to any one of Items 71 to 104, wherein the serum-free medium further contains exogenously added FLT3 ligand (FLT3L), interleukin-7 (IL-7), stem cell factor (SCF), thrombopoietin (TPO), IL-2, IL-4, IL-6, IL-15, IL-21, TNF-alpha, TGF-beta, interferon-gamma, interferon-lambda, TSLP, thymopentin, pleiotrophin, midkine, or a combination thereof. 106. The method according to any of items 71 to 105, wherein the serum-free medium further comprises vitamins. (Item 107) 107. The method of claim 106, wherein the vitamins comprise biotin, DL-alpha tocopheryl acetate, DL-alpha tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, folic acid, nicotinamide, pyridoxine, riboflavin, thiamine, inositol, vitamin B12, or combinations thereof or salts thereof. (Item 108) 107. The method of claim 106, wherein the vitamin comprises biotin, DL alpha tocopherol acetate, DL alpha tocopherol, vitamin A, or a combination or salt thereof. (Item 109) 109. The method of any of items 71 to 108, wherein the serum-free medium further comprises a protein. (Item 110) 110. The method of claim 109, wherein the protein comprises albumin or bovine serum albumin, a fraction of BSA, catalase, insulin, transferrin, superoxide dismutase, or a combination thereof. (Item 111) 111. The method of any of items 71 to 110, wherein the serum-free medium further comprises corticosterone, D-galactose, ethanolamine, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, or triiodo-I-thyronine, or a combination thereof. (Item 112) 112. The method of any of items 71 to 111, wherein the serum-free medium comprises B-27® supplement, Xeno-Free B-27® supplement, GS21™ supplement, or a combination thereof. (Item 113) 113. The method according to any of items 71 to 112, wherein the serum-free medium comprises or further comprises amino acids, monosaccharides, inorganic ions. (Item 114) 114. The method of claim 113, wherein the amino acids comprise arginine, cysteine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine, or a combination thereof. (Item 115) Item 114. The method of item 113, wherein the inorganic ions comprise sodium, potassium, calcium, magnesium, nitrogen, or phosphorus, or combinations or salts thereof. (Item 116) 116. The method of any of items 71 to 115, wherein the serum-free medium further comprises molybdenum, vanadium, iron, zinc, selenium, copper, or manganese, or a combination thereof. (Item 117) 117. The method of any of items 71 to 116, wherein the 3D cell aggregates are created by mixing CD34+ transduced cells and a selected population of the stromal cells on a physical matrix or scaffold. (Item 118) 118. The method of claim 117, further comprising centrifuging the CD34+ transduced cells and stromal cells to form a cell pellet that is placed onto the physical matrix or scaffold. (Item 119) 119. The method of any of items 71 to 118, wherein the Notch ligand expressed by the stromal cells is intact, partial, or modified DLL1, DLL4, JAG1, JAG2, or a combination thereof. (Item 120) 120. The method of claim 119, wherein the Notch ligand is a human Notch ligand. (Item 121) 121. The method of claim 120, wherein the Notch ligand is human DLL1 or DLL4. (Item 122) 122. The method of any of items 71 to 121, wherein the ratio of stromal cells to CD34+ cells is about 1:5 to 1:20. (Item 123) 123. The method of any of items 71 to 122, wherein the stromal cells are a murine stromal cell line, a human stromal cell line, a selected population of primary stromal cells, a selected population of stromal cells differentiated in vitro from pluripotent stem cells, or a combination thereof. (Item 124) 124. The method of any of items 71 to 123, wherein the stromal cells are a selected population of stromal cells differentiated in vitro from hematopoietic stem or progenitor cells. (Item 125) 125. The method of any of items 71 to 124, wherein the step of selecting iNKT cells lacking surface expression of HLA-I / II molecules comprises positive selection of iNKT cells using microbeads or flow cytometry and negative selection of HLA-I / II negative cells. (Item 126) 126. The method of any of items 71 to 125, wherein the cells are frozen. (Item 127) 127. The method of any of items 1 to 126, wherein the cells are present in a solution comprising dextrose, one or more electrolytes, albumin, dextran, and DMSO. (Item 128) 128. The method of any of items 1 to 127, wherein the cells are present in a sterile, apyrogenic and isotonic solution. (Item 129) 129. The method of any one of items 71 to 128, wherein the cells are derived from cells from a human patient without cancer. (Item 130) 130. The method of any one of items 71 to 129, wherein the cells do not express an endogenous TCR. (Item 131) The feeder cells were CD34 - 131. The method of any of items 1 to 130, comprising a cell. (Item 132) 132. The method of any of items 71 to 131, further comprising activating the selected iNKT cells. (Item 133) Item 133. The method of item 132, wherein the selected iNKT cells are activated and expanded with alpha-galactosylceramide (α-GC). (Item 134) 134. The method of claim 133, wherein the feeder cells are pulsed with α-GC. (Item 135) engineered iNKT cells at least approximately 10 2 ~10 6 135. The method of any of items 71 to 134, wherein a population of engineered iNKT cells comprising the cells is produced. (Item 136) engineered iNKT cells at least approximately 10 6 ~10 12 136. The method according to any of items 71 to 135, wherein a cell population comprising the cells is produced. (Item 137) 137. The method of any of items 71 to 136, wherein the cell population is frozen and then thawed. (Item 138) 138. The method of claim 137, further comprising the step of introducing one or more additional nucleic acids into the frozen and thawed cell population. (Item 139) 139. The method of claim 138, wherein the one or more additional nucleic acids encode one or more therapeutic gene products. (Item 140) 139. The method of any one of paragraphs 71 to 138, wherein greater than 90% of the population of engineered iNKT cells comprises one or more of high levels of the NK activators NKG2D and DNAM-1, low or undetectable levels of the NK inhibitory receptor KIR, and high levels of the cytotoxic molecules perforin and granzyme B. (Item 141) 141. The method of claim 140, wherein greater than 90% of the population of engineered iNKT cells comprises high levels of the NK activators NKG2D and DNAM-1, low or undetectable levels of the NK inhibitory receptor KIR, and high levels of the cytotoxic molecules perforin and granzyme B. (Item 142) A cell population containing engineered invariant natural killer (iNKT) T cells was explanted. A method for preparing in vivo, comprising: a) selecting CD34+ cells from human peripheral blood mononuclear cells; b) culturing the CD34+ cells in a medium containing growth factors including c-kit ligand, flt-3 ligand, and human thrombopoietin (TPO); c) transducing the selected CD34+ cells with a lentiviral vector comprising nucleic acid sequences encoding an iNKT TCR alpha chain, an iNKT TCR beta chain, and a suicide thymidine kinase / imaging reporter gene; d) introducing Cas9 and gRNA against beta 2 microglobulin (B2M) and / or CTIIA into the selected CD34+ cells to disrupt the expression of the B2M gene or CTIIA gene; e) culturing the transduced cells with an irradiated stromal cell line expressing an exogenous Notch ligand for 2 to 10 weeks to expand iNKT cells in 3D aggregate cell culture; f) selecting iNKT cells that lack surface expression of HLA-I / II molecules; g) culturing the selected iNKT cells with irradiated feeder cells; A method comprising: (Item 143) 10 from the selected CD34+ cells 8 ~10 13 Item 143. The method of Item 142, wherein iNKT cells are prepared. (Item 144) A method of treating a patient with iNKT cells, comprising administering to said patient any of the cells described in items 1 to 34 or the cell populations described in items 35 to 70. (Item 145) Item 145. The method of item 144, wherein the patient has cancer. (Item 146) Item 145. The method of item 144, wherein the patient has a disease or condition involving inflammation. (Item 147) 147. The method of claim 146, wherein the disease or condition is an autoimmune disease. (Item 148) 148. The method of any of items 144 to 147, wherein the cell or cell population is allogeneic to the patient. (Item 149) 149. The method of any of items 144 to 148, wherein the patient shows no signs of complete depletion of the cell or cell population. (Item 150) 149. The method of any of items 144 to 149, further comprising administering to the patient a compound that induces the suicide gene product. (Item 151) 146. The method of claim 145, wherein after administering the cell or cell population to the patient, tumor progression in the patient is controlled or inhibited. (Item 152) 148. The method of any of items 146 or 147, wherein inflammation is reduced. (Item 153) 1. An engineered invariant natural killer T cell that expresses at least one invariant T cell receptor (TCR) gene product and an exogenous suicide gene product, wherein the at least one invariant TCR gene product is expressed from an exogenous nucleic acid and / or from an endogenous invariant TCR gene under the transcriptional control of a recombinantly modified promoter region, wherein the iNKT cell comprises: a) selecting CD34+ cells from a plurality of hematopoietic stem or progenitor cells; b) introducing one or more nucleic acids encoding at least one human invariant natural killer (iNKT) T cell receptor (TCR); c) eliminating the surface expression of one or more HLA-I and / or HLA-II molecules on the isolated human CD34+ cells; d) culturing the isolated CD34+ cells expressing the iNKT TCR to produce iNKT cells; Invariant natural killer T cells produced by a process comprising: (Item 154) Engineered iNKT cells that express at least one invariant natural killer (iNKT) T cell receptor (TCR), including high levels of the NK activators NKG2D and DNAM-1, low or undetectable levels of the NK inhibitory receptor KIR, and high levels of the cytotoxic molecules perforin and granzyme B. (Item 155) A population of engineered invariant natural killer (iNKT) T cell receptors (TCRs), wherein greater than 90% of the population contains high levels of the NK activators NKG2D and DNAM-1, low or undetectable levels of the NK inhibitory receptor KIR, and high levels of the cytotoxic molecules perforin and granzyme B.

[0047] For a complete understanding of the present disclosure, reference is made to the following descriptions taken in conjunction with the accompanying drawings. [Brief explanation of the drawings]

[0048] [Figure 1] Figure 1 is a schematic diagram of an example of the generation and use of iNKT (UHSC-iNKT) cell adoptive therapy via manipulation of off-the-shelf universal hematopoietic stem cells (HSCs).

[0049] [Figure 2] Figures 2A-2D show the generation of iNKT cells by manipulating human HSCs in a BLT (human bone marrow-liver-thymus-engrafted NOD / SCID / γc- / - mice) humanized mouse model. (2A) Example of experimental design. (2B) FACS plot of spleen cells. HSC-iNKTBLT: iNKT cells generated in BLT mice by manipulating human HSCs. hTc: human conventional T cells. Figures 2C-2D show the generation of NY-ESO-1-specific conventional T cells by manipulating human HSCs in an artificial thymic organoid (ATO) in vitro culture system. (2C) Example of experimental design. (2D) Cell yield (n = 3-6). **p < 0.01 by Student's t-test.

[0050] [Figure 3-1] Figures 3A-3D demonstrate the first CMC study of the robust, high-yield two-stage ATO-αGC in vitro culture system for the generation of iNKT cells by engineering human HSCs (HSC-iNKTATO cells were tested as a therapeutic surrogate). HSC-iNKTATO: iNKT cells engineered from human HSCs generated in ATO culture. (3A) Two-stage ATO-αGC in vitro culture system. ATO: artificial thymic organoid; αGC: alpha-galactosylceramide, a potent agonist ligand that specifically stimulates iNKT cells. (3B) Generation of HSC-iNKTATO cells during ATO culture. 6B11 is a monoclonal antibody that specifically binds to the iNKT TCR. (3C) Expansion of HSC-iNKTATO cells during PBMC / αGC culture. (3D) HSC-iNKTATO cell production. [Figure 3-2] Same as above.

[0051] [Figure 4] Figures 4A-4B present the first pharmacological studies of the phenotype and functionality of iNKT cells engineered from human HSCs. (HSC-iNKTATO and HSC-iNKTBLT cells were tested as therapeutic surrogates.) (4A) Surface FACS staining. (4B) Intracellular FACS staining. PBMC-iNKT: in vitro expanded endogenous iNKT cells derived from healthy donor PBMCs; PBMC-Tc: endogenous conventional T cells derived from healthy donor PBMCs.

[0052] [Figure 5-1] Figures 5A-5K present initial efficacy studies of the tumor-killing efficacy of engineered human HSC iNKT cells (HSC-iNKTATO and HSC-iNKTBLT cells were tested as therapeutic surrogates). (5A-5F) Hematological cancer models. (5A) MM.1S-hCD1d-FG human multiple myeloma (MM) cell line. (5B) In vitro tumor-killing assay. (5C) In vitro tumor-killing assay using luciferase activity (n=3). (5D) In ​​vivo tumor-killing assay using the NSG mouse human MM metastasis model. (E-F) In vivo tumor-killing assay using live animal bioluminescence imaging (BLI). Representative BLI images (5E) and time-course measurements of whole-body luminescence (TBL; 5F) at day 14 are shown (n=3-4). (5G-5K) Solid tumor models. (5G) A375-hCD1d-FG human melanoma cell line. (5H) In vivo tumor killing assay using the NSG mouse human melanoma solid tumor model. (5I) Tumor weight (day 25). (5J) FACS plot showing infiltration of HSC-iNKTBLT cells into the tumor site (day 25). (5K) Quantification of J (n=4). **p<0.01, ***p<0.001 by Student's t-test. [Figure 5-2] Same as above.

[0053] [Figure 6]Figures 6A-6C show initial safety studies for toxicology / tumorigenicity (HSC-iNKTBLT cells were tested as a therapeutic surrogate). (6A) Mouse weight (n=9-10). ns, not significant, Student's t-test. (6B) Mouse survival (n=9-10). (6C) Mouse pathology. Various tissues were collected and analyzed by the UCLA Pathology Core (n=9-10).

[0054] [Figure 7] Figures 7A-7D show the first safety study of the sr39TK gene for PET imaging and safety control. (HSC-iNKTBLT cells were tested as a therapeutic surrogate.) (7A) Experimental design. (7B) PET / CT images of BLT-iNKTTK mice before and after GCV treatment (n=4-5). (7C) FACS plots showing effective and specific depletion of HSC-iNKTBLT cells after GCV treatment (n=4-5). (7D) Quantification of FACS plots in 7C (n=4-5). ns, not significant; **p<0.01; Student's t-test.

[0055] [Figure 8-1] Figures 8A-8F show an example of a manufacturing process for generating UHSC-iNKT cells. (8A) Experimental design. (8B) Lenti / iNKT-sr39TK vector-mediated iNKT TCR expression in HSCs. (8C) CRISPR-Cas9 / B2M-CIITA-gRNA complex-mediated knockout of HLA-I / II expression in HSCs. (8D) Diagram showing purification steps between stage 1 and stage 2 culture. (8E) 2M2 / Tue39 mAb-mediated MACS negative selection of HLA-I / IIIneg cells. (8F) 6B11 mAb-mediated MACS positive selection of HSC-iNKTATO cells. [Figure 8-2] Same as above.

[0056] [Figure 9-1]Figures 9A-9E present examples of mechanism of action (MOA) studies. (9A) Potential mechanisms used by iNKT cells to target tumors. (9B-9C) Studies on CD1d / TCR-mediated direct killing of tumor cells. (9B) Experimental design; (9C) Killing of MM.1S-hCD1d-FG human multiple myeloma cells (n=3). (9D-9E) Studies on CD1d-independent targeting of tumor cells by activating NK cells. (9D) Experimental design; (9E) Killing of K562 tumor cells (n=2). Irradiated PBMCs loaded with αGC were used as antigen-presenting cells (APCs). ns, not significant; *p<0.05; **p<0.01; ****p<0.0001 by one-way ANAVO. [Figure 9-2] Same as above.

[0057] [Figure 10-1] Figures 10A-10G demonstrate safety considerations. (10A) Potential GvHD and HvG responses and engineered safety management strategies. (10B) In vitro mixed lymphocyte culture (MLC) assay to test for GvHD responses. (10C) IFN-γ production in the MLC assay (n=3) indicates that HSC-iNKTATO cells do not induce GvHD responses. PBMCs from three different healthy donors were included as responders. (10D) In ​​vitro mixed lymphocyte culture (MLC) assay to test for HvG responses. (10E) IFN-γ production in the MLC assay (n=3) indicates a minimal HvG response to HSC-iNKTATO cells. PBMCs from two different healthy donors were used in this experiment. (10F) HSC-iNKTBLT cells were resistant to killing by mismatched donor NK cells in in vitro mixed NK / iNKT cultures. (10G) In vivo mixed lymphocyte adoptive transfer (MLT) assay to test GvHD and HvD responses. ns, not significant, **p<0.01, ***p<0.001, ****p<0.0001 by one-way ANAVO. [Figure 10-2] Same as above.

[0058] [Figure 11] Figures 11A-11G are diagrams demonstrating examples of combination therapy. (11A) Experimental design for testing the combination of UHSC-iNKT cell therapy and checkpoint blockade therapy. (11B) UHSCCAR-iNKT cells. (11C) A375-hCD1d-hCD19-FG human melanoma cell line. (11D) Experimental design for testing the anti-tumor efficacy of UHSCCAR-iNKT cells. (11E) UHSCTCR-iNKT cells. (11F) A375-hCD1d-A2 / ESO-FG human melanoma cell line. (11G) Experimental design for testing the anti-tumor efficacy of UHSCTCR-iNKT cells.

[0059] [Figure 12] FIG. 12 shows an example of a pharmacokinetic / pharmacodynamic (PK / PD) study.

[0060] [Figure 13] FIG. 13 shows an example of an iNKT-sr39TK lentiviral vector.

[0061] [Figure 14] FIG. 14 is a diagram showing an example of a cell production process for producing UHSC-iNKT cells.

[0062] [Figure 15] Figure 15 shows the phenotype and functionality of HSC-iNKT cells. Representative FACS plots are presented showing surface staining of NK activating receptors (NKG2D and DNAM-1) and inhibitory receptors (KIR), as well as intracellular staining of cytotoxic molecules (perforin and granzyme B). Naive NK cells isolated from peripheral blood of healthy human donors (PBMC-NK cells) were included as a control.

[0063] [Figure 16-1]Figures 16A-G show in vitro efficacy and MOA studies. (A) Experimental design for testing NK cell-like tumor cell killing by HSC-iNKT cells. This study used multiple human tumor cell lines engineered to overexpress firefly luciferase (Fluc) and enhanced green fluorescent protein (EGFP) dual reporters to enable sensitive measurement of tumor killing using a luciferase activity assay. A375-FG, engineered human melanoma tumor cell line; K562-FG, engineered human chronic myeloid leukemia cell line; MM.1S-FG, engineered human multiple myeloma cell line; H292-FG, engineered human lung cancer cell line; and PC3-FG, engineered human prostate cancer cell line. (B-F) Analysis of in vitro killing of various human tumor cells by fresh or frozen / thawed HSC-iNKT cells by luciferase activity. Fresh or frozen / thawed PBMC-NK cells were included as controls. (G) Analysis of tumor cell killing efficacy by HSC-iNKT cells in the presence of NKG2D and / or DNAM-1 blocking antibodies by luciferase activity. Representative of two experiments. Data are shown as mean ± SEM. ns, not significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 by one-way ANOVA. [Figure 16-2] Same as above. [Figure 16-3] Same as above. [Figure 16-4] Same as above.

[0064] [Figure 17] Figures 17A-D are in vivo efficacy studies. (A) Experimental design. (B) Quantification of total body luminescence (TBL) over time (n=5). (C) Measurement of tumor size over time (n=5). (D) Measurement of tumor weight at day 26 (n=5). Representative of two experiments. Data are shown as mean ± SEM. ns, not significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 by Student's t-test. DETAILED DESCRIPTION OF THE INVENTION

[0065] I. Definition Examples As used herein, "a" or "an" can mean one or more. When used in a claim(s) and in conjunction with the word "comprising," the words "a" or "an" can mean one or more than one. As used herein, "another" can mean at least a second or more. In certain embodiments, aspects of the invention can, for example, "consist essentially of" or "consist of" one or more sequences of the invention. Some embodiments of the invention can consist of or consist essentially of one or more elements, method steps, and / or methods of the invention. It is contemplated that any method or composition described herein can be practiced in conjunction with any other method or composition described herein.

[0066] The present disclosure encompasses "HSC-iNKT cells," which are invariant natural killer T (iNKT) cells engineered from hematopoietic stem cells (HSCs) and / or hematopoietic progenitor cells (HPCs), and methods of making and using them. As used herein, "HSCs" is used to refer to HSCs, HPCs, or both HSCs and HPCs.

[0067] The term "therapeutically effective amount," as used herein, refers to an amount effective to alleviate, ameliorate, or prevent at least one symptom or sign of the disease or condition being treated.

[0068] The term "exogenous TCR" refers to a TCR gene or TCR gene derivative that has been transferred (i.e., by gene transfer / transduction / transfection techniques) into a cell, or is the progeny of a cell that has received the transfer of the TCR gene or gene derivative. The exogenous TCR gene is inserted into the genome of the recipient cell. In some embodiments, the insertion is random insertion. Random insertion of TCR genes is readily achieved by methods known in the art. In some embodiments, the TCR gene is inserted into an endogenous locus (e.g., an endogenous TCR gene locus). In some embodiments, the cell comprises one or more TCR genes inserted into a locus that is not an endogenous locus. In some embodiments, the cell further comprises a heterologous sequence, such as a marker or resistance gene.

[0069] The term "chimeric antigen receptor" or "CAR" refers to engineered receptors that graft any specificity onto immune effector cells. These receptors can be used to target monoclonal antibodies. The specificity of clonal antibodies is transferred to T cells. The transfer of their coding sequences is facilitated by retroviral or lentiviral vectors. Because these receptors are composed of portions derived from different sources, they are called chimeric. The most common form of these molecules is a fusion of a single-chain variable fragment (scFv) derived from a monoclonal antibody fused to the CD3-zeta transmembrane and endodomains; the CD28 or 41BB intracellular domains; or a combination thereof. Such molecules transduce signals in response to target recognition by the scFv. An example of such a construct is 14g2a-zeta, a fusion of an scFv derived from the hybridoma 14g2a (which recognizes the disialoganglioside GD2). When T cells express this molecule (e.g., achieved by oncoretroviral vector transduction), they recognize and kill target cells expressing GD2 (e.g., neuroblastoma cells). To target malignant B cells, researchers have redirected T cell specificity using chimeric immune receptors specific for the B-lineage molecule CD19. The variable portions of immunoglobulin heavy and light chains are fused by a flexible linker to form scFvs. The scFvs are preceded by a signal peptide (which is cleaved) that directs the nascent protein to the endoplasmic reticulum and subsequent surface expression. The flexible spacer allows the scFv to orient in various directions, enabling antigen binding. The transmembrane domain is typically a typical hydrophobic alpha helix derived from the original molecule, with the signaling endodomain protruding into the cell and transmitting the desired signal.

[0070] The term "antigen" refers to any substance that can cause the immune system to produce antibodies against or T cells to respond to. In some embodiments, an antigen is a peptide that is 5-50 amino acids in length, or at least, at most, or exactly 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 250, or 300 amino acids in length, or any range of lengths derivable therein.

[0071] The term "allogeneic to the recipient" refers to cells that are not isolated from the recipient. In some embodiments, the cells are not isolated from the patient. In some embodiments, the cells are not isolated from a genetically matched individual (e.g., a genotypically compatible relative).

[0072] The term "inactive" refers to not producing undesired clinical toxicity. This can be either on-target or off-target toxicity. "Inactive" is based on known or predicted clinical safety data.

[0073] The terms "xeno-free (XF)" or "animal component-free (ACF)" or "animal-free," when used with respect to a medium, extracellular matrix, or culture condition, refer to a medium, extracellular matrix, or culture condition that is essentially free of xenogeneic animal-derived components. For the culture of human cells, any proteins from non-human animals, such as mice, become xenogeneic components. In certain embodiments, a xeno-free matrix is ​​essentially free of any non-human animal-derived components and thus may exclude mouse feeder cells or Matrigel™. Matrigel™ is a solubilized basement membrane preparation extracted from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma, a tumor rich in extracellular matrix proteins, and contains laminin (a major component), type IV collagen, heparin sulfate proteoglycan, and entactin / nidogen.

[0074] The term "defined," when used with respect to a medium, extracellular matrix, or culture conditions, refers to a medium, extracellular matrix, or culture conditions in which the nature and amount of substantially all components are known.

[0075] "Chemically defined media" refers to media in which the chemical nature and amounts of nearly all components are known. These media are also called synthetic media. An example of a chemically defined medium is TeSR™.

[0076] As used herein, cells are "substantially free" of a particular reagent or element, such as serum, signal transduction inhibitors, animal components or feeder cells, exogenous genetic or vector elements, if the cells have less than 10% of the element(s), and "essentially free" of a particular reagent or element if the cells have less than 1% of the element(s). However, even more desirable are cell populations in which less than 0.5% or less than 0.1% of the total cell population contains exogenous genetic or vector elements.

[0077] The culture, matrix or medium may contain certain reagents or elements, such as serum, signal transduction inhibitors, animal components or feeder cells, such that the levels of these reagents in the culture, matrix or medium, respectively, are below detectable levels using conventional detection methods known to those skilled in the art, or that these agents are not present in the culture, matrix or medium. A serum-free medium can be essentially free of serum if it is not exogenously added to the medium or medium.

[0078] "Peripheral blood cells" refers to the cellular components of blood, including red blood cells, white blood cells, and platelets, found within the circulating pool of blood.

[0079] "Hematopoietic stem and progenitor cells" or "hematopoietic precursor cells" refer to cells that are committed to the hematopoietic lineage but can further differentiate into hematopoiesis, including hematopoietic stem cells, multipotent hematopoietic stem cells (hemocyte blasts), myeloid progenitor cells, megakaryocytic progenitor cells, erythroid progenitor cells, and lymphoid progenitor cells. "Hematopoietic stem cells (HSCs)" are multipotent stem cells that give rise to all blood cell types, including the myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells) and lymphoid (T cells, B cells, NK cells) lineages. In this disclosure, HSCs refer to both "hematopoietic stem and progenitor cells" and "hematopoietic precursor cells."

[0080] Hematopoietic stem and progenitor cells may or may not express CD34. Hematopoietic stem cells may co-express CD133 and be negative for CD38 expression, positive for CD90, negative for CD45RA, negative for lineage markers, or a combination of these. Hematopoietic progenitor / precursor cells include CD34(+) / CD38(+) cells and CD34(+) / CD45RA(+) / lin(-)CD10+ (common lymphoid progenitor cells), CD34(+)CD45RA(+)lin(-)CD10(-)CD62L(hi) (lymphoid-stimulated multipotent progenitor cells), CD34(+)CD45RA(+)lin(-)CD10(-)CD123+ (granulocyte-monocyte progenitor cells), CD34(+)CD45RA(-)lin(-)CD10(-)CD123+ (common myeloid progenitor cells), or CD34(+)CD45RA(-)lin(-)CD10(-)CD123- (megakaryocyte-erythroid progenitor cells).

[0081] A "vector" or "construct" (sometimes called a gene delivery or gene transfer "vehicle") refers to a macromolecule, complex of molecules, or viral particle containing a polynucleotide that is delivered to a host cell either in vitro or in vivo. The polynucleotide can be a linear or circular molecule.

[0082] A "plasmid" is a general type of vector; it is an extrachromosomal DNA molecule separate from chromosomal DNA that can replicate independently of the chromosomal DNA. In certain cases, plasmids are circular, double-stranded.

[0083] "Expression construct" or "expression cassette" refers to a nucleic acid molecule capable of directing transcription. At a minimum, an expression construct contains a promoter or a structure functionally equivalent to a promoter. Additional elements, such as enhancers, and / or transcription termination signals, can also be included.

[0084] The term "exogenous," when used with reference to a protein, gene, nucleic acid, or polynucleotide in a cell or organism, refers to a protein, gene, nucleic acid, or polynucleotide that has been introduced into the cell or organism by artificial means, or when used with reference to a cell, refers to a cell that has been isolated and then introduced into another cell or organism by artificial means. An exogenous nucleic acid may be from a different organism or cell, or may be one or more additional copies of a nucleic acid that naturally occurs in the organism or cell. An exogenous cell may be from a different organism or from the same organism. As a non-limiting example, an exogenous nucleic acid is present in a chromosomal location that is different from the chromosomal location of the native cell, or is otherwise flanked by nucleic acid sequences that are different from those found in nature.

[0085] The term "corresponding to" is used herein to mean that a polynucleotide sequence is homologous (i.e., identical, although not strictly evolutionarily related) to all or a portion of a reference polynucleotide sequence, or that a polypeptide sequence is identical to a reference polypeptide sequence. In contrast, the term "complementary to" is used herein to mean that a complementary sequence is homologous to all or a portion of a reference polynucleotide sequence. By way of illustration, the nucleotide sequence "TATAC" corresponds to the reference sequence "TATAC" and is complementary to the reference sequence "GTATA."

[0086] A "gene," "polynucleotide," "coding region," "sequence," "segment," "fragment," or "transgene" that "encodes" a particular protein is a nucleic acid molecule that, when placed under the control of appropriate regulatory sequences, can be transcribed in vitro or in vivo and, if necessary, translated into a gene product, e.g., a polypeptide. The coding region may exist in either cDNA, genomic DNA, or RNA form. If present in DNA form, the nucleic acid molecule may be single-stranded (i.e., the sense strand) or double-stranded. The boundaries of a coding region are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A gene may include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and synthetic DNA sequences. A transcription termination sequence is typically located 3' to the gene sequence.

[0087] The term "cell" is used herein in its broadest sense in the art and refers to a living entity that is a structural unit of tissue in a multicellular organism, is isolated from the outside by a membrane structure, has the ability to self-replicate, and has genetic information and mechanisms for expression. As used herein, a cell may be a naturally occurring cell or an artificially modified cell (e.g., a fused cell, a genetically modified cell, etc.).

[0088] As used herein, the term "stem cell" refers to a cell that can self-renew and is pluripotent or multipotent. Generally, stem cells can regenerate damaged tissues. The stem cells herein may be, but are not limited to, embryonic stem (ES) cells, induced pluripotent stem cells, or tissue stem cells (also referred to as tissue-specific stem cells or somatic stem cells).

[0089] Embryonic stem (ES) cells are pluripotent stem cells derived from early embryos. ES cells were first established in 1981, and have been used to create knockout mice since 1989. Human ES cells were established in 1998, and are now becoming available for regenerative medicine.

[0090] Unlike ES cells, tissue stem cells have limited differentiation potential. They reside in specific locations within tissues and possess undifferentiated intracellular structures. Therefore, their pluripotency is generally low. Tissue stem cells have a high nucleus / cytoplasm ratio and few intracellular organelles. The majority of tissue stem cells have low pluripotency, a long cell cycle, and the ability to proliferate beyond the lifetime of an individual. Tissue stem cells are categorized based on the site from which they originate, such as the skin, digestive system, bone marrow, and nervous system. Skin stem cells include epidermal stem cells and hair follicle stem cells. Digestive stem cells include pancreatic (common) stem cells and hepatic stem cells. Bone marrow stem cells include hematopoietic stem cells and mesenchymal stem cells. Nervous system stem cells include neural stem cells and retinal stem cells.

[0091] "Induced pluripotent stem cells," commonly abbreviated as iPS cells or iPSCs, refer to a type of pluripotent stem cell that is artificially prepared from non-pluripotent cells, generally adult somatic cells, or terminally differentiated cells such as fibroblasts, hematopoietic cells, muscle cells, neurons, and epidermal cells, by introducing certain factors known as reprogramming factors.

[0092] As used herein, "isolated," with respect to, for example, cells and / or nucleic acids, means altered or removed by human intervention from the natural state.

[0093] "Pluripotent" refers to stem cells that have the potential to differentiate into one or more tissues or organs, or in particular, all of the cells that make up any of the three germ layers: endoderm (stomach lining, gastrointestinal tract, lungs), mesoderm (muscle, bone, blood, urogenital tract), or ectoderm (epidermal tissue and nervous system). As used herein, "pluripotent stem cells" refer to cells that can differentiate into cells of any of the three germ layers, e.g., the direct descendants of totipotent cells or induced pluripotent cells.

[0094] "Operably linked," with reference to nucleic acid molecules, means that two or more nucleic acid molecules (e.g., a nucleic acid molecule to be transcribed, a promoter, and an enhancer element) are connected in a manner that allows for transcription of the nucleic acid molecule. "Operably linked," with reference to peptide and / or polypeptide molecules, means that two or more peptide and / or polypeptide molecules are connected in a manner that results in a single polypeptide chain, i.e., a fusion polypeptide, having at least one property of each peptide and / or polypeptide component of the fusion. Fusion polypeptides are particularly chimeric, i.e., composed of heterologous molecules.

[0095] Embodiments of the present disclosure relate to HSC cells engineered to function as iNKT cells that possess NKT cell T cell receptors (TCRs), have imaging and suicide targeting capabilities, and are resistant to targeted depletion by host immune cells. Such cells are generated in an artificial thymic organoid (ATO) in vitro culture system that supports the efficient and high-yield differentiation of TCR-engineered HSCs into clonal T cells. II. Invariant NKT cells (NKT cells) generated by engineering universal hematopoietic stem cells (HSCs) U HSC-iNKT cells)

[0096] Embodiments of the present disclosure utilize cells (e.g., HSCs) that have been engineered to have one or more characteristics that make the cells modified to function as invariant NKT cells suitable for universal use (use in individuals other than those from which the original cells were obtained) without adverse immune responses in the recipient of the cells. The present disclosure encompasses engineered invariant natural killer T (iNKT) cells that contain nucleic acids comprising: i) all or a portion of an iNKT alpha T-cell receptor gene; ii) all or a portion of an iNKT beta T-cell receptor gene; and iii) a suicide gene, wherein the genome of the cells has been modified to eliminate surface expression of at least one HLA-I or HLA-II molecule. A.iNKT cells

[0097] In certain embodiments, the engineered iNKT cells of the present disclosure are generated from other types of cells to enhance their activity as iNKT cells. iNKT cells are a small subpopulation of αβ T lymphocytes that possess several unique features that make them useful for off-the-shelf cell therapies, including at least cancer treatment. Non-iNKT cells are engineered to function as iNKT cells because iNKT cells offer the following advantages:

[0098] 1) iNKT cells have the remarkable ability to target multiple types of cancer, independent of tumor antigen and MHC restriction (Fujii et al., 2013). iNKT cells recognize glycolipid antigens presented by non-polymorphic CD1d and are therefore not restricted by MHC. Although the natural ligands of iNKT cells have not yet been identified, it has been suggested that iNKT cells may recognize certain conserved glycolipid antigens from many tumor tissues. iNKT cells recognize glycolipid antigens that are presented by non-polymorphic CD1d. + It can be directly presented by tumor cells or expressed by CD1d - In the case of tumors, iNKT cells can be stimulated by recognizing these glycolipid antigens that are indirectly cross-presented by tumor-infiltrating antigen-presenting cells (APCs), such as macrophages or dendritic cells (DCs). Thus, iNKT cells express CD1d + Tumor and CD1d - Both tumors may respond.

[0099] 2) iNKT cells can use multiple mechanisms to attack tumor cells (Vivier et al., 2012; Fujii et al., 2013). iNKT cells express CD1d + tumor Although iNKT cells can directly kill tumor cells by cytotoxicity, their most potent antitumor activity comes from their immunoadjuvant effect. iNKT cells remain quiescent before stimulation, but immediately produce large amounts of cytokines, primarily IFN-γ, after stimulation. IFN-γ activates NK cells to kill MHC-negative tumor target cells. Meanwhile, iNKT cells also activate DCs, which then stimulate CTLs to kill MHC-positive tumor target cells. Therefore, iNKT cell-induced antitumor immunity can effectively target multiple types of cancers, regardless of tumor antigen and MHC restriction, thereby effectively blocking tumor immune escape and minimizing the chance of tumor relapse.

[0100] 3) iNKT cells do not cause graft-versus-host disease (GvHD). Because iNKT cells do not recognize mismatched MHC molecules and protein self-antigens, it is predicted that these cells will not cause GvHD. This concept is strongly supported by clinical data analyzing donor-derived iNKT cells in patients with hematologic cancers who underwent allogeneic bone marrow or peripheral blood stem cell transplants. These clinical data showed that the level of engrafted allogeneic iNKT cells in patients correlated positively with the graft-versus-leukemia effect and negatively with GvHD (Haraguchi et al., 2004; de Lalla et al., 2011).

[0101] 4) iNKT cells can be engineered to avoid host-versus-graft (HvG) depletion. With the availability of powerful gene editing tools such as the CRISPR-Cas9 system, it is possible to genetically modify iNKT cells to make them resistant to targeted depletion by host immune cells: knockout of the beta2-microglobulin (B2M) gene eliminates the expression of HLA-I molecules in iNKT cells, resulting in the suppression of host CD8 + T cell-mediated killing is circumvented; knockout of the CIITA gene abolishes the expression of HLA-II molecules in iNKT cells, resulting in CD4 +Evading T cell-mediated killing. Both the B2M and CIITA genes are well-established targets for CRISPR-Cas9 systems in human primary cells (Ren et al., 2017; Abrahimi et al., 2015). Removal of HLA-I expression in iNKT cells allows them to become targets for host NK cells. However, iNKT cells appear to be naturally resistant to allogeneic NK cell killing. Nevertheless, if necessary, this concern can be addressed by delivering NK-inhibitory genes, such as HLA-E, to iNKT cells.

[0102] 5) iNKT cells have a strong association with cancer. There is compelling evidence suggesting a critical role for iNKT cells in tumor surveillance in mice, where defects in iNKT cells confer cancer susceptibility and adoptive transfer or stimulation of iNKT cells can confer protection against cancer (Vivier et al., 2012; Berzins et al., 2011). In humans, the frequency of iNKT cells is increased in solid tumors (melanoma, iNKT cell counts are decreased in patients with cancers of the colon, lung, breast, and head and neck, as well as hematological malignancies (including leukemia, multiple myeloma, and myelodysplastic syndromes), whereas increased numbers of iNKT cells are associated with a better prognosis (Berzins et al., 2011). In patients with lung cancer and head and neck cancer, α-GalCer loading Although administration of ex vivo expanded autologous iNKT cells and DCs has led to promising clinical benefits in some cases, the increase in iNKT cells was transient and the clinical benefit was short-lived. This may be due to the limited number of iNKT cells used for transfer and their subsequent depletion (Fujii et al., 2014). 2012; Yamasaki et al., 2011). It is therefore reasonable to advocate for an "off-the-shelf" iNKT cell product that allows patients to receive multiple doses of sufficient iNKT cells, providing them with the best opportunity to harness the full potential of their iNKT cells to fight disease.

[0103] However, the development of allogeneic, off-the-shelf iNKT cell products is significantly hindered by their availability: these cells are present in very low numbers in humans and are highly variable (approximately 0.001-1% in human blood), making it extremely difficult to grow therapeutic numbers of iNKT cells from allogeneic human donor blood cells. Therefore, novel methods that can reliably generate homogenous populations of iNKT cells in large quantities are key to the development of off-the-shelf iNKT cell therapies.

[0104] Given the lack of sufficient iNKT cells for this clinical application, embodiments of the present disclosure encompass engineering non-iNKT cells such that the resulting engineered cells function as iNKT cells. In certain embodiments, the cells that function as iNKT cells are further modified to have one or more desired characteristics. In certain embodiments, the non-iNKT cells are genetically modified by transducing them to express an iNKT T cell receptor (TCR). B. iNKT cells generated from HSCs

[0105] In embodiments of the present disclosure, iNKT cells generated from other types of cells are engineered to have one or more characteristics that make them suitable for universal use. In certain embodiments, the cells are genetically modified to contain at least one exogenous invariant natural killer T cell receptor (iNKT TCR) nucleic acid molecule. In some embodiments, the cells are hematopoietic stem cells. In some embodiments, the cells are hematopoietic progenitor cells. In some embodiments, the cells are human cells. In some embodiments, the cells are CD34 + In some embodiments, the cells are human CD34+ In some embodiments, the cell is a recombinant cell. In some embodiments, the cell is in culture.

[0106] In some embodiments, the iNKT TCR nucleic acid molecule is derived from a human invariant natural killer T cell. In some embodiments, the iNKT TCR nucleic acid molecule comprises one or more nucleic acid sequences obtained from a human iNKT TCR. In some embodiments, the iNKT TCR nucleic acid sequence can be obtained from any subset of iNKT cells, such as the CD4 / DN / CD8 subset or subsets that produce Th1, Th2, or Th17 cytokines and include double-negative iNKT cells. In some embodiments, the iNKT The TCR nucleic acid sequences are obtained from iNKT cells derived from donors who had or have cancer, e.g., melanoma, kidney cancer, lung cancer, prostate cancer, breast cancer, lymphoma, leukemia, hematological malignancies, etc. In some embodiments, the iNKT TCR nucleic acid molecule has a TCR-beta sequence from an iNKT cell that is different from the TCR-alpha sequence from one iNKT cell. In some embodiments, the iNKT cell from which the TCR-alpha sequence is obtained and the iNKT cell from which the TCR-beta sequence is obtained are derived from the same donor. In some embodiments, the donor of the iNKT cell from which the TCR-alpha sequence is obtained is different from the donor of the iNKT cell from which the TCR-beta sequence is obtained. In some embodiments, the TCR-alpha and / or TCR-beta sequences are codon-optimized for expression. In some embodiments, the TCR-alpha and / or TCR-beta sequences are modified to encode a polypeptide having one or more amino acid substitutions, deletions, and / or truncations compared to the polypeptide encoded by the unmodified sequences. In some embodiments, the iNKT TCR nucleic acid molecule encodes a T cell receptor that recognizes alpha-galactosylceramide (alpha-GalCer) presented on CD1d. In some embodiments, the iNKT TCR nucleic acid molecule comprises one or more sequences selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:63, and SEQ ID NO:64.In some embodiments, the iNKT TCR nucleic acid molecule encodes a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 32, SEQ ID NO: 35, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 47, SEQ ID NO: 50, SEQ ID NO: 53, SEQ ID NO: 56, SEQ ID NO: 59, SEQ ID NO: 62, and SEQ ID NO: 65. In some embodiments, the engineered cells lack an exogenous oncogene, such as, for example, Oct4, Sox2, Klf, c-Myc, etc.

[0107] In some embodiments, the engineered cells are functional iNKT cells. In some embodiments, the engineered cells are capable of producing one or more cytokines and / or chemokines, such as, for example, IFN-gamma, TNF-alpha, TGF-beta, GM-CSF, IL-2, IL-4, IL-5, IL-6, IL-10, IL-13, IL-17, IL-21, RANTES, eotaxin, MIP-1-alpha, MIP-1-beta, etc.

[0108] Donor HSPCs can be obtained from the donor's bone marrow, peripheral blood, amniotic fluid, or umbilical cord blood. The donor can be an autologous donor, i.e., a donor different from the subject receiving treatment with HSPC-iNKT cells, or an allogeneic donor, i.e., a donor different from the subject receiving treatment with HSPC-iNKT cells. In embodiments where the donor is an allogeneic donor, it is preferred that the tissue (HLA) type of the allogeneic donor matches that of the subject receiving treatment with HSPC-iNKT cells derived from the donor HSPCs.

[0109] According to the present disclosure, HSPCs are transduced with one or more exogenous iNKT TCR nucleic acid molecules. As used herein, an "iNKT TCR nucleic acid molecule" is a nucleic acid molecule encoding the alpha chain of the iNKT T cell receptor (TCR-alpha), the beta chain of the iNKT T cell receptor (TCR-beta), or both. As used herein, an "iNKT T cell receptor" is one that is expressed in iNKT cells and recognizes alpha-GalCer presented on CD1d. The TCR-alpha and TCR-beta sequences of iNKT TCRs can be cloned and / or recombinantly engineered using methods in the art. For example, iNKT cells can be obtained from a donor, and the TCR-alpha and TCR-beta genes of the iNKT cells can be cloned as described herein. The cloned iNKT TCR can be obtained from any mammal, including humans, non-human primates such as monkeys, mice, rats, hamsters, guinea pigs, and other rodents, rabbits, cats, dogs, horses, cows, sheep, goats, pigs, etc. In some embodiments, the cloned iNKT TCR is a human iNKT TCR. In some embodiments, the iNKT TCR clone comprises a human iNKT TCR sequence and a non-human iNKT TCR sequence.

[0110] In some embodiments, the cloned TCR may have a TCR-alpha chain derived from one iNKT cell and a TCR-beta chain derived from a different iNKT cell. In some embodiments, the iNKT cells from which the TCR-alpha chain is derived and the iNKT cells from which the TCR-beta chain is derived are derived from the same donor. In some embodiments, the donor of the iNKT cells from which the TCR-alpha chain is derived is different from the donor of the iNKT cells from which the TCR-beta chain is derived. In some embodiments, the sequence encoding the TCR-alpha chain and / or the sequence encoding the TCR-beta chain of the TCR clone is modified. In some embodiments, the modified sequence may encode the same polypeptide sequence as the unmodified TCR clone, e.g., the sequence is codon-optimized for expression. In some embodiments, the modified sequence may encode a polypeptide having a different sequence from the unmodified TCR clone, e.g., the modified sequence encodes a polypeptide sequence with one or more amino acid substitutions, deletions, and / or truncations. C. Imaging and depletion characteristics of HLA-negative HSC-iNKT cells

[0111] In certain embodiments, iNKT cells generated from HSPC cells are further modified to have one or more characteristics, including making the cells suitable for allogeneic use, or more suitable for allogeneic use than if the cells were not further modified to have one or more characteristics. U The present invention encompasses HSC-iNKT cells. In some embodiments, the HSC-iNKT cells are non-allo-reactive and express exogenous iNTK TCR. These cells are useful for "off-the-shelf" cell therapy, and do not require the use of the patient's own iNKT and other cells. Therefore, this method provides a more cost-effective and less labor-intensive cellular immunotherapy.

[0112] In certain embodiments, HSC-iNKT cells are engineered to be HLA-negative to achieve safe and successful allogeneic engraftment without causing graft-versus-host disease (GvHD) and without being rejected by host immune cells (HvG rejection). In certain embodiments, expression of the transgenic iNKT TCR gene blocks recombination of endogenous TCRs by allelic exclusion, so allogeneic HSC-iNKT cells do not express endogenous TCRs and do not cause GvHD. In certain embodiments, allogeneic HSC-iNKT cells U HSC-iNKT cells do not express HLA-I and / or HLA-II molecules on their cell surface and do not bind to host CD8 + and CD4 + Resistance to T cell-mediated allograft depletion and sr39TK immunogen-targeted depletion.

[0113] Thus, in certain embodiments, the engineered iNKT cells do not express surface HLA-I and surface HLA-II molecules, which is achieved by disrupting genes encoding proteins associated with HLA-I / II expression, including, but not limited to, beta2-microglobulin (B2M), major histocompatibility complex II transactivator (CIITA), or HLA-I / II molecules. In some cases, the iNKT cells do not express HLA-I or HLA-II on their surface because they have been engineered by gene editing, which may or may not involve CRISPR-Cas9.

[0114] In cases where the iNKT cells have been modified to exhibit any type of one or more characteristics, the iNKT cells may contain nucleic acid sequences from a recombinant vector introduced into the cells. The vector may be a non-viral vector, such as a plasmid, or a viral vector, such as a lentivirus, retrovirus, adeno-associated virus (AAV), herpesvirus, or adenovirus.

[0115] The iNKT cells of the present disclosure may or may not have been exposed to one or more specific conditions before, during, or after their production. In certain cases, the cells are not or have not been exposed to a medium containing animal serum. The cells can be frozen. The cells can be present in a solution containing dextrose, one or more electrolytes, albumin, dextran, and / or DMSO. Any solution in which the cells are present can be sterile, non-pyrogenic, and isotonic. The cells can be activated and expanded by any suitable method, such as activation with alpha-galactosylceramide (α-GC).

[0116] Aspects of the present disclosure include β2 microglobulin (B2M), CIITA, T The present invention relates to a human cell comprising: i) an exogenous expression or activity inhibitor of one or more of RAC, TRBC1, or TRBC2; or ii) a genomic mutation. In some embodiments, the cell comprises a genomic mutation. In some embodiments, the genomic mutation comprises a mutation of one or more endogenous genes in the genome of the cell, the one or more endogenous genes comprising B2M, CIITA, TRAC, TRBC1, or TRBC2 genes. In some embodiments, the mutation comprises a loss-of-function mutation. In some embodiments, the inhibitor is an expression inhibitor. In some embodiments, the inhibitor comprises an inhibitory nucleic acid. In some embodiments, the inhibitory nucleic acid comprises one or more of an siRNA, shRNA, miRNA, or antisense molecule. In some embodiments, the cell comprises an activity inhibitor. In some embodiments, after modification, the cell lacks any detectable expression of one or more of B2M, CIITA, TRAC, TRBC1, or TRBC2 proteins. In some embodiments, the cell comprises an inhibitor or genomic mutation of B2M. In some embodiments, the cell comprises an inhibitor or genomic mutation of CIITA. In some embodiments, the cells comprise an inhibitor or genomic mutation of TRAC. In some embodiments, the cells comprise an inhibitor or genomic mutation of TRBC1. In some embodiments, the cells comprise an inhibitor or genomic mutation of TRBC2. In some embodiments, at least 90% of the genomic DNA encoding B2M, CIITA, TRAC, TRBC1, and / or TRBC2 is deleted. In some embodiments, at least or up to 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% (or any range derivable therein) of the genomic DNA encoding B2M, CIITA, TRAC, TRBC1, and / or TRBC2 is deleted. In other embodiments, deletions, insertions, and / or substitutions are made within the genomic DNA. In some embodiments, the cells are progeny of human stem or progenitor cells.

[0117] modified to become HLA-negative UHSC-iNKT cells can be genetically modified in any suitable manner. Genetic mutations of the present disclosure, such as those in the CIITA and / or B2M genes, can be introduced by methods known in the art. In certain embodiments, engineered nucleases can be used to introduce exogenous nucleic acid sequences for genetic modification of any cell mentioned herein. Genome editing, or genome editing with engineered nucleases (GEEN), is a type of genetic engineering that uses artificially engineered nucleases, or "molecular scissors," to insert, replace, or remove DNA from a genome. The nuclease creates a specific double-strand break (DSB) at a desired location in the genome, and the cell's endogenous mechanisms are used to repair the induced break through the natural processes of homologous recombination (HR) and non-homologous end joining (NHEJ). Non-limiting examples of engineered nucleases include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), CRISPR / Cas9 systems, and homing endonucleases re-engineered from engineered meganucleases. Any engineered nuclease known in the art can be used in certain embodiments of the methods and compositions.

[0118] Engineered iNKT cells can be modified using methods that employ RNA interference. This is commonly performed in genetic analysis to understand gene or protein function, interfering with it in a sequence-specific manner, and monitoring its effects on the organism. However, in some organisms, site-directed mutagenesis is difficult or impossible to perform, and therefore more indirect methods, such as silencing the gene of interest with small RNA interference (siRNA), must be used. However, gene disruption by siRNA can be variable and incomplete. Genome editing with nucleases such as ZFNs differs from siRNA in that the DNA binding specificity of the engineered nuclease can be modified, thus, in principle, cleaving any targeted location within the genome and introducing endogenous sequence modifications into genes that cannot be specifically targeted by conventional RNAi. Furthermore, the specificity of ZFNs and TALENs is enhanced because two ZFNs are required to recognize their target moiety and then direct them to adjacent sequences.

[0119] Meganucleases can be used to modify engineered iNKT cells. Meganucleases are generally found in microbial species and have the unique property of having very long recognition sequences (>14 bp), making them naturally highly specific. This can be utilized to perform site-specific DSBs in genome editing. However, there is a challenge that sufficient meganucleases that cover all possible target sequences are not known, or may not become known at any time. To overcome this challenge, mutagenesis and high-throughput screening methods have been used to create meganuclease variants that recognize unique sequences. Others have been able to fuse various meganucleases to create hybrid enzymes that recognize new sequences. Still others have attempted to design sequence-specific meganucleases by modifying the amino acids of meganucleases that interact with DNA in a method called rationally designed meganuclease (U.S. Patent No. 8,021,867, incorporated herein by reference). Meganuclease has the advantage that it causes less toxicity in cells compared to methods such as ZFN, possibly due to the more stringent DNA sequence recognition.However, constructing sequence-specific enzymes for all possible sequences is costly and time-consuming, and does not benefit from the combinatorial possibilities that methods such as ZFN and TALEN utilize.Therefore, there are both advantages and disadvantages.

[0120] In contrast to meganucleases, the concept behind ZFNs and TALENs is largely based on nonspecific DNA-cleaving enzymes linked to peptides that recognize specific DNA sequences, such as zinc fingers and transcription activator-like effectors (TALEs). One approach has been to find endonucleases in which the DNA recognition site and cleavage site are separated from each other, a situation that is uncommon among restriction enzymes. Once this enzyme is found, the cleavage portion, which is highly nonspecific because it has no recognition ability, can be separated. This portion can then be linked to a peptide that recognizes a sequence, which can result in extremely high specificity. An example of a restriction enzyme with such properties is FokI. Furthermore, FokI has the advantage that it requires dimerization for nuclease activity, which means that specificity is dramatically increased because each nuclease partner recognizes a unique DNA sequence. To enhance this effect, FokI nucleases have been engineered that function only as heterodimers and have increased catalytic activity. Nucleases that function as heterodimers avoid the possibility of unwanted homodimeric activity and therefore increase DSB specificity.

[0121] Although the nuclease moieties of both ZFNs and TALENs have similar properties, the difference between these engineered nucleases is their DNA recognition peptide. ZFNs rely on Cys2-His2 zinc fingers, while TALENs rely on TALEs. Both of these DNA recognition peptide domains have the characteristic of naturally occurring in combination in their proteins. Cys2-His2 zinc fingers generally exist as repeats spaced 3 bp apart, and are found in various combinations in various nucleic acid-interacting proteins, such as transcription factors. On the other hand, TALEs are found as repeats with a 1:1 recognition ratio between amino acids and recognized nucleotide pairs. Both zinc fingers and TALEs exist in repeat patterns, and different combinations can be tried to create a wide variety of sequence specificities. Zinc fingers are more established in this regard, and other methods such as modular assembly (sequential attachment of zinc fingers that correlate with triplet sequences to cover the required sequence), OPEN (low stringency selection of peptide domains versus triplet nucleotides, followed by high stringency selection of peptide combinations versus the final target in a bacterial system), and bacterial one-hybrid screening of zinc finger libraries have been used to generate site-specific nucleases.

[0122] Therefore, embodiments of the present disclosure may or may not include targeting endogenous sequences to reduce or knock out the expression of one or more specific endogenous sequences.In certain embodiments, endogenous TCR rearrangement can be blocked by disrupting one or more of the following genes.For example, to produce guide RNA or iRNA, the sequences of the following genes are provided as examples:

[0123] B-2 microglobulin (B2M) (also known as IMD43) is located at 15q21.1 and has the following mRNA sequence:

[0124] [ka] [ka]

[0125] The human class II major histocompatibility complex transactivator (CIITA) gene is located at 16p13.13 and has the following mRNA sequence: [ka] [ka] [ka]

[0126] The human T cell receptor alpha chain (TRAC) mRNA sequence is as follows: [ka]

[0127] The human T cell receptor beta chain (TRBC1) mRNA sequence is as follows: [ka] [ka]

[0128] The human TRBC2 T cell receptor beta constant 2 (TCRB2) sequence is as follows: [ka] [ka]

[0129] In certain embodiments, inhibitory nucleic acids or any manner known in the art that inhibit gene expression of CIITA and / or B2M are contemplated. Examples of inhibitory nucleic acids include, but are not limited to, siRNA (small interfering RNA), short hairpin RNA (shRNA), double-stranded RNA, antisense oligonucleotides, ribozymes, and nucleic acids encoding them. Inhibitory nucleic acids can inhibit gene transcription in cells or prevent translation of gene transcripts. Inhibitory nucleic acids can be 16 to 1000 nucleotides in length, and in certain embodiments, 18 to 100 nucleotides in length. A nucleic acid can have at least or at most 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, 40, 50, 60, 70, 80, 90 nucleotides, or any range derivable therein. siRNAs that occur naturally in living animals are not "isolated," but synthetic siRNAs, or siRNAs that are partially or completely separated from coexisting materials in their natural state, are "isolated." An isolated siRNA can exist in a substantially purified form, or can exist in a non-native environment, such as, for example, a cell into which the siRNA has been delivered.

[0130] Inhibitory nucleic acid is well known in the art.For example, siRNA and double-stranded RNA are described in United States Patent (USP) No. 6,506,559 and No. 6,573,099, and United States Patent Publication No. 2003 / 0051263, No. 2003 / 0055020, No. 2004 / 0265839, No. 2002 / 0168707, No. 2003 / 0159161 and No. 2004 / 0064842, all of which are incorporated herein by reference.

[0131] In particular, an inhibitory nucleic acid may be one that is capable of reducing protein or mRNA expression by at least 10%, 20%, 30%, or 40%, more particularly at least 50%, 60%, or 70%, and most particularly at least 75%, 80%, 90%, 95% or more, or any range or value therebetween.

[0132] In further embodiments, there are synthetic nucleic acids that are protein inhibitors. The inhibitors can be 17-25 nucleotides in length and comprise a 5' to 3' sequence that is at least 90% complementary to the 5' to 3' sequence of a mature mRNA. In certain embodiments, the inhibitor molecule is 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, or any range derivable therein. Furthermore, the inhibitor molecule may be 90% or at least 90%, 91% or at least 91%, 92% or at least 92%, 93% or at least 93%, 94% or at least 94%, 95% or at least 95%, 96% or at least 96%, 97% or at least 97%, 98% or at least 98%, 99% or at least The sequence (5' to 3') is 99%, 99.1% or at least 99.1%, 99.2% or at least 99.2%, 99.3% or at least 99.3%, 99.4% or at least 99.4%, 99.5% or at least 99.5%, 99.6% or at least 99.6%, 99.7% or at least 99.7%, 99.8% or at least 99.8%, 99.9% or at least 99.9%, or 100% or at least 100% complementary, or any extent derivable therein. Those skilled in the art can use the part of the probe sequence that is complementary to the sequence of mature mRNA as the sequence of mRNA inhibitor. Furthermore, this part of the probe sequence can be modified so that it is still 90% complementary to the sequence of mature mRNA.

[0133] In cases where the engineered iNKT cells contain one or more suicide genes for subsequent depletion when needed, the suicide genes may be of any suitable type. The iNKT cells of the present disclosure may express a suicide gene product, which may be, for example, enzyme-based. Examples of suicide gene products include herpes simplex virus thymidine kinase (HSV-TK), purine nucleoside phosphorylase (PNP), cytosine deaminase (CD), carboxypeptidase G2, cytochrome P450, linamarase, beta-lactamase, nitroreductase (NTR), carboxypeptidase A, or inducible caspase 9. Thus, in certain cases, the suicide gene may encode a thymidine kinase (TK). In certain cases, the TK gene is a viral TK gene, such as the herpes simplex virus TK gene. In certain embodiments, the suicide gene product is activated by a substrate such as ganciclovir, penciclovir, or a derivative thereof.

[0134] In a particular embodiment, the suicide gene is sr39TK, and an example of the corresponding sequence is as follows:

[0135] sr39TK cDNA sequence (codon optimized): [ka]

[0136] sr39TK amino acid sequence: [ka] [ka]

[0137] In some embodiments, engineered iNKT cells can be detected by imaging or other methods. In certain cases, the cells contain exogenous nucleic acids encoding polypeptides with substrates that can be labeled for imaging, and the imaging can be fluorescent imaging, radioactive imaging, colorimetric imaging, etc. In certain cases, the cells are detected by positron emission tomography. In at least some cases, the cells express the sr39TK gene, which is a positron emission tomography (PET) reporter / thymidine kinase gene, allowing these genetically modified cells to be tracked by PET imaging and eliminated by the function of the sr39TK suicide gene.

[0138] A population of engineered iNKT cells is encompassed by the present disclosure. In certain embodiments, the iNKT clonal cells contain an exogenous nucleic acid encoding an iNKT T cell receptor (T cell receptor) and lack surface expression of one or more HLA-I or HLA-II molecules. The iNKT cells may contain an exogenous nucleic acid encoding a suicide gene, including a suicide gene based on an enzyme such as thymidine kinase (TK). The TK gene may be a viral TK gene, such as the herpes simplex virus TK gene. In the cells of the population, the suicide gene may be activated by a substrate, such as ganciclovir, penciclovir, or a derivative thereof. The cells may contain an exogenous nucleic acid encoding a polypeptide having a substrate that can be labeled for imaging; in some cases, the suicide gene product is a polypeptide having a substrate that can be labeled for imaging. In certain embodiments, the suicide gene is sr39TK.

[0139] In certain embodiments of the iNKT cell population, the iNKT cells do not express surface HLA-I and HLA-II molecules, for example, due to disruption of the expression of beta2-microglobulin (B2M), major histocompatibility complex class II transactivator (CIITA), and / or genes encoding HLA-I or HLA-II molecules. In certain cases, HLA-I or HLA-II molecules are not expressed on the cell surface of the iNKT cells because the iNKT cells have been engineered by gene editing. The gene editing may or may not involve CRISPR-Cas9.

[0140] In certain cases involving iNKT cell populations, the iNKT cells have been introduced with a nucleic acid sequence from a recombinant vector, such as a viral vector (including at least a lentivirus, retrovirus, adeno-associated virus (AAV), herpesvirus, or adenovirus).

[0141] In certain embodiments, the cells of the iNKT cell population may or may not have been exposed to one or more certain conditions. In certain cases, for example, the cells of the population have not been exposed to or have not been exposed to a medium containing animal serum. The cells of the population may or may not be frozen. In some cases, the cells of the population are present in a solution containing dextrose, one or more electrolytes, albumin, dextran, and / or DMSO. The solution may include dextrose, one or more electrolytes, albumin, dextran, and DMSO. The cells may be present in a sterile, non-pyrogenic, and isotonic solution. In certain cases, the iNKT cells are activated, such as activated with alpha-galactosylceramide (α-GC). In certain aspects, the cell population comprises at least about 10 clonal cells. 2 ~10 6 In some cases, the cell population comprises at least about 10 total cells. 6 ~10 12 It may include pieces.

[0142] In certain embodiments, an invariant natural killer T (iNKT) cell population comprising clonal iNKT cells comprising one or more exogenous nucleic acids encoding an iNKT T cell receptor (T cell receptor) and suicide thymidine kinase, wherein the clonal iNKT cells have been engineered not to express functional beta 2-microglobulin (B2M), major histocompatibility complex class II transactivator (CIITA), and / or HLA-I and HLA-II molecules, and wherein the cell population comprises at least about 10 total cells. 6 ~10 12 and the cloned cells are at least about 10 2 ~10 6 In some cases, the cells are frozen in solution. III. Cell Formulation and Culture

[0143] In certain embodiments, U At any stage in the process of generating HSC-iNKT cells, U The HSC-iNKT cells and / or their precursors can be specially formulated and / or cultured in a particular medium (whether or not present in an in vitro ATO culture system). The cells can be formulated in a manner suitable for delivery to a recipient without adverse effects.

[0144] In certain embodiments, the medium can be prepared using a medium used to culture animal cells as the basal medium, such as AIM V, X-VIVO-15, NeuroBasal, EGM2, TeSR, BME, BGJb, CMRL 1066, Glasgow MEM, Improved MEM Zinc Option, IMDM, Medium 199, Eagle's MEM, αMEM, DMEM, Ham's, RPMI-1640, and Fisher's medium, as well as any combination thereof, but the medium need not be particularly limited thereto as long as it can be used to culture animal cells. In particular, the medium can be xeno-free or chemically defined.

[0145] The medium may be serum-containing, serum-free, or xeno-free. To prevent contamination with components derived from different species of animals, the serum may be derived from the same animal as the stem cell(s). Serum-free medium refers to a medium that does not contain untreated or unpurified serum, and therefore may include a medium containing purified blood-derived components or animal tissue-derived components (such as growth factors).

[0146] The medium may or may not contain any serum substitute. Serum substitutes may include materials that suitably contain albumin (e.g., lipid-rich albumin, bovine albumin, albumin substitutes such as recombinant or humanized albumin, plant starch, dextran, and protein hydrolysates), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, or equivalents thereof. Serum substitutes can be prepared, for example, by the methods disclosed in International Publication No. 98 / 30679 (incorporated herein in its entirety). Alternatively, any commercially available material can be used for added convenience. Commercially available materials include Knockout Serum Replacer (KSR), Chemically Defined Lipid Concentrate (Gibco), and Glutamax (Gibco).

[0147] In further embodiments, the medium can be a serum-free medium suitable for cell development. For example, the medium can be supplemented with B-27® supplement, Xenofree B-27® supplement (available on the World Wide Web at thermofisher.com / us / en / home / technical-resources / media-formulation.250.html), NS21 supplement (Chen et al., J Neurosci Methods, 2008 Jun 30; 171 (2): 239-247, incorporated herein in its entirety). ), GS21™ supplement (available on the world wide web at amsbio.com / B-27.aspx), or a combination thereof, at a concentration effective to generate T cells from the 3D cell aggregates.

[0148] In certain embodiments, the medium may contain one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more of the following: vitamins such as biotin; DL-alpha tocopherol acetate; DL-alpha tocopherol; vitamin A (acetate); proteins such as BSA (bovine serum albumin) or human albumin, fatty acid free fraction V; catalase; human recombinant insulin; human transferrin; superoxide dismutase; other components such as corticosterone; D-galactose; ethanolamine HCl; glutathione (reduced); L-carnitine HCl; linoleic acid; linolenic acid; progesterone; putrescine 2HCl; sodium selenite; and / or T3 (triiodo-l-thyronine).

[0149] In some embodiments, the medium further comprises a vitamin. In some embodiments, the medium comprises one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or thirteen of the following (and any range derivable therein): biotin, DL-alpha tocopherol acetate, DL-alpha tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, folic acid, nicotinamide, pyridoxine, riboflavin, thiamine, inositol, vitamin B12, or the medium comprises a combination thereof or a salt thereof. In some embodiments, the medium comprises or consists essentially of biotin, DL-alpha tocopherol acetate, DL-alpha tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, folic acid, nicotinamide, pyridoxine, riboflavin, thiamine, inositol, and vitamin B12. In some embodiments, the vitamin comprises or consists essentially of biotin, DL-alpha tocopherol acetate, DL-alpha tocopherol, vitamin A, or a combination or salt thereof. In some embodiments, the medium further comprises a protein. In some embodiments, the protein comprises albumin or bovine serum albumin, a fraction of BSA, catalase, insulin, transferrin, superoxide dismutase, or a combination thereof. In some embodiments, the medium further comprises one or more of the following: corticosterone, D-galactose, ethanolamine, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, or triiodo-L-thyronine, or a combination thereof. In some embodiments, the medium comprises one or more of the following: B-27® supplement, XenoFree B-27® supplement, GS21™ supplement, or a combination thereof. In some embodiments, the medium comprises or further comprises amino acids, simple sugars, and inorganic ions. In some embodiments, the amino acids include arginine, cystine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine, or a combination thereof.In some embodiments, the inorganic ions include sodium, potassium, calcium, magnesium, nitrogen, or phosphorus, or combinations or salts thereof. In some embodiments, the medium further includes one or more of the following: molybdenum, vanadium, iron, zinc, selenium, copper, or manganese, or combinations thereof. In certain embodiments, the medium comprises or consists essentially of one or more vitamins discussed herein and / or one or more proteins discussed herein, and / or one or more of the following: corticosterone, D-galactose, ethanolamine, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, or triiodo-I-thyronine, B-27® supplement, XenoFree B-27® supplement, GS21™ supplement, amino acids (e.g., arginine, cystine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine), simple sugars, inorganic ions (e.g., sodium, potassium, calcium, magnesium, nitrogen, and / or phosphorus) or salts thereof, and / or molybdenum, vanadium, iron, zinc, selenium, copper, or manganese.

[0150] In further embodiments, the medium may include exogenously added ascorbic acid. The medium may also include one or more exogenously added fatty acids or lipids, amino acids (e.g., non-essential amino acids), vitamin(s), growth factors, cytokines, antioxidant substances, 2-mercaptoethanol, pyruvic acid, buffering agents, and / or inorganic salts.

[0151] One or more of the media components can be added at a concentration of at least, at most, or about 0.1, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 180, 200, 250 ng / L, ng / ml, μg / ml, mg / ml, or any range derivable therein.

[0152] The medium used can be supplemented with at least one exogenously added cytokine at a concentration of about 0.1 ng / mL to about 500 ng / mL, more particularly 1 ng / mL to 100 ng / mL, or at least, up to, or about 0.1, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 180, 200, 250 ng / L, ng / ml, μg / ml, mg / ml, or any range derivable therein. Suitable cytokines include, but are not limited to, FLT3 ligand (FLT3L), interleukin 7 (IL-7), stem cell factor (SCF), thrombopoietin (TPO), IL-2, IL-4, IL-6, IL-15, IL-21, TNF-alpha, TGF-beta, interferon-gamma, interferon-lambda, TSLP, thymopentin, pleiotrophin, and / or midkine. In particular, the culture medium may contain at least one of FLT3L and IL-7. More particularly, the culture may contain both FLT3L and IL-7.

[0153] Other culture conditions can be defined as appropriate. For example, the culture temperature can be about 20-40°C, e.g., at least, at most, or about 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, or 40°C (or any range derivable therein), although the temperature can be above or below these values. The CO2 concentration can be about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% (or any range derivable therein), e.g., about 2%-10%, e.g., about 2-5%, or any range derivable therein. The oxygen partial pressure can be at least or about 1%, 5%, 8%, 10%, 20%, or any range derivable therein.

[0154] In certain embodiments, the HLA-negative iNKT cells engineered from allogeneic HSCs are specially formulated. The HLA-negative iNKT cells engineered from allogeneic HSCs may or may not be formulated as a cell suspension. In certain cases, the HLA-negative iNKT cells engineered from allogeneic HSCs are formulated into a single-dose form. The HLA-negative iNKT cells engineered from allogeneic HSCs can be formulated for systemic or local administration. In some cases, the cells are formulated for storage prior to use, and the cell formulation may include one or more cryopreservatives, such as DMSO (e.g., 5% DMSO). The cell formulation may include albumin, including human albumin, with certain formulations including 2.5% human albumin. The cells can be formulated specifically for intravenous administration. For example, the cells are formulated for intravenous administration over a period of less than one hour. In certain embodiments, the cells are present as a formulated cell suspension that is stable at room temperature for 1 hour, 2 hours, 3 hours, or 4 hours or longer from the time of thawing.

[0155] In some embodiments, the method further comprises priming the T cells. In some embodiments, the T cells are primed with antigen-presenting cells. In some embodiments, the antigen-presenting cells present a tumor antigen.

[0156] In certain embodiments, U The exogenous TCR of the HSC-iNKT cells can have any defined antigen specificity. U The exogenous TCR of HSC-iNKT cells can be selected based on the absence or reduced alloreactivity of the intended recipient (e.g., certain virus-specific, xeno-specific, or cancer / testis antigen-specific TCRs). In instances where the exogenous TCR is non-alloreactive, rearrangement and / or expression of the endogenous TCR locus is suppressed during T cell differentiation by a developmental process called allelic exclusion, resulting in T cells that express only the non-alloreactive exogenous TCR and are therefore non-alloreactive. In some embodiments, the selection of the exogenous TCR may not necessarily be defined based on the absence of alloreactivity. In some embodiments, the endogenous TCR gene is modified by genome editing so that it does not express the protein. Methods for gene editing, such as those using the CRISPR / Cas9 system, are known in the art and are described herein.

[0157] In some embodiments, the isolated U The HSC-iNKT cells or populations thereof contain one or more chimeric antigen receptors (CARs). Examples of tumor cell antigens to which CARs can be directed include, at least, 5T4, 8H9, α vβ6 integrin, BCMA, B7-H3, B7-H6, CAIX, CA9, CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, ERBB3, ERBB4, ErbB3 / 4, EPCAM, EphA2, EpCAM, folate receptor-a, FAP, FBP, fetal AchR, FRα, GD2, G250 / CAIX, GD3, glypican-3 (GPC3), Her2, IL-13Rα2, lambda, Lewis-Y, kappa, KDR, MAGE, MCSP, mesothelin, Muc1, Muc16, NCAM, NKG2D ligand, NY-ESO-1, PRAME, PSC1, PSCA, PSMA, ROR1, SP17, survivin, TAG72, TEM, carcinoembryonic antigen, HMW-MAA, AFP, CA-125, ETA, tyrosinase, MAGE, laminin receptor, HPV CARs include E6, E7, BING-4, calcium-activated chloride channel 2, cyclin-B1, 9D7, EphA3, telomerase, SAP-1, BAGE family, CAGE family, GAGE ​​family, MAGE family, SAGE family, XAGE family, NY-ESO-1 / LAGE-1, PAME, SSX-2, MelanA / MART-1, GP100 / pmel17, TRP-1 / -2, P. polypeptide, MC1R, prostate-specific antigen, β-catenin, BRCA1 / 2, CML66, fibronectin, MART-2, TGF-βRII, or VEGF receptor (e.g., VEGFR2). CARs can be first-generation, second-generation, third-generation, or later-generation CARs. CARs can be bispecific to any two non-identical antigens, or CARs can be specific to more than two non-identical antigens. IV. Additional Modifications and Polypeptide Embodiments

[0158] Additionally, the polypeptides of the present disclosure can be chemically modified, for example, to alter the glycosylation of a polypeptide by altering one or more sites of glycosylation within the polypeptide sequence to increase the affinity of the polypeptide for an antigen (U.S. Patent Nos. 5,714,350 and 6,350,861).

[0159] Polypeptides of the disclosure or regions or fragments of nucleic acids of the disclosure encoding polypeptides can be substituted with any of SEQ ID NOs: 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, or 71, or with any of SEQ ID NOs: 1-19, 21, 22, 24, 25, 27, 28, 30, 31, 33, 34, 36, 37, 39, 40, 42, 43, 45, 46, 48, 49, 51, 52, 54, 55, 57, 58, 60, 61, 63, 64, 66-70, or 72-74. 1, at least 1 or at most 1, 2, at least 2 or at most 2, 3, at least 3 or at most 3, 4, at least 4 or at most 4, 5, at least 5 or at most 5, 6, at least 6 or at most 6, 7, at least 7 or at most 7, 8, at least 8 or at most 8, 9, at least 9 or at most 9, 10, at least 10 or at most 10, 11, or at least 11 or a maximum of 11, 12, at least 12 or a maximum of 12, 13, at least 13 or a maximum of 13, 14, at least 14 or a maximum of 14, 15, at least 15 or a maximum of 15, 16, at least 16 or a maximum of 16, 17, at least 17 or a maximum of 17, 18, at least 18 or a maximum of 18, 19, at least 19 or a maximum of 19, 20, at least 20 or a maximum At most 20, 21, at least 21 or a maximum of 21, 22, at least 22 or a maximum of 22, 23, at least 23 or a maximum of 23, 24, at least 24 or a maximum of 24, 25, at least 25 or a maximum of 25, 26, at least 26 or a maximum of 26, 27, at least 27 or a maximum of 27, 28, at least 28 or a maximum of 28, 29, at least 29 or a maximum of 29, 30,At least 30 or a maximum of 30, 31, at least 31 or a maximum of 31, 32, at least 32 or a maximum of 32, 33, at least 33 or a maximum of 33, 34, at least 34 or a maximum of 34, 35, at least 35 or a maximum of 35, 36, at least 36 or a maximum of 36, 37, at least 37 or a maximum of 37, 38, at least 38 or a maximum of 38, 39, at least 39 or Up to 39 locations, 40 locations, at least 40 locations or up to 40 locations, 41 locations, at least 41 locations or up to 41 locations, 42 locations, at least 42 locations or up to 42 locations, 43 locations, at least 43 locations or up to 43 locations, 44 locations, at least 44 locations or up to 44 locations, 45 locations, at least 45 locations or up to 45 locations, 46 locations, at least 46 locations or up to 46 locations, 47 locations, at least 47 locations or up to 47 locations, 48 ​​locations, at least 48 locations or up to 48 locations, 49 locations, At least 49 or a maximum of 49, 50, at least 50 or a maximum of 50, 51, at least 51 or a maximum of 51, 52, at least 52 or a maximum of 52, 53, at least 53 or a maximum of 53, 54, at least 54 or a maximum of 54, 55, at least 55 or a maximum of 55, 56, at least 56 or a maximum of 56, 57, at least 57 or a maximum of 57, 58, at least 58 or At most 58 locations, 59 locations, at least 59 locations or a maximum of 59 locations, 60 locations, at least 60 locations or a maximum of 60 locations, 61 locations, at least 61 locations or a maximum of 61 locations, 62 locations, at least 62 locations or a maximum of 62 locations, 63 locations, at least 63 locations or a maximum of 63 locations, 64 locations, at least 64 locations or a maximum of 64 locations, 65 locations, at least 65 locations or a maximum of 65 locations, 66 locations, at least 66 locations or a maximum of 66 locations, 67 locations, at least 67 locations or a maximum of 67 locations, 68 locations,At least 68 locations or a maximum of 68 locations, 69 locations, at least 69 locations or a maximum of 69 locations, 70 locations, at least 70 locations or a maximum of 70 locations, 71 locations, at least 71 locations or a maximum of 71 locations, 72 locations, at least 72 locations or a maximum of 72 locations, 73 locations, at least 73 locations or a maximum of 73 locations, 74 locations, at least 74 locations or a maximum of 74 locations, 75 locations, at least 75 locations or a maximum of 75 locations, 76 locations , at least 76 or a maximum of 76, 77, at least 77 or a maximum of 77, 78, at least 78 or a maximum of 78, 79, at least 79 or a maximum of 79, 80, at least 80 or a maximum of 80, 81, at least 81 or a maximum of 81, 82, at least 82 or a maximum of 82, 83, at least 83 or a maximum of 83, 84 , at least 84 or a maximum of 84, 85, at least 85 or a maximum of 85, 86, at least 86 or a maximum of 86, 87, at least 87 or a maximum of 87, 88, at least 88 or a maximum of 88, 89, at least 89 or a maximum of 89, 90, at least 90 or a maximum of 90, 91, at least 91 or a maximum of 91, 9 2, at least 92 or a maximum of 92, 93, at least 93 or a maximum of 93, 94, at least 94 or a maximum of 94, 95, at least 95 or a maximum of 95, 96, at least 96 or a maximum of 96, 97, at least 97 or a maximum of 97, 98, at least 98 or a maximum of 98, 99, at least 99 or a maximum of 99, 100 locations, at least 100 locations or a maximum of 100 locations, 101 locations, at least 101 locations or a maximum of 101 locations, 102 locations, at least 102 locations or a maximum of 102 locations, 103 locations, at least 103 locations or a maximum of 103 locations, 104 locations, at least 104 locations or a maximum of 104 locations, 105 locations, at least 105 locations or a maximum of 105 locations, 106 locations, at least 106 locations or a maximum of 106 locations, 107 locations, at least 107 locations or a maximum of 107 locations, 108 locations, at least 108 locations or at most 108, 109, at least 109 or at most 109, 110, at least 110 or at most 110, 111, at least 111 or at most 111, 112, at least 112 or at most 112, 113, at least 113 or at most 113, 114, at least 114 or at most 114, 115, at least 115 or at most 115, 116, at least 116 or at most 116, 117 , at least 117 or a maximum of 117, 118, at least 118 or a maximum of 118, 119, at least 119 or a maximum of 119, 120, at least 120 or a maximum of 120, 121, at least 121 or a maximum of 121, 122, at least 122 or a maximum of 122, 123, at least 123 or a maximum of 123, 124, at least 124 or a maximum of 124, 125, at least 125 or is at most 125, 126, at least 126 or at most 126, 127, at least 127 or at most 127, 128, at least 128 or at most 128, 129, at least 129 or at most 129, 130, at least 130 or at most 130, 131, at least 131 or at most 131, 132, at least 132 or at most 132, 133, at least 133 or at most 133, 134,At least 134 or a maximum of 134, 135, at least 135 or a maximum of 135, 136, at least 136 or a maximum of 136, 137, at least 137 or a maximum of 137, 138, at least 138 or a maximum of 138, 139, at least 139 or a maximum of 139, 140, at least 140 or a maximum of 140, 141, at least 141 or a maximum of 141, 142, at least 142 or At most 142 locations, 143 locations, at least 143 locations or at most 143 locations, 144 locations, at least 144 locations or at most 144 locations, 145 locations, at least 145 locations or at most 145 locations, 146 locations, at least 146 locations or at most 146 locations, 147 locations, at least 147 locations or at most 147 locations, 148 locations, at least 148 locations or at most 148 locations, 149 locations, at least 149 locations or at most 149 locations, 150 locations, at least 150 locations or at most 150 locations, 151 locations, At least 151 or a maximum of 151, 152, at least 152 or a maximum of 152, 153, at least 153 or a maximum of 153, 154, at least 154 or a maximum of 154, 155, at least 155 or a maximum of 155, 156, at least 156 or a maximum of 156, 157, at least 157 or a maximum of 157, 158, at least 158 ​​or a maximum of 158, 159, at least 159 or At most 159 locations, 160 locations, at least 160 locations or a maximum of 160 locations, 161 locations, at least 161 locations or a maximum of 161 locations, 162 locations, at least 162 locations or a maximum of 162 locations, 163 locations, at least 163 locations or a maximum of 163 locations, 164 locations, at least 164 locations or a maximum of 164 locations, 165 locations, at least 165 locations or a maximum of 165 locations, 166 locations, at least 166 locations or a maximum of 166 locations, 167 locations, at least 167 locations or a maximum of 167 locations, 168 locations,At least 168 locations or a maximum of 168 locations, 169 locations, at least 169 locations or a maximum of 169 locations, 170 locations, at least 170 locations or a maximum of 170 locations, 171 locations, at least 171 locations or a maximum of 171 locations, 172 locations, at least 172 locations or a maximum of 172 locations, 173 locations, at least 173 locations or a maximum of 173 locations, 174 locations, at least 174 locations or a maximum of 174 locations, 175 locations, at least 175 locations or a maximum of 175 locations, 176 locations, at least 176 locations or at most 176, 177, at least 177 or at most 177, 178, at least 178 or at most 178, 179, at least 179 or at most 179, 180, at least 180 or at most 180, 181, at least 181 or at most 181, 182, at least 182 or at most 182, 183, at least 183 or at most 183, 184, at least 184 or at most 184, 18 5 locations, at least 185 locations or a maximum of 185 locations, 186 locations, at least 186 locations or a maximum of 186 locations, 187 locations, at least 187 locations or a maximum of 187 locations, 188 locations, at least 188 locations or a maximum of 188 locations, 189 locations, at least 189 locations or a maximum of 189 locations, 190 locations, at least 190 locations or a maximum of 190 locations, 191 locations, at least 191 locations or a maximum of 191 locations, 192 locations, at least 192 locations or a maximum of 192 locations, 193 locations, at least 193 locations or at most 193, 194, at least 194 or at most 194, 195, at least 195 or at most 195, 196, at least 196 or at most 196, 197, at least 197 or at most 197, 198, at least 198 or at most 198, 199, at least 199 or at most 199, 200, at least 200 or at most 200 or more amino acid substitutions, consecutive amino acid additions,Alternatively, it is intended that the amino acid sequence may have a sequence of consecutive amino acid deletions.

[0160] Alternatively, a region or fragment of a polypeptide of the present disclosure may be 50%, at least 50%, or at most identical to any of SEQ ID NOs: 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, or 71, or to a polypeptide encoded by any of SEQ ID NOs: 1-19, 21, 22, 24, 25, 27, 28, 30, 31, 33, 34, 36, 37, 39, 40, 42, 43, 45, 46, 48, 49, 51, 52, 54, 55, 57, 58, 60, 61, 63, 64, 66-70, or 72-74. 50%, 51%, at least 51% or at most 51%, 52%, at least 52% or at most 52%, 53%, at least 53% or at most 53%, 54%, at least 54% or at most 54%, 55%, at least 55% or at most 55%, 56%, at least 56% or at most 56%, 57%, at least 57% or at most 57%, 58%, at least 58% or at most 58%, 59%, at least 59% or at most 59%, 60%, at least 60% or at most 60%, 61%, at least 61% or or at most 61%, 62%, at least 62% or at most 62%, 63%, at least 63% or at most 63%, 64%, at least 64% or at most 64%, 65%, at least 65% or at most 65%, 66%, at least 66% or at most 66%, 67%, at least 67% or at most 67%, 68%, at least 68% or at most 68%, 69%, at least 69% or at most 69%, 70%, at least 70% or at most 70%, 71%, at least 71% or at most 71%, 72%, at least 72% or at most 72%, 73%, at least 73% or at most 73%, 74%, at least 74% or at most 74%, 75%, at least 75% or at most 75%, 76%, at least 76% or at most 76%, 77%, at least 77% or at most 77%, 78%, at least 78% or at most 78%, 79%, at least 79% or at most 79%, 80%, at least 80% or at most 80%, 81%, at least 81% or at most 81%, 82%, at least 82% or at most 82%, 83%,at least 83% or at most 83%, 84%, at least 84% or at most 84%, 85%, at least 85% or at most 85%, 86%, at least 86% or at most 86%, 87%, at least 87% or at most 87%, 88%, at least 88% or at most 88%, 89%, at least 89% or at most 89%, 90%, at least 90% or at most 90%, 91%, at least 91% or at most 91%, 92%, at least 92% or at most 92%, 93%, at least or may have an amino acid sequence that comprises or consists of an amino acid sequence that is 93% or at most 93%, 94%, at least 94% or at most 94%, 95%, at least 95% or at most 95%, 96%, at least 96% or at most 96%, 97%, at least 97% or at most 97%, 98%, at least 98% or at most 98%, 99%, at least 99% or at most 99%, 100%, at least 100% or at most 100% (or any range derivable therein) identical to the amino acid sequence of the present invention. Furthermore, in some embodiments, the region or fragment is selected from the group consisting of positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 24, 25, 27, 28, 30, 31, 33, 34, 36, 37, 39, 40, 42, 43, 45, 46, 48, 49, 51, 52, 54, 55, 57, 58, 60, 61, 63, 64, 66-70, and 72-74. 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, 30th, 31st, 32nd, 33rd, 34th, 35th, 36th, 37th, 38th, 39th, 40th, 41st, 42nd, 43rd, 44th, 45th, 46th , 47th, 48th, 49th, 50th, 51st, 52nd, 53rd, 54th, 55th, 56th, 57th, 58th, 59th, 60th, 61st, 62nd, 6 3rd place, 64th place, 65th place, 66th place, 67th place, 68th place, 69th place, 70th place, 71st place, 72nd place, 73rd place, 74th place, 75th place, 76th place, 77th place, 78th place, 79th place,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, 134th, 135th, 136th, 137th, 138th, 139th, 140th, 141st, 142nd, 143rd, 144th, 145th, 146th, 147th, 148th, 149th, 150th, 151st, 152nd, 153rd, 154th, 155th, 156th, 157th, 158th, 159th, 160th, 161st, 162nd, 163rd, 164th, 165th, 166th, 167th, 168th, 169th, 170th, 171st, 172nd, 173rd, 174th, 175th, 176th, 177th, 178th, 179th, 180th, 181st, 182nd, 183rd, 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,Starting from 484th, 485th, 486th, 487th, 488th, 489th, 490th, 491st, 492nd, 493rd, 494th, 495th, 496th, 497th, 498th, 499th, 500th, 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, 1 24, 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, 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 pieces, 425 pieces, 426 pieces, 427 pieces, 428 pieces, 429 pieces, 430 pieces, 431 pieces, 432 pieces, 433 pieces, 434 pieces, 435 pieces, 436 pieces, 437 pieces, 438 pieces, 439 pieces, 440 pieces, 441 pieces, 442 pieces, 443 pieces, 444 pieces, 445 pieces, 446 pieces, 447 pieces, 448 pieces, 449 pieces, 450 pieces, 451 pieces, 452 pieces, 453 pieces, 454 pieces, 455 pieces, 456 pieces, 457 pieces, 458 pieces, 459 pieces, 460 pieces, 461 pieces, 462 pieces, 463 pieces, 464 pieces, 465 pieces, 466 pieces, 467 pieces, 468 pieces, 469 pieces, and / or a region of amino acids that is 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, 500 or more consecutive amino acids (position 1 is the N-terminus of the SEQ ID NO or the N-terminus of the polypeptide encoded by the SEQ ID NO). The polypeptides of the present disclosure may have 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, 7, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 or more bars or may contain at least or up to 3, 4, 5, 6, 7, 8, 9, 10, 11, or more of a variant amino acid or nucleic acid substitution in a polypeptide encoded by any of SEQ ID NOs: 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, or 71, or any of SEQ ID NOs: 1-19, 21, 22, 24, 25, 27, 28, 30, 31, 33, 34, 36, 37, 39, 40, 42, 43, 45, 46, 48, 49, 51, 52, 54, 55, 57, 58, 60, 61, 63, 64, 66-70, or 72-74,12, 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 4, 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, 1 04, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 1 29, 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, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 300, 400, 500, 550, 1000, 1500, or 2000 or more consecutive amino acids, or any range derivable therein; is at least 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% similar, identical, or homologous to the nucleic acid.

[0161] Polypeptides of the disclosure may contain 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, 1 47 locations, 48 ​​locations, 49 locations, 50 locations, 51 locations, 52 locations, 53 locations, 54 locations, 55 locations, 56 locations, 57 locations, 58 locations, 59 locations, 60 locations, 61 locations, 62 locations, 63 locations, 64 locations, 65 locations, 66 locations, 67 locations, 68 locations, 69 locations, 70 locations, 71 locations, 72 locations, 73 locations, 74 locations, 75 locations, 76 locations, 77 locations, 78 locations, 79 locations, 80 locations, 81 locations, 82 locations, 83 locations, 84 locations, 85 locations, 86 locations, 87 locations, 88 locations, 89 locations, 90 locations, 91 locations, 92 locations, 93 locations, 94 locations, 95 locations, 96 locations, 97 locations, 98 locations, 99 locations, 100 locations, 101 locations, 102 locations, 103 locations, 104 locations, 105 locations, 106 locations, 107 locations, 108 locations, 109 locations, 110 locations, 111 locations, 112 locations, 113 locations, 114 locations, 115 locations, 116 locations, 117 locations, 11 8 locations, 119 locations, 120 locations, 121 locations, 122 locations, 123 locations, 124 locations, 125 locations, 126 locations, 127 locations, 128 locations, 129 locations, 130 locations, 131 locations, 132 locations, 133 locations, 134 locations, 135 locations, 136 locations, 137 locations, 138 locations, 1 39 locations, 140 locations, 141 locations, 142 locations, 143 locations, 144 locations, 145 locations, 146 locations, 147 locations, 148 locations, 149 locations, 150 locations, 151 locations, 152 locations, 153 locations, 154 locations, 155 locations, 156 locations, 157 locations, 158 locations, 159 locations, 160 locations, 161 locations, 162 locations, 163 locations, 164 locations, 165 locations, 166 locations, 167 locations, 168 locations, 169 locations, 170 locations, 171 locations, 172 locations, 173 locations, 174 locations, 175 locations, 176 locations, 177 locations, 178 locations, 179 locations, 180 locations,181 locations, 182 locations, 183 locations, 184 locations, 185 locations, 186 locations, 187 locations, 188 locations, 189 locations, 190 locations, 191 locations, 192 locations, 193 locations, 194 locations, 195 locations, 196 locations, 197 locations, 198 locations, 199 locations, 200 locations, 201 locations , 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222 223 locations, 224 locations, 225 locations, 226 locations, 227 locations, 228 locations, 229 locations, 230 locations, 231 locations, 232 locations, 233 locations, 234 locations, 235 locations, 236 locations, 237 locations, 238 locations, 239 locations, 240 locations, 241 locations, 242 locations, 2 43 locations, 244 locations, 245 locations, 246 locations, 247 locations, 248 locations, 249 locations, 250 locations, 251 locations, 252 locations, 253 locations, 254 locations, 255 locations, 256 locations, 257 locations, 258 locations, 259 locations, 260 locations, 261 locations, 262 locations, 263 locations, 264 locations, 265 locations, 266 locations, 267 locations, 268 locations, 269 locations, 270 locations, 271 locations, 272 locations, 273 locations, 274 locations, 275 locations, 276 locations, 277 locations, 278 locations, 279 locations, 280 locations, 281 locations, 282 locations, 283 locations, 284 locations 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, 3 26 locations, 327 locations, 328 locations, 329 locations, 330 locations, 331 locations, 332 locations, 333 locations, 334 locations, 335 locations, 336 locations, 337 locations, 338 locations, 339 locations, 340 locations, 341 locations, 342 locations, 343 locations, 344 locations, 345 locations, 346 locations,347 locations, 348 locations, 349 locations, 350 locations, 351 locations, 352 locations, 353 locations, 354 locations, 355 locations, 356 locations, 357 locations, 358 locations, 359 locations, 360 locations, 361 locations, 362 locations, 363 locations, 364 locations, 365 locations, 366 locations, 367 locations , 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388 389 places, 390 places, 391 places, 392 places, 393 places, 394 places, 395 places, 396 places, 397 places, 398 places, 399 places, 400 places, 401 places, 402 places, 403 places, 404 places, 405 places, 406 places, 407 places, 408 places, 4 09 locations, 410 locations, 411 locations, 412 locations, 413 locations, 414 locations, 415 locations, 416 locations, 417 locations, 418 locations, 419 locations, 420 locations, 421 locations, 422 locations, 423 locations, 424 locations, 425 locations, 426 locations, 427 locations, 428 locations, 429 locations, 430 locations, 431 locations, 432 locations, 433 locations, 434 locations, 435 locations, 436 locations, 437 locations, 438 locations, 439 locations, 440 locations, 441 locations, 442 locations, 443 locations, 444 locations, 445 locations, 446 locations, 447 locations, 448 locations, 449 locations, 450 locations 451 locations, 452 locations, 453 locations, 454 locations, 455 locations, 456 locations, 457 locations, 458 locations, 459 locations, 460 locations, 461 locations, 462 locations, 463 locations, 464 locations, 465 locations, 466 locations, 467 locations, 468 locations, 469 locations, 470 locations, 471 472 locations, 473 locations, 474 locations, 475 locations, 476 locations, 477 locations, 478 locations, 479 locations, 480 locations, 481 locations, 482 locations, 483 locations, 484 locations, 485 locations, 486 locations, 487 locations, 488 locations, 489 locations, 490 locations, 491 locations, 4 92 locations, 493 locations, 494 locations, 495 locations, 496 locations, 497 locations, 498 locations, 499 locations, 500 locations, 501 locations, 502 locations, 503 locations, 504 locations, 505 locations, 506 locations, 507 locations, 508 locations, 509 locations, 510 locations, 511 locations, 512 locations,513 locations, 514 locations, 515 locations, 516 locations, 517 locations, 518 locations, 519 locations, 520 locations, 521 locations, 522 locations, 523 locations, 524 locations, 525 locations, 526 locations, 527 locations, 528 locations, 529 locations, 530 locations, 531 locations, 532 locations, 533 locations, 534 locations, 535 locations, 536 locations, 537 locations, 538 locations, 539 locations, 540 locations, 541 locations, 542 locations, 543 locations, 544 locations, 545 locations, 546 locations, 547 locations, 548 locations, 549 locations, 550 locations, 551 locations, 552 locations, 553 locations, 554 locations, 555 locations, 556 locations, 557 locations, 558 locations, 559 locations, 560 locations, 561 locations, 562 locations, 563 locations, 564 locations, 565 locations, 566 locations, 567 locations, 568 locations, 569 locations, 570 locations, 571 locations, 572 locations, 573 locations, 574 locations, 575 locations, 576 locations, 577 locations, 578 locations, 579 locations, 580 locations, 581 locations, 582 locations, 583 locations, 584 locations, 585 locations, 586 locations, 587 locations, 588 locations, 589 locations, 590 locations, 591 locations, 592 locations, 593 locations, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, or 615 substitutions (or any range derivable therein).

[0162] The substitutions are at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 24, 25, 27, 28, 30, 31, 33, 34, 36, 37, 39, 40, 42, 43, 45, 46, 48, 49, 51, 52, 54, 55, 57, 58, 60, 61, 63, 64, 66-70, or 72-74 of a polypeptide encoded by any of SEQ ID NOs: 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, or 71, or any of SEQ ID NOs: 1-19, 21, 22, 24, 25, 27, 28, 30, 31, 33, 34, 36, 37, 39, 40, 42, 43, 45, 46, 48, 49, 51, 52, 54, 55, 57, 58, 60, 61, 63, 64, 66-70, or 72-74. 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th 30th place, 31st place, 32nd place, 33rd place, 34th place, 35th place, 36th place, 37th place, 38th place, 39th place, 40th place, 41st place, 42nd place, 43rd place, 44th place, 4th place 5th, 46th, 47th, 48th, 49th, 50th, 51st, 52nd, 53rd, 54th, 55th, 56th, 57th, 58th, 59th, 60th , 61st, 62nd, 63rd, 64th, 65th, 66th, 67th, 68th, 69th, 70th, 71st, 72nd, 73rd, 74th, 75th, 76th 77th, 78th, 79th, 80th, 81st, 82nd, 83rd, 84th, 85th, 86th, 87th, 88th, 89th, 90th, 91st, 92nd, 93rd, 94th, 95th, 96th, 97th, 98th, 99th, 100th, 101st, 102nd, 103rd, 104th, 105th, 106th, 107th, 108th, 109th, 110th, 111th, 112th, 113th, 114th, 115th, 116th, 117th, 11th 8th, 119th, 120th, 121st, 122nd, 123rd, 124th, 125th, 126th, 127th, 128th, 129th, 130th, 131st, 132nd, 133rd, 134th, 135th, 136th, 137th, 138th, 139th, 140th, 141st, 142nd, 14 3rd place, 144th place, 145th place, 146th place, 147th place, 148th place, 149th place, 150th place, 151st place, 152nd place, 153rd place, 154th place, 155th place, 156th, 157th, 158th, 159th, 160th, 161st, 162nd, 163rd, 164th, 165th, 166th, 167th, 16th 8th, 169th, 170th, 171st, 172nd, 173rd, 174th, 175th, 176th, 177th, 178th, 179th, 180th,181st, 182nd, 183rd, 184th, 185th, 186th, 187th, 188th, 189th, 190th, 191st, 192nd, 193rd, 194th, 195th, 196th, 197th, 198th, 199th, 200th, 201st, 202nd, 203rd, 204th, 205th, 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-bit, 257-bit, 258-bit, 259-bit, 260-bit, 261-bit, 262-bit, 263-bit, 264-bit, 265-bit, 266-bit, 267-bit, 268-bit, 269-bit, 270-bit, 271-bit, 272-bit, 273-bit, 274-bit, 275-bit, 276-bit, 277-bit, 278-bit, 279-bit, 280-bit, 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, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580,The amino acid sequence may be at amino acid positions 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 650, 700, 750, 800, 850, 900, 1000, 1500, or 2000 (or any range derivable therein).

[0163] The polypeptides described herein may comprise at least, at most, or exactly 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 24, 25, 27, 28, 30, 31, 33, 34, 36, 37, 39, 40, 42, 43, 45, 46, 48, 49, 51, 52, 54, 55, 57, 58, 60, 61, 63, 64, 66-70, or 72-74. 1 piece, 12 pieces, 13 pieces, 14 pieces, 15 pieces, 16 pieces, 17 pieces, 18 pieces, 19 pieces, 20 pieces, 21 pieces, 22 pieces, 23 pieces, 24 pieces, 25 pieces, 26 pieces , 27 pieces, 28 pieces, 29 pieces, 30 pieces, 31 pieces, 32 pieces, 33 pieces, 34 pieces, 35 pieces, 36 pieces, 37 pieces, 38 pieces, 39 pieces, 40 pieces, 41 pieces, 42 pieces, 43 pieces, 44 pieces, 45 pieces, 46 pieces, 47 pieces, 48 ​​pieces, 49 pieces, 50 pieces, 51 pieces, 52 pieces, 53 pieces, 54 pieces, 55 pieces, 56 pieces, 57 pieces, 58 pieces, 59 pieces, 60 pieces, 61 pieces, 62 pieces, 63 pieces, 64 pieces, 65 pieces, 66 pieces, 67 pieces, 68 pieces, 69 pieces, 70 pieces, 71 pieces, 72 pieces, 73 pieces , 74 pieces, 75 pieces, 76 pieces, 77 pieces, 78 pieces, 79 pieces, 80 pieces, 81 pieces, 82 pieces, 83 pieces, 84 pieces, 85 pieces, 86 pieces, 87 pieces, 88 pieces, 8 9 pieces, 90 pieces, 91 pieces, 92 pieces, 93 pieces, 94 pieces, 95 pieces, 96 pieces, 97 pieces, 98 pieces, 99 pieces, 100 pieces, 101 pieces, 102 pieces, 103 pieces, 104 pieces, 105 pieces, 106 pieces, 107 pieces, 108 pieces, 109 pieces, 110 pieces, 111 pieces, 112 pieces, 113 pieces, 114 pieces, 115 pieces, 11 6 pieces, 117 pieces, 118 pieces, 119 pieces, 120 pieces, 121 pieces, 122 pieces, 123 pieces, 124 pieces, 125 pieces, 126 pieces, 127 pieces, 128 pieces, 129 pieces, 130 pieces, 131 pieces, 132 pieces, 133 pieces, 134 pieces, 135 pieces, 136 pieces, 137 pieces, 138 pieces, 139 pieces, 140 pieces, 14 1 piece, 142 pieces, 143 pieces, 144 pieces, 145 pieces, 146 pieces, 147 pieces, 148 pieces, 149 pieces, 150 pieces, 151 pieces, 152 pieces, 153 pieces, 154 pieces, 155 pieces, 156 pieces, 157 pieces, 158 pieces, 159 pieces, 160 pieces, 161 pieces, 162 pieces, 163 pieces, 164 pieces, 165 pieces, 16 6 pieces, 167 pieces, 168 pieces, 169 pieces, 170 pieces, 171 pieces, 172 pieces, 173 pieces, 174 pieces, 175 pieces, 176 pieces, 177 pieces, 178 pieces,179 pieces, 180 pieces, 181 pieces, 182 pieces, 183 pieces, 184 pieces, 185 pieces, 186 pieces, 187 pieces, 188 pieces, 189 pieces, 190 pieces, 191 pieces, 192 pieces, 193 pieces, 194 pieces, 195 pieces, 196 pieces, 197 pieces, 198 pieces, 199 pieces, 200 pieces , 201 pieces, 202 pieces, 203 pieces, 204 pieces, 205 pieces, 206 pieces, 207 pieces, 208 pieces, 209 pieces, 210 pieces, 211 pieces, 212 pieces, 213 pieces, 214 pieces, 215 pieces, 216 pieces, 217 pieces, 218 pieces, 219 pieces, 220 pieces, 221 pieces, 222 pieces , 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, 300, 400, 500, 550, 1000 or more amino acids (or any range derivable therein).

[0164] Substitutional variants generally involve the exchange of one amino acid for another at one or more sites within the protein and can be designed so that one or more properties of the polypeptide are modulated, with or without loss of other functions or properties. Substitutions can be conservative, i.e., one amino acid is replaced with one of similar shape and charge. Conservative substitutions are well known in the art and include, for example, alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartic acid to glutamic acid; cysteine ​​to serine; glutamine to asparagine; glutamic acid to aspartic acid; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine, or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine. Alternatively, the substitution can be non-conservative, such that the function or activity of the polypeptide is affected. Non-conservative changes generally involve replacing a residue with a chemically different residue, such as substituting a polar or charged amino acid for a non-polar or uncharged amino acid, or conversely, substituting a non-polar or uncharged amino acid for a polar or charged amino acid.

[0165] The protein can be recombinant or synthesized in vitro. Alternatively, non-recombinant or recombinant proteins can be isolated from bacteria. It is also contemplated that bacteria containing such variants can function in the compositions and methods. Therefore, it is not necessary to isolate the protein.

[0166] The term "functionally equivalent codon" is used herein to refer to codons that encode the same amino acid, e.g., the six codons for arginine or serine, and also refers to codons that encode biologically equivalent amino acids.

[0167] It will also be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids, or 5' or 3' sequences, respectively, and still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the above criteria, including, in the case of expression of the protein, maintenance of biological activity of the protein. The addition of terminal sequences particularly applies to nucleic acid sequences, which may include, for example, various non-coding sequences adjacent to either the 5' or 3' portion of the coding region.

[0168] The following is a discussion based on changing the amino acids of proteins to create equivalent or even improved second-generation molecules. For example, in protein structures, certain amino acids can be substituted for other amino acids without appreciable loss of interactive binding ability. For example, structures such as enzymatic catalytic domains or interacting components can have substituted amino acids so that such function is maintained. Because the interacting ability and properties of proteins define the biological functional activity of proteins, certain amino acid substitutions can be made in protein sequences and in the underlying DNA coding sequence, and still produce proteins with similar properties. Therefore, the inventors intend that various changes can be made to the DNA sequences of genes without appreciable loss of their biological usefulness or activity.

[0169] In other embodiments, it is contemplated that the function of a polypeptide can be changed by introducing one or more substitutions. For example, certain amino acids can be substituted for other amino acids in a protein structure to alter the interactive binding ability of interacting components. For example, structures such as protein interaction domains, nucleic acid interaction domains, and catalytic sites can have amino acid substitutions to change such functions. Because the biological functional activity of a protein is determined by the interaction ability and properties of the protein, certain amino acid substitutions can be made in the protein sequence and in the underlying DNA coding sequence, and still result in proteins with different properties. Therefore, the present inventors contemplate that various changes can be made to the DNA sequence of the gene with appreciable changes in the biological usefulness or activity of the gene.

[0170] In making such changes, the hydropathic index of amino acids can be considered. The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte and Doolittle, 1982). The relative hydropathic properties of amino acids can influence the resulting protein secondary structure. It is recognized that secondary structure contributes to the function of the protein, which in turn determines the interactions of the protein with other molecules, such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc.

[0171] It is also understood in the art that similar amino acids can be effectively substituted based on hydrophilicity. U.S. Patent No. 4,554,101, incorporated herein by reference, describes that the maximum local average hydrophilicity of a protein is governed by the hydrophilicity of adjacent amino acids, and therefore correlates with the biological properties of the protein. It is understood that an amino acid can be substituted for another amino acid with a similar hydrophilicity value, and still produce a biologically equivalent and immunologically equivalent protein.

[0172] As outlined above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, e.g., their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions that take into consideration various of the foregoing characteristics are well known and include: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

[0173] In certain embodiments, all or part of the proteins described herein can be synthesized in solution or on a solid support according to conventional techniques. A variety of automated synthesizers are commercially available and can be used according to known protocols. See, for example, Stewart and Young, (1984); Tam et al., (1983); Merrifield, (1986); and Barany and Merrifield (1979), each of which is incorporated herein by reference. Alternatively, proteins can be synthesized by cleavage or by cleavage. Recombinant DNA technology can be used in which a nucleotide sequence encoding the peptide or polypeptide is inserted into an expression vector and appropriate host cells are transformed or transfected and cultured under conditions suitable for expression.

[0174] One embodiment involves the use of gene transfer into cells, including microorganisms, for protein production and / or display. The gene for the protein of interest is transferred into a suitable host cell, which can then be cultured under appropriate conditions. Nucleic acids encoding virtually any polypeptide can be used. The generation of recombinant expression vectors and the elements contained therein are discussed herein. Alternatively, the protein produced can be an endogenous protein normally synthesized by the cells used for protein production. V. U Method for generating HSC-iNKT cells

[0175] UHSC-iNKT cells can be generated by any suitable method(s). The method(s) may utilize one or more sequential steps for one or more modifications to the cells and / or one or more simultaneous steps for one or more modifications to the cells. In certain embodiments, a starting source of cells is modified to be functional as iNKT cells, followed by one or more steps to confer one or more additional characteristics to the cells, such as the ability to be imaged, and / or the ability to be selectively killed, and / or the ability to be allogeneically usable. In certain embodiments, U At least part of the process for generating HSC-iNKT cell is carried out in specific in vitro culture system.The example of specific in vitro culture system is that it allows certain cells to differentiate with high efficiency and high yield.In certain embodiments, the in vitro culture system is artificial thymus organoid (ATO) system.

[0176] In certain cases, U HSC-iNKT cells can be generated by: 1) genetically modifying donor HSCs to express iNKT TCR (e.g., by lentiviral vector) and abolishing expression of HLA-I / II molecules (e.g., by CRISPR / Cas9-based gene editing); 2) differentiating into iNKT cells in vitro by ATO culture; 3) purifying and expanding iNKT cells in vitro; and 4) formulating, cryopreserving, and / or using.

[0177] Certain embodiments of the present disclosure provide a method for preparing a population of clonal invariant natural killer T (iNKT) cells, comprising: a) selecting CD34+ cells from human peripheral blood cells (PBMCs); b) introducing one or more nucleic acids encoding a human iNKT T cell receptor (TCR); c) eliminating expression of one or more HLA-I / II genes in the isolated human CD34+ cells; and d) culturing the isolated CD34+ cells expressing the iNKT TCR in an artificial thymic organoid (ATO) system to produce iNKT cells, the ATO system comprising 3D cell aggregates containing a selected population of stromal cells expressing Notch ligands and serum-free medium. The method may further comprise isolating CD34- cells. In alternative embodiments, the method may further comprise culturing the iNKT cells in a 2D culture system or other forms of 3D culture system (e.g., FTOC-like culture, metrigel-aided culture), other than the ATO system. A culture system is used.

[0178] Certain aspects of the present disclosure relate to a novel three-dimensional cell culture system for generating iNKT cells from less differentiated cells, such as embryonic stem cells, pluripotent stem cells, hematopoietic stem or progenitor cells, induced pluripotent stem (iPS) cells, or stem or progenitor cells. Any type of stem cell derived from a variety of sources can be utilized, including, for example, fetal liver, umbilical cord blood, and peripheral blood CD34+ cells (either G-CSF-mobilized or non-G-CSF-mobilized).

[0179] In certain embodiments, the system involves the use of serum-free media. In certain aspects, the system uses serum-free media suitable for cell development to culture the three-dimensional cell aggregates. In such systems, a sufficient amount of U In embodiments of the present disclosure, 3D cell aggregates are grown from stem or progenitor cells in serum-free medium containing insulin. U to or from precursors of HSC-iNKT cells UThe cells are cultured for a period sufficient for in vitro differentiation into HSC-iNKT cells.

[0180] Embodiments of cell culture compositions include ATO 3D cultures, which use highly standardized serum-free components and stromal cell lines to facilitate robust and highly reproducible differentiation of human HSCs into T cells. In certain embodiments, cell differentiation in ATO closely mimics endogenous thymopoiesis and, in contrast to monolayer co-cultures, results in functional T cell differentiation. U Efficient positive selection of HSC-iNKT is supported. Certain embodiments of the 3D culture composition use serum-free conditions, avoid the use of human thymus tissue or proprietary scaffold materials, and produce fully functional mature human HSC-iNKT cells from the source cells. U Facilitate the positive selection and robust generation of HSC-iNKT cells.

[0181] In certain embodiments, this ATO 3D culture system can involve the aggregation of human HSCs in a 3D structure with stromal cells expressing Notch ligands in the presence of an optimized medium containing FLT3 ligand (FLT3L), interleukin-7 (IL-7), B27, and ascorbic acid. Conditions allowing culture at an air-fluid interface can also be present. Combinatorial signaling within ATO from soluble factors (cytokines, ascorbic acid, B27 components, and stromal cell-derived factors), along with 3D cell-cell interactions between hematopoietic cells and stromal cells, has been confirmed to facilitate human T lineage commitment, positive selection, and efficient differentiation into functional, mature T cells.

[0182] In certain embodiments, the 3D cell aggregates are created by mixing a selected population of CD34+ transduced cells and stromal cells on a physical matrix or scaffold. The method may further include centrifuging the CD34+ transduced cells and stromal cells to form a cell pellet that is placed on the physical matrix or scaffold. The Notch ligand expressed by the stromal cells may be intact, partial, or modified DLL1, DLL4, JAG1, JAG2, or a combination thereof. In certain cases, the Notch ligand is a human Notch ligand, such as human DLL1.

[0183] The ATO system used to generate iNKT cells can have a specific ratio of stromal cells to CD34+ cells. In certain cases, the ratio of stromal cells to CD34+ cells is approximately 1:5 to 1:20. The stromal cells can be a mouse stromal cell line, a human stromal cell line, a selected population of primary stromal cells, a selected population of stromal cells differentiated in vitro from pluripotent stem cells, or a combination thereof. The stromal cells can be a selected population of stromal cells differentiated in vitro from hematopoietic stem or progenitor cells.

[0184] In methods for preparing a population of clonal iNKT cells, selecting iNKT cells lacking surface expression of HLA-I and HLA-II molecules can include contacting the iNKT cells with magnetic beads that bind to the iNKT cells, positively selecting them, and negatively selecting HLA-I / II-negative cells. In certain embodiments, the magnetic beads are coated with monoclonal antibodies that recognize the human iNKT TCR, HLA-I, or HLA-II molecules. In certain embodiments, the monoclonal antibodies are clone 6B11 (recognizing human TCR Vα24-Jα18 and thus recognizing the human iNKT invariant TCR alpha chain), clone 2M2 (recognizing human B2M and thus recognizing human HLA-I molecules displayed on the cell surface), clone W6 / 32 (recognizing HLA-A, B, and C and thus recognizing human HLA-I molecules), and clone Tu39 (recognizing human HLA-DR, DP, and DQ and thus recognizing human HLA-II molecules).

[0185] The cells produced by the preparation method can be frozen. The cells can be placed in a solution containing dextrose, one or more electrolytes, albumin, dextran, and DMSO. The solution can be sterile, non-pyrogenic, and isotonic.

[0186] In a specific embodiment, the ATO system - Feeder cells are utilized that may contain the cells.

[0187] The preparation method may further include activating and expanding the selected iNKT cells. For example, the selected iNKT cells may be activated with alpha-galactosylceramide (α-GC). The feeder cells may be pulsed with α-GC.

[0188] In the preparation method of the present disclosure, at least about 10 clonal iNKT cells are 2 ~10 6 The method can generate a population of clonal iNKT cells comprising at least about 10 total cells.6 ~10 12 A cell population containing the cells can be produced. The produced cell population can be frozen and then thawed. In some preparation methods, the method further comprises introducing one or more additional nucleic acids into the frozen and thawed cell population, such as one or more additional nucleic acids encoding one or more therapeutic gene products.

[0189] In a specific embodiment, the MS-5 murine stromal cell line transduced with human DLL1 (hereafter MS5-hDLL1) was transfected with CD34 IL-16 cells isolated from human umbilical cord blood, bone marrow, or G-CSF-mobilized peripheral blood, as developed. + Methods for 3D culture compositions (e.g., ATO production) involving aggregation with HSPCs may be provided. 6 HSPCs are mixed with MS5-hDLL1 cells at an optimized ratio (generally 1:10 HSPCs to stromal cells).

[0190] For example, aggregation can be achieved by centrifugation of the mixed cell suspension ("compression aggregation"), followed by aspiration of the cell-free supernatant. In certain embodiments, the cell pellet can then be aspirated as a slurry in 5-10 μl of differentiation medium and transferred as a droplet to a 0.4 μm nylon transwell culture insert, which is allowed to float within the well of differentiation medium, allowing the bottom of the insert to be in contact with the medium and the top to be in contact with air.

[0191] For example, the differentiation medium can contain RPMI-1640, 5 ng / ml human FLT3L, 5 ng / ml human IL-7, 4% serum-free B27 supplement, and 30 μM L-ascorbic acid. The medium can be completely replaced around the culture insert every 3–4 days. During the first 2 weeks of culture, cell aggregates can self-organize into ATOs, allowing for early T-lineage commitment and differentiation. In certain embodiments, ATOs are cultured for at least 6 weeks to allow for optimal T-cell differentiation. Hematopoietic cell removal from ATOs is achieved by disaggregating the ATOs using pipetting.

[0192] Variations in the protocol allow for the use of alternative ingredients with varying effects on efficacy, particularly:

[0193] The basal medium RPMI can be used in place of some commercially available alternatives (eg, IMDM).

[0194] The stromal cell line used was a previously described murine bone marrow cell line (Itoh et al., 1989 ), but MS-5 can be substituted for similar murine stromal cell lines (e.g., OP9, S17), human stromal cell lines (e.g., HS-5, HS-27a), primary human stromal cells, or human pluripotent stem cell-derived stromal cells.

[0195] Stromal cell lines are transduced with lentivirus encoding human DLL1 cDNA. However, the method of gene delivery and the Notch ligand gene can be varied. Alternative Notch ligand genes include DLL4, JAG1, JAG2, etc. Notch ligands also include those described in U.S. Patent Nos. 7,795,404 and 8,377,886, which are incorporated herein by reference. Notch ligands also include Delta 1, 3, and 4, and Jagged 1 and 2.

[0196] Types and sources of HSCs may include HSCs derived from bone marrow, umbilical cord blood, peripheral blood, thymus, or other primary sources; or from human embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs).

[0197] Cytokine conditions can be varied: for example, FLT3L and IL-7 levels can be altered to modify T cell differentiation kinetics; other hematopoietic cytokines such as stem cell factor (SCF / Kit-ligand), thrombopoietin (TPO), IL-2, and IL-15 can be added.

[0198] Genetic modifications can also be introduced into certain components to generate antigen-specific T cells and model positive and negative selection. Examples of these modifications include transducing HSCs with lentiviral vectors encoding antigen-specific T cell receptors (TCRs) or chimeric antigen receptors (CARs) to generate antigen-specific, allelically excluded naive T cells; transducing HSCs with a gene or genes to direct lineage commitment to specialized lymphoid cells. For example, transducing HSCs with a TCR associated with invariant natural killer T cells (iNKTs) to generate functional iNKT cells in ATO; transducing ATO stromal cell lines (e.g., MS5-hDLL1) with human MHC genes (e.g., human CD1d genes) to enhance the positive selection and maturation of both TCR-engineered and non-engineered T cells in ATO; and / or transducing ATO stromal cell lines with antigens and costimulatory molecules or cytokines to enhance the positive selection of CAR T cells in ATO.

[0199] In generating engineered iNKT cells, CD34+ cells derived from human peripheral blood cells (PBMCs) can be modified by introducing certain exogenous gene(s) and knocking out certain endogenous gene(s). The method may further include culturing the selected CD34+ cells in a medium before introducing one or more nucleic acids into the cells. The culturing step may include incubating the selected CD34+ cells with a medium containing one or more growth factors, and in some cases, the one or more growth factors may include, for example, c-kit ligand, flt-3 ligand, and / or human thrombopoietin (TPO). The growth factors may or may not be at a certain concentration, such as between about 5 ng / ml and about 500 ng / ml.

[0200] In certain methods, the nucleic acid(s) introduced into the cells are one or more nucleic acids comprising nucleic acid sequences encoding α-TCR and β-TCR. The method may further comprise introducing a nucleic acid encoding a suicide gene into the selected CD34+ cells. In certain embodiments, one nucleic acid encodes both α-TCR and β-TCR, or one nucleic acid encodes α-TCR, β-TCR, and the suicide gene. The suicide gene may be based on an enzyme, such as thymidine kinase (TK), including viral TK genes, such as those derived from herpes simplex virus TK gene. The suicide gene may be activated by a substrate, such as ganciclovir, penciclovir, or a derivative thereof. The cells may be engineered to contain an exogenous nucleic acid encoding a polypeptide having a substrate that can be labeled for imaging. In some cases, the suicide gene product is a polypeptide having a substrate that can be labeled for imaging, e.g., sr39TK.

[0201] Cells can be engineered to lack surface expression of HLA-I and / or HLA-II molecules, for example, by disrupting the functional expression of genes encoding beta2-microglobulin (B2M), major histocompatibility complex class II transactivator (CIITA), and / or HLA-I and HLA-II molecules. In the production method, eliminating surface expression of one or more HLA-I / II molecules in isolated human CD34+ cells can include introducing CRISPR and one or more guide RNAs (gRNAs) corresponding to B2M, CIITA, or individual HLA-I or HLA-II molecules into the cells. In some cases, CRISPR or one or more gRNAs are transfected into the cells by electroporation or lipid-mediated transfection. In certain embodiments, a nucleic acid encoding a TCR receptor is introduced into the cells using a recombinant vector, such as a viral vector, including at least a lentivirus, retrovirus, adeno-associated virus (AAV), herpesvirus, or adenovirus.

[0202] In producing engineered iNKT cells, the cells may be present in certain serum-free media, including those containing exogenously added ascorbic acid. In certain embodiments, the serum-free media further contains exogenously added FLT3 ligand (FLT3L), interleukin 7 (IL-7), stem cell factor (SCF), thrombopoietin (TPO), IL-2, IL-4, IL-6, IL-15, IL-21, TNF-alpha, TGF-beta, interferon-gamma, interferon-lambda, TSLP, thymopentin, pleiotrophin, midkine, or a combination thereof. The serum-free medium may further comprise vitamins, including biotin, DL-alpha tocopherol acetate, DL-alpha tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, folic acid, nicotinamide, pyridoxine, riboflavin, thiamine, inositol, vitamin B12, or combinations or salts thereof. The serum-free medium may further comprise one or more exogenously added (or non-exogenously added) proteins, such as albumin or bovine serum albumin, a fraction of BSA, catalase, insulin, transferrin, superoxide dismutase, or combinations thereof. The serum-free medium may further comprise corticosterone, D-galactose, ethanolamine, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, or triiodo-L-thyronine, or combinations thereof. The serum-free medium may contain B-27® supplement, Xenofree B-27® supplement, GS21™ supplement, or a combination thereof. Amino acids (including arginine, cysteine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine, or a combination thereof), simple sugars, and / or inorganic ions (including, for example, sodium, potassium, calcium, magnesium, nitrogen, or phosphorus, or a combination or salt thereof) may be present in the serum-free medium.The serum-free medium may further comprise molybdenum, vanadium, iron, zinc, selenium, copper, or manganese, or a combination thereof.

[0203] Cell culture conditions can be provided for culturing the 3D cell aggregates described herein, as well as for generating T cells and / or their positive / negative selection. In certain embodiments, the starting cells of the selected population are at least or about 10 4 pieces, 10 5 pieces, 10 6 pieces, 10 7 pieces, 10 8 pieces, 10 9 pieces, 10 10 pieces, 10 11 pieces, 10 12 pieces, 10 13 The seeding density of the starting cell population can be at least or about 10 cells per ml, 10 1 pieces, 10 2 pieces, 10 3 pieces, 10 4 pieces, 10 5 pieces, 10 6 pieces, 10 7 pieces, 10 8 It can be a number, or any range that can be derived therein.

[0204] Culture vessels used to culture the 3D cell aggregates or their progeny may include, but are not limited to, flasks, tissue culture flasks, dishes, Petri dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, multi-plates, multiwell plates, microslides, chamber slides, tubes, trays, CellSTACK® chambers, culture bags, and roller bottles, so long as stem cells can be cultured therein. Stem cells can be cultured in volumes of at least or about 0.2 ml, 0.5 ml, 1 ml, 2 ml, 5 ml, 10 ml, 20 ml, 30 ml, 40 ml, 50 ml, 100 ml, 150 ml, 200 ml, 250 ml, 300 ml, 350 ml, 400 ml, 450 ml, 500 ml, 550 ml, 600 ml, 800 ml, 1000 ml, 1500 ml, or any range derivable therein, depending on the needs of the culture. In certain embodiments, the culture vessel may be a bioreactor, which may refer to any device or system that supports a biologically active environment. The volume of the bioreactor may be at least or about 2 liters, 4 liters, 5 liters, 6 liters, 8 liters, 10 liters, 15 liters, 20 liters, 25 liters, 50 liters, 75 liters, 100 liters, 150 liters, 200 liters, 500 liters, 1 cubic meter, 2 cubic meters, 4 cubic meters, 6 cubic meters, 8 cubic meters, 10 cubic meters, 15 cubic meters, or any range derivable therein.

[0205] The culture vessel can be cell-adhesive or non-adhesive, and is selected according to the purpose. The cell-adhesive culture vessel can be coated with any substrate for cell adhesion, such as an extracellular matrix (ECM), to improve the adhesion of the vessel surface to cells. The substrate for cell adhesion can be made of any material intended to attach stem cells or feeder cells (if used). Substrates for cell adhesion include collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, and fibronectin, and mixtures thereof, such as Matrigel™, and dissolved cell membrane preparations.

[0206] A variety of defined matrix components can be used in the culture methods or compositions. For example, as described in Ludwig et al. (2006a; 2006b), which are incorporated by reference in their entireties, a combination of recombinant type IV collagen, fibronectin, laminin, and vitronectin can be used to coat a culture surface as a means of providing a solid support for growing pluripotent cells.

[0207] The matrix composition can be immobilized on a surface to provide support for cells. The matrix composition can include one or more extracellular matrix (ECM) proteins and an aqueous solvent. The term "extracellular matrix" is recognized in the art. Its components include one or more of the following proteins: fibronectin, laminin, vitronectin, tenascin, entactin, thrombospondin, elastin, gelatin, collagen, fibrillin, merosin, anchorin, chondronectin, link protein, bone sialoprotein, osteocalcin, osteopontin, epinectin, hyaluronectin, undulin, epiligrin, and kalinin. Other extracellular matrix proteins are described in Kleinman et al., (1993), which is incorporated herein by reference. Currently unknown extracellular matrices that may be discovered in the future are also intended to be encompassed by the term "extracellular matrix," as their characterization as extracellular matrices can be readily determined by one of skill in the art.

[0208] In some aspects, the total protein concentration in the matrix composition can be about 1 ng / mL to about 1 mg / mL. In some embodiments, the total protein concentration in the matrix composition is about 1 μg / mL to about 300 μg / mL. In more preferred embodiments, the total protein concentration in the matrix composition is about 5 μg / mL to about 200 μg / mL.

[0209] Extracellular matrix (ECM) proteins may be of natural origin and purified from human or animal tissue. Alternatively, ECM proteins may essentially be genetically engineered recombinant or synthetic proteins. ECM proteins may be whole proteins or in the form of peptide fragments, native forms, or engineered forms. Examples of ECM proteins that may be useful as matrices for cell culture include laminin, type I collagen, type IV collagen, fibronectin, and vitronectin. In some embodiments, the matrix composition comprises synthetically produced peptide fragments of fibronectin or recombinant fibronectin.

[0210] In yet another embodiment, the matrix composition comprises a mixture of at least fibronectin and vitronectin.In some other embodiments, the matrix composition preferably comprises laminin.

[0211] Preferably, the matrix composition contains a single type of extracellular matrix protein. In some embodiments, the matrix composition contains fibronectin, particularly for use in culturing progenitor cells. For example, a suitable matrix composition can be prepared by diluting human fibronectin, such as human fibronectin (Cat. #354008) sold by Becton, Dickinson & Co. of Franklin Lakes, NJ (BD), in Dulbecco's phosphate-buffered saline (DPBS) to a protein concentration of 5 μg / mL to approximately 200 μg / mL. In a specific example, the matrix composition contains a fibronectin fragment, such as RetroNectin®. RetroNectin® is an approximately 63 kDa protein (574 amino acids) containing the central cell-binding domain (type III repeats 8, 9, and 10) of human fibronectin, the high-affinity heparin-binding domain II (type III repeats 12, 13, and 14), and the CS1 site within the alternatively spliced ​​IIICs region.

[0212] In some other embodiments, the matrix composition may include laminin. For example, a suitable matrix composition can be prepared by diluting laminin (Sigma-Aldrich (St. Louis, Mo.); Cat# L6274 and L2020) in Dulbecco's phosphate-buffered saline (DPBS) to a protein concentration of 5 μg / ml to about 200 μg / ml.

[0213] In some embodiments, the matrix composition is xeno-free, in that the matrix or its component proteins are exclusively of human origin. This is desirable for certain research applications. For example, a xeno-free matrix for culturing human cells can use matrix components of human origin and exclude any non-human animal components. In certain aspects, Matrigel™ can be excluded as a substrate for culturing the composition. Matrigel™ is a gelatinous protein mixture secreted by mouse tumor cells and is available from BD Biosciences (New York, NY). This mixture resembles the complex extracellular environment found in many tissues and is frequently used by cell biologists as a substrate for cell culture, although it can introduce unwanted xenoantigens or contaminants.

[0214] In certain embodiments, cells containing exogenous nucleic acids can be identified in vitro or in vivo by including a marker in the expression vector or exogenous nucleic acid. Such a marker confers a distinguishable change to the cells, thereby allowing easy identification of cells containing the expression vector. Generally, a selection marker can confer a characteristic that allows selection. A positive selection marker can be one whose presence allows for its selection, while a negative selection marker is one whose presence prevents its selection. An example of a positive selection marker is a drug resistance marker.

[0215] Typically, the inclusion of a drug selection marker aids in the cloning and identification of transformants; for example, genes conferring resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin, and histidinol are useful selection markers. In addition to markers that confer a phenotype that allows for the identification of transformants based on the performance of conditions, other types of markers are contemplated, including screenable markers such as GFP based on colorimetric analysis. Alternatively, screenable enzymes can be used as negative selection markers, such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT). Those skilled in the art will also know how to use immunological markers, optionally in conjunction with FACS analysis. The marker used is not believed to be important, so long as it can be expressed simultaneously with the nucleic acid encoding the gene product. Further examples of selectable and screenable markers are well known to those skilled in the art.

[0216] Selectable markers can include a type of reporter gene used in experimental microbiology, molecular biology, and genetic engineering to indicate the success of transfection or other procedures intended to introduce foreign DNA into cells. Selectable markers are often antibiotic resistance genes. Cells subjected to a procedure to introduce foreign DNA are grown in a medium containing an antibiotic; cells that are able to grow indicate that the introduced genetic material has been successfully incorporated and expressed. Examples of selectable markers include the Abicr gene or Neo gene from Tn5, which confers antibiotic resistance to geneticin.

[0217] Screenable markers may include reporter genes that allow researchers to distinguish between desirable and undesirable cells. In certain embodiments of the present invention, reporter genes are utilized to indicate specific cell lineages. For example, reporter genes can be located within expression elements and placed under the control of ventricular or atrial selective regulatory elements that are typically associated with the coding regions of ventricular or atrial selective genes for co-expression. Reporters allow cells of specific lineages to be isolated without placing them under drug or other selection pressure or otherwise compromising cell viability.

[0218] Examples of such reporters include genes encoding cell surface proteins (e.g., CD4, HA epitope), fluorescent proteins, antigenic determinants, and enzymes (e.g., β-galactosidase). Cells containing the vector can be isolated by FACS, for example, using fluorescently tagged antibodies against a substrate that can be converted to a fluorescent product by the cell surface protein or vector-encoded enzyme.

[0219] In certain embodiments, the reporter gene is a fluorescent protein. A wide range of fluorescent protein gene variants have been developed, featuring fluorescence emission spectral profiles spanning nearly the entire visible light spectrum. Mutagenesis attempts in the original Aequorea victoria jellyfish green fluorescent protein resulted in new fluorescent probes ranging from blue to yellow, which are some of the most widely used in vivo reporter molecules in biological research. Longer wavelength fluorescent proteins emitting in the orange and red spectral regions have been discovered in the marine anemone, Discosomus jellyfish. Fluorescent proteins have been developed from the reef-building corals A. striata and Anthozoa. Additional species are being investigated to produce similar proteins with blue-green, green, yellow, orange, and deep red fluorescence. Research efforts are underway to improve the brightness and stability of fluorescent proteins and, therefore, their overall utility.

[0220] In certain embodiments, cells can be engineered to contain one or more genetic modifications before or after differentiation (US2002 / 0168766). Cells can be said to be "genetically altered," "genetically modified," or "transgenic" if an exogenous nucleic acid or polynucleotide has been introduced into the cell by any suitable means of artificial manipulation, or if the cell is the progeny of an originally altered cell that inherits the polynucleotide. For example, cells can be engineered to increase their replicative potential by genetically modifying the cell to express telomerase reverse transcriptase before or after progression to a restricted developmental lineage or terminally differentiated cell (US2003 / 0022367).

[0221] In certain embodiments, cells containing the exogenous nucleic acid construct can be identified in vitro or in vivo by including a marker, such as a selectable or screenable marker, in the expression vector. Such a marker confers a distinguishable change to the cells, thereby allowing for easy identification of cells containing the expression vector, or by using tissue-specific promoters to facilitate enrichment or identification of differentiated cardiac cells. For example, in cardiomyocyte differentiation embodiments, cardiac-specific promoters such as those for cardiac troponin I (cTnI), cardiac troponin T (cTnT), sarcomeric myosin heavy chain (MHC), GATA-4, Nkx2.5, N-cadherin, □1-adrenergic receptor, ANF, the MEF-2 family of transcription factors, creatine kinase MB (CK-MB), myoglobin, or atrial natriuretic factor (ANF) can be used. In neuronal differentiation embodiments, neuronal-specific promoters can be used, including, but not limited to, TuJ-1, Map-2, Dcx, or synapsin. In hepatocyte differentiation embodiments, definitive endoderm-specific promoters and / or hepatocyte-specific promoters can be used, including but not limited to ATT, Cyp3a4, ASGPR, FoxA2, ​​HNF4a, or AFP.

[0222] Generally, a selectable marker confers a property that allows for selection. A positive selectable marker is one whose presence allows for its selection, while a negative selectable marker is one whose presence prevents its selection. An example of a positive selectable marker is a drug resistance marker.

[0223] Typically, the inclusion of a drug selection marker aids in the cloning and identification of transformants; for example, genes that confer resistance to blasticidin, neomycin, puromycin, hygromycin, DHFR, GPT, zeocin, and histidinol are useful selectable markers. In addition to markers that confer phenotypes that allow for the identification of transformants based on the performance of conditions, other types of markers are also contemplated, including colorimetrically based screenable markers such as GFP. Alternatively, screenable enzymes such as chloramphenicol acetyltransferase (CAT) can be utilized. Those skilled in the art will also know how to use immunological markers, optionally in conjunction with FACS analysis. The marker used is not believed to be critical, so long as it can be expressed simultaneously with the nucleic acid encoding the gene product. Further examples of selectable and screenable markers are well known to those skilled in the art.

[0224] For example, in the embodiment of genetically modifying cells to add or reduce one or more characteristics, genetic modification can be carried out by any suitable method.For example, any genetic modification composition or method can be used to introduce exogenous nucleic acid into cells or edit genomic DNA, such as gene editing, homologous recombination or non-homologous recombination, RNA-mediated gene delivery or any conventional nucleic acid delivery method.Non-limiting examples of genetic modification methods can include gene editing methods such as CRISPR / CAS9, zinc finger nuclease or TALEN technology.

[0225] Genetic modification can also include the introduction of selectable or screenable markers to aid in in vitro or in vivo selection, screening, or imaging. In particular, in vivo imaging agents or suicide genes can be exogenously expressed or added to the starting cells or progeny cells. In a further aspect, the method can involve image-guided adoptive cell therapy.

[0226] Specific Embodiments

[0227] In certain embodiments of the present disclosure, a method for preparing a cell population comprising clonal invariant natural killer (iNKT) T cells comprises the steps of: a) selecting CD34+ cells from human peripheral blood cells (PBMCs); b) culturing the CD34+ cells in a medium containing growth factors including c-kit ligand, flt-3 ligand, and human thrombopoietin (TPO); c) transducing the selected CD34+ cells with a lentiviral vector comprising nucleic acid sequences encoding α-TCR, β-TCR, and thymidine kinase; and d) transducing the selected CD34+ cells with Cas9 and beta2 microglobulin (B2 e) culturing the transduced cells with an irradiated stromal cell line expressing an exogenous Notch ligand for 2-12 weeks (or 2-10 weeks or 6-12 weeks) to expand iNKT cells in 3D aggregate cell culture; f) selecting iNKT cells lacking surface expression of HLA-I / II molecules; and g) culturing the selected iNKT cells with irradiated feeder cells. In certain embodiments, a method is provided that includes the steps of: (a) introducing gRNA against B2M and / or CTIIA to disrupt expression of the B2M gene or CTIIA gene, thereby eliminating surface expression of HLA-I and / or HLA-II molecules; (b) culturing the transduced cells with an irradiated stromal cell line expressing an exogenous Notch ligand for 2-12 weeks (or 2-10 weeks or 6-12 weeks) to expand iNKT cells in 3D aggregate cell culture; (c) selecting iNKT cells lacking surface expression of HLA-I / II molecules; and (d) culturing the selected iNKT cells with irradiated feeder cells. In certain embodiments, a method is provided that includes the steps of: (i) culturing 10 iNKT cells from the selected CD34+ cells; (ii) culturing 10 iNKT cells from the selected CD34+ cells; and (iii) culturing 10 iNKT cells from the selected CD34+ cells. 8 ~10 13 iNKT cells are prepared.

[0228] Thus, the present disclosure encompasses a universal, off-the-shelf, advanced HSC-based iNKT cell therapy (Figure 1). Specifically, G-CSF-mobilized CD34 + HSCs can be collected from healthy donors or from cell repositories. From a single donor, approximately 1-5 x 10 8 In certain cases, these HSCs can be harvested in These cells are engineered in vitro with lentivirus / iNKT-sr39TK lentiviral vectors and CRISPR-Cas9 / B2M-CIITA-gRNA complexes and then differentiated into iNKT cells in artificial thymic organoid (ATO) culture for 8 weeks. iNKT cells are then purified and further expanded in vitro for an additional 2-4 weeks before cryopreservation and lot release. In a specific embodiment, approximately 10 iNKT cells are generated from HSCs from a single donor. 12 iNKT cells are generated and administered in 1,000-10,000 doses (e.g., approximately 10 cells per dose). 8 ~10 9 The resulting cryopreserved cell product can then be formulated into iNKT ( U HSC-iNKT cells can be easily stored and distributed to treat cancer patients off-the-shelf by allogeneic adoptive cell transfer. iNKT cells can target multiple types of cancer without tumor antigen restriction or major histocompatibility complex (MHC) restriction. U HSC-iNKT therapy is useful as a universal cancer treatment to treat a large number of cancers and a large population of cancer patients, and therefore to address unmet medical needs (Figure 1) (Vivier et al., 2012; Berzins et al., 2011). The presented HSC-iNKT therapy is useful for treating many types of cancers that have been clinically implicated as being regulated by iNKT cells, including hematological cancers (leukemia, multiple myeloma, and myelodysplastic syndromes), and solid tumors (melanoma, colon cancer, lung cancer, breast cancer, and head and neck cancer) ( Berzins et al., 2011 ).

[0229] UThe scientific embodiments underlying HSC-iNKT therapy are as follows: 1) HSCs are programmed to differentiate into iNKT cells through lentiviral vector-mediated expression of the human iNKT T cell receptor (TCR) gene; 2) the inclusion of the sr39TK PET imaging / suicide gene allows for the identification of iNKT cells in patients using PET imaging. U It allows for monitoring of HSC-iNKT cells and depletion of these cells by ganciclovir (GCV) administration if necessary for safety; 3) CRISPR-Cas9 / B2M-CIITA-gRNA-based gene editing of HSCs knocks out the B2M and CIITA genes, resulting in an HLA-I / II-negative cell product suitable for allogeneic infusion; 4) the ATO culture system supports efficient in vitro development of human iNKT cells; and 5) the manufacturing process is high-yielding and pure. Data supporting these scientific embodiments are presented in the Examples section herein.

[0230] In certain cases, U The production of HSC-iNKT involves 1) harvesting leukopaks mobilized by G-CSF; 2) purifying G-CSF-leukopaks to express CD34 + 1) differentiation of iNKT cells into HSCs; 2) transduction of HSCs with the lentiviral vector lenti / iNKT-sr39TK; 3) transduction of HSCs with the lentiviral vector lenti / iNKT-sr39TK; 4) gene editing of B2M and CIITA by CRISPR / Cas9; 5) differentiation into iNKT cells in vitro by ATO; 6) purification of iNKT cells; 7) expansion of cells in vitro; and 8) harvesting, formulation, and cryopreservation of cells. In certain embodiments, two drug substances (lenti / iNKT-sr39TK vector and U HSC-iNKT cells) are present, and the final drug product is, in certain cases, formulated and cryopreserved in an infusion bag. U It may be HSC-iNKT.

[0231] UAn example of an efficient protocol for generating HSC-iNKT cells is presented herein. Efficient gene editing of HSCs to eliminate cell surface expression of class I HLA by knocking out B2M is demonstrated herein. By utilizing multiplex editing CRISPR / Cas9, for example, it is also possible to simultaneously disrupt cell surface class II HLA expression by knocking out the gene for class II transactivator of HLA-II (CIITA), a key regulator of HLA-II expression, using validated gRNA sequences (Abrahimi et al., 2015) (Steimle et al., 1994). Thus, cell By incorporating this gene editing step to disrupt surface HLA-I and cell surface HLA-II expression as well as a microbead purification step, U Generate HSC-iNKT cells. Flow cytometry analysis can be used to measure the purity and surface phenotype of these engineered iNKT cells. Cell purity is determined by the expression of TCR Vα24 + Jα18 + HLA-I - HLA-II - In certain embodiments, the iNKT cell population can be characterized by CD45RO + CD161 + , which exhibits memory and NK phenotypes and CD4 + CD8 - (CD4 single positive), CD4 - CD8 + (CD8 single positive), and CD4 - CD8 - (double negative, DN) (Kronenberg and Gapin, 2002). Since CD62L expression has been shown to be associated with the in vivo persistence of iNKT cells and their antitumor activity (Tian et al., 2016), CD62L expression can be analyzed for these phenotypes. UHSC-iNKT can be compared with PBMC-derived iNKT using RNA sequencing. U Comparative gene expression analysis can be performed on HSC-iNKT and PBMC iNKT cells.

[0232] PBMC iNKT were used as a benchmark control, and IFN-γ production and cytotoxicity assays were used to assess U The functional properties of HSC-iNKT can be assessed. U HSC-iNKT cells can be simulated with irradiated PBMCs pulsed with αGC, and supernatants collected from 1 day of stimulation can be subjected to IFN-γ ELISA (Smith et al., 2015). iNKT cells were stimulated for 6 hours followed by IFN-γ ELISA. Intracellular cytokine staining (ICCS) for γ effectors can also be performed. U Cytotoxicity assays were performed by incubating HSC-iNKT cells with αGC-loaded A375.CD1d target cells engineered to express luciferase and GFP for 4 hours, and cytotoxicity was measured using a plate reader based on the luminescence intensity. Since sr39TK is introduced as a PET / suicide gene, U The functionality of HSC-iNKT can be confirmed by incubating them with ganciclovir (GCV), and cell viability can be measured, for example, by MTT assays and annexin V-based flow cytometry assays.

[0233] Pharmacokinetic / pharmacodynamic (PK / PD) studies can be performed. PK / PD studies can determine the following in vivo in animal models: 1) the efficacy and safety of an injected U 1) Kinetics and persistence of HSC-iNKT expansion in various tissues / organs U Biodistribution of HSC-iNKT; 3) UThe ability of HSC-iNKT to traffic to tumors and how this filtration relates to tumor growth. Immunodeficient NSG mice bearing A375.CD1d (A375.CD1d) tumors can be utilized as a solid tumor animal model. The study design is outlined in Figure 12. Two cell dose groups (1 x 10 6 pieces and 10x10 6 (n=8) can be studied. Tumors can be inoculated (sc) on day -4, and baseline PET imaging and blood draws can be performed on day 0. U HSC-iNKT cells are infused intravenously (i.v.) and can be monitored by: 1) PET imaging in live animals on days 7 and 21; 2) periodic blood sampling on days 7, 14, and 21; and 3) endpoint tissue collection after animal sacrifice on day 21. Cells collected from the various blood samplings can be analyzed by flow cytometry; iNKT cells express CD161 + 6B11 + To understand how iNKT subsets fluctuate over time, the expression of other markers such as CD45RO, CD62L, and CD4 can be examined. PET imaging via sr39TK makes it possible to track the presence of iNKT cells in tumors and other tissues / organs such as bone, liver, spleen, and thymus. At the end of the study, U To examine the distribution of HSC-iNKT cells, tumor and mouse tissues including spleen, liver, brain, heart, kidney, lung, stomach, bone marrow, ovary, intestine, etc. can be collected for qPCR analysis.

[0234] The mechanism of action (MOA) of iNKT cells can be characterized. iNKT cells are known to target tumor cells either by direct killing or by releasing large amounts of IFN-γ, directing NK cells and CD8 T cells to eradicate tumors (Fujii et al., 2013). In vitro pharmacological studies have demonstrated direct cytotoxicity. The role of NK cells and CD8 T cells in supporting anti-tumor responses in vivo can now be investigated. Tumor-bearing NSG mice (A375.CD1d or MM.1S.Luc) were U HSC-iNKT alone (as in dose selected based on in vivo studies) or PBMCs (mismatched donor, 5 × 10 6 can be injected in combination with U Because HSC-iNKT are MHC negative, U An allogeneic immune response may not occur between HSC-iNKT and unrelated PBMCs. Tumor growth can be monitored and compared with and without PBMC groups (n=8 per group). In certain embodiments, if a greater anti-tumor response is observed in the combination group, it may indicate that components of PBMCs, such as NK and / or CD8 T cells, play a role in enhancing therapeutic efficacy. To further determine their individual roles, PBMCs depleted of NK cells (by CD56 beads), PBMCs depleted of CD8 T cells (by CD8 beads), or PBMCs depleted of myeloid cells (by CD14 beads) were used. U They can be co-injected with HSC-iNKT cells into tumor-bearing mice. It has been suggested that immune checkpoint inhibitors such as PD-1 and CTLA-4 regulate iNKT cell function (Pilones et al., 2012; Durgan et al., 2013). et al., 2011). Treatment with anti-PD-1 or anti-CTLA-4 U By adding these molecules to HSC-iNKT therapy, U How HSC-iNKT therapy is modulated can be determined and inform the design of combination cancer therapies.

[0235] UCertain vectors are available for the generation and / or use of HSC-iNKT cells. For example, the HIV-1-derived lentiviral vector lenti / iNKT-sr39TK, encoding the human iNKT TCR gene along with the sr39TK PET imaging / suicide gene, can be used to genetically engineer HSCs into iNKT cells (Figure 13). The components of this third-generation self-inactivating (SIN) vector are: 1) 3' self-inactivating long-terminal repeats (ΔLTR); 2) the ψ region vector genome packaging signal; and 3) the supra- 3) a Rev response element (RRE) to enhance nuclear export of uncised vector RNA; 4) a middle gene to facilitate nuclear import of the vector genome (unclear import). 5) expression cassettes for the α-chain gene (TCRα) and β-chain gene (TCRβ) of human iNKT TCR, and the PET / suicide gene sr39TK (Gscheng et al., 2014), driven by an internal promoter derived from murine stem cell virus (MSCV). All genes were codon-optimized and linked with 2A self-cleaving sequences (T2A and P2A) to ensure their optimal co-expression (Gscheng et al., 2014).

[0236] Regarding vector quality control, a series of QC assays can be performed to ensure the vector product is of high quality. Standard assays such as vector identity, vector physical titer, and vector purity (sterility, mycoplasma, viral contamination, replication-competent lentivirus (RCL) testing, endotoxin, residual DNA, and benzonase) can be performed at IU VPF and presented on the Certificate of Analysis (COA). Additional QC assays that can be performed include: 1) transduction / biological titer (by transducing HT29 cells at serial dilutions and performing ddPCR, ≥ 1 x 10); 6These include: 1) vector proviral integrity (by sequencing the vector-integrated portion of the genomic DNA of transduced HT29 cells, which is identical to the original vector plasmid sequence); and 3) vector function. Vector function can be measured by transducing human PBMC T cells (Chodon et al., 2014). Expression of the iNKT TCR gene can be detected by staining with 6B11, which is specific for the iNKT TCR (Montoya et al., 2007). Functionality of the expressed iNKT TCR can be confirmed by αGa The expression and functionality of the sr39TK gene will be analyzed by IFN-γ production in response to stimulation with IFN-γ (Watarai et al., 2008). The vector stock can be analyzed by PCR and GCV killing assays (Gschweng et al., 2014). The stability of the vector stock (stored in a -80°C freezer) can be tested by measuring its transduction titer every three months. VI. Specific Cell Manufacturing and Product Formulation

[0237] U An overview and specific manufacturing processes for HSC-iNKT cells are provided. In certain embodiments, U HSC-iNKT cells are important drug substances that function as "living drugs" to target and combat diseases in mammals, including, for example, combating tumor cells. In certain embodiments, U HSC-iNKT cells are generated by in vitro differentiation and expansion of genetically modified donor HSCs. The data demonstrate a novel, efficient protocol for producing cells at laboratory scale, and in certain embodiments, the cells are produced as an "off-the-shelf" cell product in a GMP-compliant manufacturing process. In certain cases, production scales can be as low as 10 cells per batch. 12 This is estimated to treat between 1000 and 10,000 patients.

[0238] An example of a cell manufacturing process is provided. One cell manufacturing process is outlined in Figure 14, along with an example timeline and, in at least some cases, "In-Process-Control" (IPC) measurements for each process step. Step 1 is the collection of G-CSF-mobilized donor PBSCs at a blood collection facility, which has become a routine procedure in many hospitals (Deotare et al., 2015). Fresh PBSCs can be obtained in Leukopaks from HemaCare for the transfection; HemaCare has IRB-approved collection protocols and donor consents to support clinical trials and commercial product manufacturing. Step 2 involves extracting CD34 cells from PBSCs using the CliniMACS system. + The primary goal of this study is to enrich for HSCs; in certain embodiments, this step can be completed using a system such as that found in the UCLA GMP facility, and at least 10 8 CD34 + CD34 cells can be obtained. - Cells can also be harvested and stored (and used as PBMC feeders in step 7).

[0239] Step 3 involves the culture and vector transduction of HSCs. + Cells can be cultured in retronectin-coated flasks in X-VIVO15 medium supplemented with 1% HAS (USP) and a growth factor cocktail (c-kit ligand, flt-3 ligand, and tpo; 50 ng / ml each) for 12 hours, after which the lenti / iNKT-sr39TK vector can be added and cultured for an additional 8 hours (Gschweng et al., 2014). Transduced cells can be measured for vector integration copies (VCN) by sampling approximately 50 colonies formed in a methylcellulose assay, and the average vector copy number per cell can be determined using ddPCR (Nolta et al., 1994). In certain cases, procedures can be optimized to achieve >50% transduction. Transfection is routinely achieved with VCN = 1-3 per cell.

[0240] Step 4 is to utilize the powerful CRISPR / Cas9 multiplex gene editing method to target both B2M and CIITA genomic loci in HSCs and disrupt their gene expression (Ren et al., 2017; Liu et al., 2017). iNKT cells derived from the edited HSCs lack MHC / HLA expression, thereby avoiding rejection by the host immune system. Initial data suggest that electroporation of Cas9 / B2M-gRNA induces CD34 + High-efficiency (approximately 75% success rate) B2M disruption in HSCs has been demonstrated. B2M / CIITA double knockout can be achieved by electroporation of a mixture of RNPs (Cas9 / B2M-gRNA and Cas9 / CIITA-gRNA) (Abrahimi et al., 2015). This process can be optimized and validated by varying electroporation parameters, the ratio of the two RNPs, and the stem cell culture time before electroporation (24, 48, or 72 hours after transduction) (Gundry et al., 2016). The high-fidelity Cas9 protein from IDT (Slaymaker) was also used. et al., 2016;Tsai and Joung, 2016) to investigate "off-target" effects. Exemplary evaluation parameters can be viability, deletion (indel) frequency (on-target efficiency) measured by T7E1 assay targeting B2M and CIITA sites, and hematopoietic function of edited HSCs measured by next-generation sequencing (NGS), MHC expression by flow cytometry, and colony-forming unit (CFU) assay.

[0241] Step 5: Artificial Thymic Organoid (ATO) Culture 1 Modified CD34 +The goal is to differentiate HSCs into iNKT cells in vitro. Initial studies have shown that functional iNKT cells can be efficiently generated from HSCs engineered to express the iNKT TCR. Based on this data, we are investigating the feasibility of iNKT cells in vitro. 8 Modified CD34 + HSC to 10 10 The 8-week GMP-compliant ATO culture process for generating iNKT cells can be tested and validated. ATO can be cultured from HSCs (5 x 10 4 ) and irradiated (80 Gy) MS5-hDLL1 stromal cells (10 6 The cell slurry (5 μl) containing the mixture of 1000 cells / ml was pipetted dropwise onto a 0.4 μm Millicell transwell insert, and the insert was then filled with RB27 medium. 1 This involves placing the ATO droplets into a 6-well plate containing 1 ml of medium; the medium may be changed every 4 days for 8 weeks. Considering three ATOs per insert, approximately 170 6-well plates can be utilized for each batch production. An automated, programmable pipetting / dispensing system (Eppendorf epMontion 5070f) located in a biosafety cabinet can be used for plating ATO droplets and changing the medium; completing 170 plates per round can require 2 hours of operation. At the end of the ATO culture, iNKT cells can be collected and characterized. In a specific embodiment, a component of the ATO is the MS5-hDLL1 stromal cell line, constructed by lentiviral transduction to express human DLL1 followed by cell sorting. In preparation for a specific GMP process, this polyclonal cell population can be subjected to a single-cell clonal selection process to establish several clonal MS5-hDLL1 cell lines, from which efficient ones can be selected (as assessed by ATO culture) and used to generate a master cell bank. Such a bank can be used to supply irradiated stromal cells for future clinical-grade ATO cultures.

[0242] Step 6 is the purification of ATO-derived iNKT cells using the CliniMACS system. This purification step was performed by MHCI + Cells and MHCII + Depleting iNKT cells + Anti-MHCI and anti-MHCII beads can be prepared by incubating Miltenyi anti-biotin beads with commercially available biotinylated anti-MHCI (clone W6 / 32, HLA-A, B, C), anti-B2M (clone 2M2), and anti-MHCII (clone Tu39, HLA-DR, DP, DQ), and anti-TCR Vα24-Jα18 (clone 6B11). Microbeads directly coated with 6B11 are also available from Miltenyi; anti-iNKT beads are available from Miltenyi Biotec. Collected iNKT cells can be labeled with the anti-MHC bead mixture, washed twice, and depleted using the CliniMACS depletion program. + and / or MHCII + Cells can be depleted; if necessary, this depletion step can be repeated to remove any remaining MHC + Cells can be further removed. Then, iNKT cells can be further purified using standard anti-iNKT beads and CliniMACS enrichment program. Cell purity can be measured, for example, by flow cytometry.

[0243] Step 7 is the in vitro expansion of purified iNKT cells. We used a validated PBMC feeder-based in vitro expansion protocol to expand iNKT cells over a 10 10 Starting from 10 cells 12The G-Rex-based bioprocess can be evaluated for this cell expansion. G-Rex is a cell growth flask with a gas-permeable membrane at the bottom, allowing for more efficient gas exchange; G-Rex 500M flasks are capable of supporting a 100-fold cell expansion in 10 days (Vera et al., 2010; Bajgain et al., 2014; Jin et al., 2012). The CD34 cells stored in step 1 can be expanded to 100 cells per 1000-fold. - The cells (used as feeder cells) can be thawed, pulsed with αGalCer (100 ng / ml), and irradiated (40 Gy). iNKT cells are mixed with irradiated feeder cells (1:4 ratio) and seeded into G-Rex flasks (1.25 x 10 cells each). 8 iNKT cells (80 flasks) can be expanded over a 2-week period. IL-2 (200 U / ml) is added every 2–3 days, with medium changes occurring on day 7; all media manipulations can be accomplished with a peristaltic pump. This expansion process is GMP-compliant, as similar PBMC feeder-based expansion procedures (referred to as rapid expansion protocols) have already been utilized to generate therapeutic T cells for numerous clinical trials (Dudley et al., 2008; Rosenberg et al., 2008).

[0244] Step 8 is the formulation of the collected iNKT cells (active drug component) from step 7 as a cell suspension for direct infusion. After at least three rounds of extensive washing, the cells from step 7 can be counted and suspended in an infusion / cold storage compatible solution consisting of Plasma-Lyte A Injection (31.25% v / v), Dextrose and Sodium Chloride Injection (31.25% v / v), Human Albumin (20% v / v), Dextran 40 in Dextrose Injection (10%, v / v), and Cryoserv DMSO (7.5%, v / v) (10 cells per ml).7 ~10 8 This solution is used to formulate Novartis' approved T cell product, tisagenlecleucel (Grupp et al., 2013). Once filled into freezing bags (e.g., Miltenyi Biotec's CryoMACS freezing bags), the product can be frozen in a controlled rate freezer and stored in a liquid nitrogen freezer. U Validation and / or optimization studies can be performed by measuring viability and recovery to ensure suitability as an HSC-iNKT cell product.

[0245] To ensure high-quality production, the proposed bioprocess can incorporate various IPC assays, such as cell count, viability, sterility, mycoplasma, identity, purity, and VCN. Tests can include: 1) appearance (color, opacity); 2) cell viability and count; 3) identity and VCN by qPCR for iNKT TCR; 4) purity by iNKT positivity and B2M negativity; 5) endotoxin; 6) sterility; 7) mycoplasma; 8) potency as measured by IFN-γ release in response to stimulation with αGalCer; and 9) RCL (replication-competent lentivirus) (Cornetta et al., 2011). Most of these assays are either standard biological assays or specific assays unique to this product. Product stability testing can be performed by periodically thawing LN storage bags and measuring their cell viability, purity, recovery, potency (IFN-γ release), and sterility. In certain embodiments, the product is stable for at least one year. A. Source of Starting Cells

[0246] Certain compositions or methods for differentiation along selected T cell lineages can use starting cells such as pluripotent stem cells or hematopoietic stem or progenitor cells. Stromal cells can be used to co-culture with the stem or progenitor cells. B. Stromal cells

[0247] Stromal cells are connective tissue cells of any organ, such as bone marrow, thymus, uterine mucosa (endometrium), prostate, and ovary. Stromal cells are cells that support the function of the parenchymal cells of that organ. Fibroblasts (also known as mesenchymal stromal cells / MSCs) and pericytes are among the most common types of stromal cells.

[0248] It is known that the interaction between stromal cells and tumor cells plays a key role in the growth and progression of cancer. Furthermore, bone marrow stromal cells have been reported to be involved in human hematopoiesis and inflammatory processes by regulating local cytokine networks (e.g., M-CSF, LIF).

[0249] Stromal cells in the bone marrow, thymus, and other hematopoietic organs regulate hematopoietic and immune cell development through cell-cell ligand-receptor interactions and through the release of soluble factors, including cytokines and chemokines. Stromal cells within these tissues form niches that regulate stem cell maintenance, lineage specification and commitment, and differentiation into effector cell types.

[0250] The stroma is composed of non-malignant host cells that provide tissue-specific cell types and, in some cases, an extracellular matrix upon which tumors can grow. C. Hematopoietic stem and progenitor cells

[0251] Due to the significant medical potential of hematopoietic stem and progenitor cells, substantial research has been conducted to improve methods for differentiating hematopoietic progenitors from embryonic stem cells. In adult humans, hematopoietic stem cells, primarily present in the bone marrow, produce a heterogeneous population of hematopoietic (CD34+) progenitors that differentiate into all cells of the blood lineage. In adult humans, hematopoietic progenitors proliferate and differentiate, resulting in the daily generation of hundreds of billions of mature blood cells. Hematopoietic progenitor cells are also present in umbilical cord blood. Human embryonic stem cells can be differentiated into hematopoietic progenitor cells in vitro. Hematopoietic progenitor cells can also be expanded or enriched from samples of peripheral blood as described below. Hematopoietic cells can be of human origin, murine origin, or any other mammalian species.

[0252] Isolation of hematopoietic progenitor cells includes any method of choice, including cell sorters, magnetic separation using antibody-coated magnetic beads, packed columns; affinity chromatography; cytotoxic agents conjugated to or used in conjunction with monoclonal antibodies, including, but not limited to, complement and cytotoxins; and "panning" with antibodies attached to a solid matrix, e.g., a plate, or any other convenient technique.

[0253] The use of separation or isolation techniques includes, but is not limited to, those based on differences in physical properties (density gradient centrifugation and counterflow centrifugation elution), those based on differences in cell surface properties (lectin and antibody affinity), and those based on differences in vital staining properties (mitochondrial-binding dye rhol23 and DNA-binding dye Hoechst 33342). Techniques that result in precise separation include, but are not limited to, FACS (fluorescence-activated cell sorting) or MACS (magnetic-activated cell sorting), which can vary in sophistication, for example, multiple color channels, small-angle and obtuse-angle light scattering detection channels, impedance channels, etc.

[0254] Antibodies utilized in the preceding techniques or techniques for assessing the purity of cell types (e.g., flow cytometry) can be conjugated with distinguishable agents, including, but not limited to, enzymes, magnetic beads, colloidal magnetic beads, haptens, fluorescent dyes, metal compounds, radioactive compounds, drugs, or haptens. Enzymes that can be conjugated to antibodies include, but are not limited to, alkaline phosphatase, peroxidase, urease, and β-galactosidase. Fluorescent dyes that can be conjugated to antibodies include, but are not limited to, fluorescein isothiocyanate, tetramethylrhodamine isothiocyanate, phycoerythrin, allophycocyanin, and Texas Red. For additional fluorescent dyes that can be conjugated to antibodies, see Haugland, Molecular Probes: Handbook of Fluorescent Probes and Research Chemicals (1992-1994). Antibodies and conjugates Metal compounds that can be conjugated include, but are not limited to, ferritin, colloidal gold, and especially colloidal superparamagnetic beads. Haptens that can be conjugated to antibodies include, but are not limited to, biotin, digoxigenin, oxazalone, and nitrophenol. Radioactive compounds that can be conjugated to or incorporated into antibodies are known in the art and include, but are not limited to, technetium-99m (99TC), 125I, and amino acids containing any radionuclide, including, but not limited to, 14C, 3H, and 35S.

[0255] Other techniques for positive selection that allow for accurate separation can be used, such as affinity columns, etc. The method should allow for removal of non-target cell populations to a residual amount of less than about 20%, preferably less than about 5%.

[0256] Cells can be selected based on their light-scattering properties and their expression of various cell surface antigens. FACS analysis of purified stem cells reveals a low side-scatter profile and a low-to-moderate forward-scatter profile. Cytospin preparations show that the enriched stem cells have a size between mature lymphoid cells and mature granulocytes.

[0257] For example, the methods described in Sutherland et al. (1992) and U.S. Pat. No. 4,714,680. Before culturing the inoculated population using the CD34 + It is also possible to enrich for specific cells. For example, cells are subjected to negative selection to remove cells expressing lineage-specific markers. In an exemplary embodiment, a cell population is subjected to negative selection to deplete non-CD34+ hematopoietic cells and / or specific hematopoietic cell subsets. Negative selection can be performed based on the cell surface expression of various molecules, including T cell markers such as CD2, CD4, and CD8; B cell markers such as CD10, CD19, and CD20; monocyte marker CD14; NK cell markers CD2, CD16, and CD56, or any lineage-specific marker. Negative selection can be performed based on the cell surface expression of various molecules, such as a cocktail of antibodies (e.g., CD2, CD3, CD11b, CD14, CD15, CD16, CD19, CD56, CD123, and CD235a), which can be used to separate other cell types, for example, by MACS or column separation.

[0258] As used herein, lineage-negative (LIN-) refers to cells that lack at least one marker associated with lineage-committed cells, such as a marker associated with T cells (e.g., CD2, 3, 4, and 8), a marker associated with B cells (e.g., CD10, 19, and 20), a marker associated with myeloid cells (e.g., CD14, 15, 16, and 33), a marker associated with natural killer ("NK") cells (e.g., CD2, 16, and 56), a marker associated with RBCs (e.g., glycophorin A), a marker associated with megakaryocytes (CD41), a marker associated with mast cells, a marker associated with eosinophils or basophils, or other markers such as CD38, CD71, and HLA-DR. Lineage-specific markers preferably include, but are not limited to, at least one of CD2, CD14, CD15, CD16, CD19, CD20, CD33, CD38, HLA-DR, and CD71. More preferably, LIN- includes at least CD14 and CD15. Further purification can be achieved, for example, by positive selection for c-kit+ or Thy-1+. Further enrichment can be achieved by using the mitochondria-binding dye rhodamine 123 to select rhodamine+ cells by methods known in the art. Highly enriched compositions can be obtained by selectively isolating cells that are CD34+, preferably CD34+LIN-, and most preferably CD34+Thy-1+LIN-. Highly enriched stem cell populations and methods for obtaining them are well known to those skilled in the art, see, for example, the methods described in PCT Patent Application Nos. PCT / US94 / 09760; PCT / US94 / 08574; and PCT / US94 / 10501.

[0259] Various techniques can be used to separate cells by first removing cells of a dedicated lineage. Monoclonal antibodies are particularly useful for identifying markers associated with specific cell lineages and / or stages of differentiation. Antibodies can be attached to a solid support to allow for crude separation. The separation technique used should maximize the retention of viability of the collected fraction. Various techniques with varying effectiveness can be used to obtain "relatively crude" separations. Such separations involve retaining unwanted cells along with the retained cell population, with the remaining cells representing up to 10%, usually no more than about 5%, and preferably no more than about 1% of the total cells present. The particular technique used depends on the efficiency of the separation, the associated cytotoxicity, the ease and speed of implementation, and the need for sophisticated equipment and / or technical skill.

[0260] Selection of progenitor cells need not be achieved solely by cell-specific markers: a combination of negative and positive selection can be used to obtain enriched cell populations. D. Source of blood cells

[0261] Hematopoietic stem cells (HSCs) normally reside in the bone marrow but can be released into the blood; this process is called mobilization and is used clinically to collect large numbers of HSCs into peripheral blood. One example of a mobilizing agent of choice is granulocyte colony-stimulating factor (G-CSF).

[0262] CD34+ hematopoietic stem or progenitor cells circulating in peripheral blood can be collected by apheresis either in an undisturbed state or after mobilization following the external administration of hematopoietic growth factors such as G-CSF. The number of stem or progenitor cells collected after mobilization is greater than that obtained after apheresis in an undisturbed state. In certain embodiments of the present invention, the source of the cell population is a subject whose cells have not been mobilized by externally applied factors, due to the lack of need for in vivo enrichment of hematopoietic stem or progenitor cells.

[0263] The cell population for use in the methods described herein can be mammalian cells, such as human cells, non-human primate cells, rodent cells (e.g., mouse or rat), bovine cells, ovine cells, porcine cells, equine cells, ovine cells, canine cells, and feline cells, or a mixture thereof. Non-human primate cells include rhesus monkey cells. The cells can be obtained from an animal, such as a human patient, or can be derived from a cell line. When the cells are obtained from an animal, they can be used as such, for example, as unseparated cells (i.e., a mixed population); the cells can be initially established in culture, for example, by transformation; or the cells can be subjected to a preliminary purification method. For example, the cell population can be manipulated by positive or negative selection based on the expression of cell surface markers; stimulated with one or more antigens in vitro or in vivo; treated with one or more biological modifiers in vitro or in vivo; or a combination of any or all of these.

[0264] Populations of cells include peripheral blood mononuclear cells (PBMCs), whole blood or its fractions containing mixed populations, spleen cells, bone marrow cells, tumor-infiltrating lymphocytes, cells obtained by leukapheresis, biopsy tissue, and lymph nodes, e.g., lymph nodes draining a tumor. Suitable donors include immunized donors, non-immunized (naive) donors, treated donors, or untreated donors. A "treated" donor is a donor that has been exposed to one or more biological modifiers. An "untreated" donor is a donor that has not been exposed to one or more biological modifiers.

[0265] For example, peripheral blood mononuclear cells (PBMCs) can be obtained as described in accordance with methods known in the art. Examples of such methods are discussed by Kim et al. (1992); Biswas et al. (1990); Biswas et al. (1991).

[0266] The method of obtaining precursor cells from a cell population is also well known in the art.Precursor cells can be expanded using various cytokines such as hSCF, hFLT3, and / or IL-3 (Akkina et al., 1996), or can be enriched with CD34+ cells using MACS or FACS.As mentioned above, negative selection techniques can also be used to enrich CD34+ cells.

[0267] It is also possible to obtain a cell sample from a subject and then enrich it for desired cell types.For example, PBMC and / or CD34+ hematopoietic cells can be isolated from blood as described herein.Cells can also be isolated from other cells using various techniques, such as isolation and / or activation with an antibody that binds to an epitope on the cell surface of the desired cell type.Another method that can be used includes negative selection using an antibody against a cell surface marker to selectively enrich for a specific cell type without activating cells by receptor association.

[0268] Bone marrow cells can be obtained from the iliac crest, femur, tibia, vertebrae, ribs, or other medullary cavities. Bone marrow can be removed from a patient and isolated by various separation and washing procedures. An exemplary procedure for isolating bone marrow cells includes the following steps: a) centrifuging the bone marrow suspension into three fractions and collecting the middle fraction, or buffy coat; b) centrifuging the buffy coat fraction from step (a) again in a separation fluid, typically Ficoll (a trademark of Pharmacia Fine Chemicals AB), to collect the middle fraction containing bone marrow cells; and c) washing the fraction collected in step (b) to recover bone marrow cells that can be reinfused. E. Pluripotent stem cells

[0269] Cells suitable for the compositions and methods described herein may be hematopoietic stem or progenitor cells, which may also be prepared by in vitro differentiation of pluripotent stem cells. In some embodiments, the cells used in the methods described herein are pluripotent stem cells (embryonic stem cells or induced pluripotent stem cells) directly seeded onto ATO. In further embodiments, the cells used in the methods and compositions described herein are derivatives or progeny of PSCs, such as, but not limited to, mesodermal progenitors, hemogenic endothelial progenitors, or hematopoietic progenitors.

[0270] The term "pluripotent stem cells" refers to cells that can produce cells of all three germ layers, i.e., endoderm, mesoderm, and ectoderm. Although pluripotent stem cells can theoretically differentiate into any cell of the body, experimental determination of pluripotency is generally based on the differentiation of pluripotent cells into several cell types of each germ layer. In some embodiments, pluripotent stem cells are embryonic stem (ES) cells derived from the inner cell mass of a blastocyst. In other embodiments, pluripotent stem cells are induced pluripotent stem cells obtained by reprogramming somatic cells. In certain embodiments, pluripotent stem cells are embryonic stem cells obtained by somatic cell nuclear transfer.

[0271] Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of a blastocyst. ES cells can be isolated by removing the outer trophectoderm layer of a developing embryo and then culturing the inner cell mass on a feeder layer of undeveloped cells. Under appropriate conditions, a colony of growing, undifferentiated ES cells is generated. The colony is removed, dissociated into individual cells, and then replated on a fresh feeder layer. The replated cells are allowed to continue to proliferate, thereby generating a new colony of undifferentiated ES cells. The new colony is then removed, dissociated, replated, and grown again. This process of "subculture" or "passaging" of undifferentiated ES cells can be repeated multiple times to generate cell lines containing undifferentiated ES cells (U.S. Patent Nos. 5,843,780; 6,200,806; and 7,029,913). A "primary cell culture" is a culture of cells obtained directly from a tissue, such as the inner cell mass of a blastocyst. A "subculture" is any culture derived from a primary cell culture.

[0272] Methods for obtaining mouse ES cells are well known. In one method, preimplantation blastocysts from 129 mouse strains are treated with mouse antisera to remove the trophectoderm, and the inner cell mass is cultured on a feeder cell layer of chemically inactivated mouse embryonic fibroblasts in medium containing fetal bovine serum. Colonies of undifferentiated ES cells that arise are subcultured on the mouse embryonic fibroblast feeder layer in the presence of fetal bovine serum to generate a population of ES cells. In some methods, mouse ES cells can be grown in the absence of a feeder layer by adding the cytokine leukemia inhibitory factor (LIF) to serum-containing culture medium (Smith, 2000). In another method, mouse ES cells can be grown in serum-free medium in the presence of bone morphogenetic protein and LIF (Ying et al., 2003).

[0273] Human ES cells can be obtained from blastocysts using previously described methods (Thomson et al., 1995; Thomson et al., 1998; Thomson and Marshall, 1998; Reubinoff et al., 2000). In one method, day 5 human blastocysts are incubated with rabbit anti-human ES cells. Trophectoderm cells are lysed by exposure to spleen cell antisera, followed by a 1:5 dilution of guinea pig complement. After removal of the lysed trophectoderm cells from the intact inner cell mass, the inner cell mass is cultured on a feeder layer of gamma-inactivated mouse embryonic fibroblasts in the presence of fetal bovine serum. After 9–15 days, clumps of cells derived from the inner cell mass can be chemically dissociated (i.e., exposed to trypsin) or mechanically dissociated and replated in fresh medium containing fetal bovine serum and a feeder layer of mouse embryonic fibroblasts. Following further expansion, colonies with undifferentiated morphology are selected with a micropipette, mechanically dissociated into clumps, and replated (see U.S. Patent No. 6,833,269). ES-like morphology is characterized by compact colonies with a clearly high nuclear-to-cytoplasm ratio and prominent nucleoli. The resulting ES cells can be routinely passaged by simple trypsinization or by selecting individual colonies with a micropipette. In some methods, human ES cells can be grown without serum by culturing them on a fibroblast feeder layer in the presence of basic fibroblast growth factor (Amit et al., 2000). In other methods, human ES cells can be grown without a feeder cell layer by culturing the cells on a protein matrix such as Matrigel™ or laminin in the presence of a "conditioned" medium containing basic fibroblast growth factor (Xu et al., 2001). The medium is preconditioned by co-culturing with fibroblasts.

[0274] Methods for isolating rhesus monkey and common marmoset ES cells are also known (Thomson and Marshall, 1998; Thomson et al., 1995; Thomson and Odorico, 2000).

[0275] Another source of ES cells is established ES cell lines.Various mouse cell lines and human ES cell lines are known, and the conditions for their growth and propagation have been defined.For example, the mouse CGR8 cell line was established from the inner cell mass of mouse strain 129 embryos, and the culture of CGR8 cells can be grown in the presence of LIF without using a feeder layer.As another example, human ES cell lines H1, H7, H9, H13 and H14 were established by Thompson et al.In addition, subclones H9.1 and H9.2 of the H9 line have been developed.

[0276] The source of ES cells can be blastocysts, cells derived from the inner cell mass culture of blastocysts, or cells obtained by culturing established cell lines.Therefore, as used herein, the term "ES cells" can refer to inner cell mass cells of blastocysts, ES cells obtained by culturing inner mass cells, and ES cells obtained by culturing ES cell lines.

[0277] Induced pluripotent stem (iPS) cells are cells that have the characteristics of embryonic stem cells but are obtained by reprogramming differentiated somatic cells. Induced pluripotent stem cells have been obtained by a variety of methods. In one method, human adult dermal fibroblasts are transfected with the transcription factors Oct4, Sox2, c-Myc, and Klf4 using retroviral transduction (Takahashi et al., 2007). The transfected cells are called basal fibroblasts. The cells are plated onto SNL feeder cells (a mouse fibroblast cell line that produces LIF) in medium supplemented with growth factor (bFGF). After approximately 25 days, colonies resembling human ES cell colonies appear in the culture. ES cell-like colonies are selected and expanded on feeder cells in the presence of bFGF.

[0278] Based on cellular characteristics, the cells in the ES cell-like colonies are induced pluripotent stem cells. Induced pluripotent stem cells are morphologically similar to human ES cells and express various human ES cell markers. Furthermore, when grown under conditions known to result in the differentiation of human ES cells, induced pluripotent stem cells differentiate accordingly. For example, induced pluripotent stem cells can differentiate into cells with neuronal structures and neural cell markers.

[0279] In another method, human fetal or neonatal fibroblasts are transfected with four genes, Oct4, Sox2, Nanog, and Lin28, using lentiviral transduction (Yu et al., 2007). Twelve to twenty days after infection, colonies with human ES cell morphology become visible. Colonies are selected and expanded. The induced pluripotent stem cells that comprise the colonies are morphologically similar to human ES cells, express various human ES cell markers, and, after injection into mice, form teratomas containing neural tissue, cartilage, and gastrointestinal epithelium.

[0280] Methods for preparing induced pluripotent stem cells from mice are also known (Takahashi and Yamanaka, 2006). In order to derive iPS cells, it is generally necessary to clone at least one member of the Sox family. Expression of or exposure to at least one member of the Bar and Oct families is required.Sox and Oct are thought to be at the center of the transcriptional regulatory hierarchy that determines ES cell identity.For example, Sox can be Sox-1, Sox-2, Sox-3, Sox-15, or Sox-18; Oct can be Oct-4.Additional factors such as Nanog, Lin28, Klf4, or c-Myc can increase reprogramming efficiency; a specific set of reprogramming factors can be a set that includes Sox-2, Oct-4, Nanog, and optionally Lin-28; or a set that includes Sox-2, Oct4, Klf, and optionally c-Myc.

[0281] iPS cells, like ES cells, were cultured using antibodies against SSEA-1, SSEA-3, and SSEA-4 (Developmental Studies Hybridoma Bank, National Institute of Child Health and Human Development, Bethesda, MD), and TRA-1-60. and TRA-1-81 antibodies (Andrews et al., 1987). They have characteristic antigens that can be identified or confirmed by chemical testing or flow cytometry. The pluripotency of embryonic stem cells is approximately 0.5–10 × 10 6 This can be confirmed by injecting 1000 cells into the hind limb muscles of 8-12 week old male SCID mice. Teratomas displaying at least one cell type from each of the three germ layers develop. VII. Methods of Using the Cells

[0282] The present disclosure U The HSC-iNKT cells may or may not be utilized immediately after generation. U The HSC-iNKT cells are stored for later purposes. U The HSC-iNKT cells can be utilized for therapeutic or prophylactic applications in mammalian subjects, such as patients (humans, dogs, cats, horses, etc.), who may be in need of cell therapy, including allogeneic cell therapy, for any type of medical condition.

[0283] A therapeutically effective amount of the present disclosure UA method of treating a patient with HSC-iNKT cells includes administering the cells or a clonal population thereof to the patient. The cells or cell population can be allogeneic to the patient. In certain embodiments, the patient shows no signs of depletion of the cells or cell population. The patient may or may not have a disease or condition involving cancer and / or inflammation. In certain embodiments, where the patient has cancer, after administering the cells or cell population to the cancer patient, tumor cells in the patient are killed. In certain cases, where the patient has inflammation, after administering the cells or cell population to the patient, the inflammation is reduced. In certain embodiments of the treatment method, the method further includes administering to the patient a compound that induces a suicide gene product.

[0284] For patients with cancer, it is anticipated that this cell product, once infused into the patient, will be able to target and eradicate tumor cells using multiple mechanisms. + They can directly recognize tumor cells and kill them by cytotoxicity. The injected cells can secrete cytokines such as IFN-γ, which can activate NK cells to kill HLA-negative tumor cells, and also activate DCs, which then stimulate cytotoxic T cells to kill HLA-positive tumor cells. Therefore, a series of in vitro and in vivo tests are planned to demonstrate the pharmacological efficacy of this cell product for cancer treatment.

[0285] U HSC-iNKT cells can target a wide range of cancers without tumor antigen or MHC restriction, making them suitable for off-the-shelf development. U HSC-iNKT cell products are useful as general cancer immunotherapy for treating any type of cancer and a large population of cancer patients. In certain cases, the therapy is useful for patients with cancers that have been clinically shown to be subject to iNKT cell regulation, including, for example, many types of solid tumors (melanoma, colon cancer, lung cancer, breast cancer, and head and neck cancer) and blood cancers (leukemia, multiple myeloma, and myelodysplastic syndrome).

[0286] In some embodiments of any of the methods disclosed above, the subject has or is at risk of having an autoimmune disease, graft-versus-host disease (GVHD), or graft rejection. The subject may be a subject who has been diagnosed with such a disease or a subject who has been determined to have a predisposition to such a disease based on genetic or family history analysis. The subject may also be a subject preparing for or who has undergone a transplant. In some embodiments, the method is for treating an autoimmune disease, GVHD, or graft rejection.

[0287] The individual treated with the cell therapy is U The patient may or may not have received treatment for the particular medical condition prior to receiving HSC-iNKT cell therapy. If the individual has cancer, the cancer may be primary, metastatic, refractory, etc. Patients who have exhausted conventional treatment options.

[0288] In certain embodiments, cells are administered to a patient at a dose of 10 cells per dose. 7 ~10 9 In certain embodiments, the administration regimen comprises lymphodepleting conditioning followed by allogeneic U A single administration of HSC-iNKT cells. For example, cells can be administered intravenously after lymphodepletion conditioning with fludarabine and cyclophosphamide.

[0289] To characterize the in vivo antitumor efficacy for subsequent in vivo therapeutic use, the in vivo pharmacological response was assessed by administering increasing doses (1 x 10) of IL-16 to tumor-bearing NSG mice. 6 pieces, 5×10 6 pieces, 10×10 6 (pieces) U Treatment with HSC-iNKT cells (n=8 per group) can be used to measure the response; treatment with PBS can be included as a control. As an example, two tumor models can be used: A375.CD1d (1×10 6 , sc) can be used as a solid tumor model, and MM.1S.Luc (5 × 106 (iv) can be used as a hematological malignancy model. Tumor growth can be monitored by either measuring size (A375.CD1d) or bioluminescence imaging (MM.1S.Luc). Antitumor immune responses can be measured by PET imaging, periodic blood sampling, and end-point tumor collection followed by flow cytometry and qPCR. U Inhibition of tumor growth in response to treatment with HSC-iNKT U The therapeutic efficacy of HSC-iNKT cell therapy may be demonstrated. Correlation of tumor inhibition with iNKT dose may confirm the therapeutic role of iNKT cells and indicate an effective therapeutic window for human therapy. Detection of iNKT cell responses against tumors may demonstrate the pharmacological antitumor activity of these cells in vivo.

[0290] The methods can be used on individuals who test positive for a medical condition, who have one or more symptoms of the medical condition, or who are thought to be at risk for developing such a condition. In some embodiments, the compositions and methods described herein are used to treat the inflammatory or autoimmune components of disorders listed herein and / or known in the art.

[0291] Certain aspects of the present disclosure relate to the treatment of cancer and / or the use of cancer antigens. The cancer or antigen to be treated can be any cancer known in the art, or an antigen associated with, for example, epithelial cancer (e.g., breast cancer, digestive cancer, lung cancer), prostate cancer, bladder cancer, lung cancer (e.g., small cell lung cancer), colon cancer, ovarian cancer, brain cancer, stomach cancer, renal cell carcinoma, pancreatic cancer, liver cancer, esophageal cancer, head and neck cancer, or colorectal cancer. In some embodiments, the cancer or antigen being treated is from one of the following cancers: adrenocortical carcinoma, idiopathic myeloid metaplasia, AIDS-related cancer (e.g., AIDS-related lymphoma), anal cancer, appendiceal cancer, astrocytoma (e.g., cerebellar astrocytoma and cerebral astrocytoma), basal cell carcinoma, bile duct cancer (e.g., extrahepatic bile duct carcinoma), 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, meningeal sarcoma, craniopharyngioma, hemangioblastoma, medulloblastoma, supratentorial primitive neuroectodermal tumor, optic tract and hypothalamic glioma, and glioblastoma), breast cancer, bronchial adenoma / carcinoid, carcinoid tumors (e.g., gastrointestinal carcinoid tumors), cancer of unknown primary site, central nervous system lymphoma, cervical cancer, colon cancer, colorectal cancer, chronic myeloproliferative disorder, 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 tumors, gastrointestinal stromal tumors (GIST), germ cell tumors (e.g., extracranial germ cell tumors, extragonadal germ cell tumors, ovarian germ cell tumors), gestational trophoblastic tumors, head and neck cancer, hepatocellular carcinoma (liver cancer) (e.g., hepatocellular carcinoma and hepatoma), hypopharyngeal cancer, islet cell carcinoma (pancreatic endocrine cancer), laryngeal cancer, leukemia, lip and oral cavity cancer, oral cancer cancer), liver cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), lymphoid neoplasms (e.g., lymphoma), medulloblastoma, ovarian cancer, mesothelioma, metastatic squamous cell cervical cancer, oral cancercancer), multiple endocrine neoplasia syndrome, myelodysplastic syndrome, myelodysplastic / myeloproliferative disorders, 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 peritoneum, pharyngeal cancer, pheochromocytoma, pineoblastoma and supratentorial primitive neuroectodermal tumor, pituitary tumor, pleuropulmonary blastoma, lymphoma, primary central nervous system lymphoma (microglioma), pulmonary lymphangioleiomyomatosis, rectal cancer, renal cancer cancer), renal pelvis and ureter cancer (transitional cell carcinoma), 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 carcinoma, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, tuberous sclerosis complex, urethral cancer, vaginal cancer, vulvar cancer, Wilms tumor, and post-transplant lymphoproliferative disorder (PTLD), abnormal blood vessel growth associated with nevus syndrome, edema (e.g., associated with a brain tumor), or Meigs syndrome.

[0292] Certain aspects of the present disclosure relate to the treatment of autoimmune conditions and / or the use of autoimmune-associated antigens. The autoimmune disease or antigen to be treated can be any autoimmune condition known in the art, or, for example, an antigen associated with diabetes, transplant rejection, GVHC, arthritis (rheumatoid arthritis, e.g., acute arthritis, chronic rheumatoid arthritis, gout or gouty arthritis, acute gouty arthritis, acute immune-mediated arthritis, chronic inflammatory arthritis, osteoarthritis, type II collagen-induced arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, Still's disease, spondyloarthritis, and juvenile-onset rheumatoid arthritis, osteoarthritis, chronic progressive arthritis, osteoarthritis, chronic primary polyarthritis, reactive arthritis, and ankylosing spondylitis), inflammatory hyperproliferative skin diseases, psoriasis such as plaque psoriasis, guttate psoriasis, pustular psoriasis, and psoriasis of the nails, atopic dermatitis such as hay fever and Job's syndrome. Atopic dermatitis including eczema, contact dermatitis, chronic contact dermatitis, exfoliative dermatitis, allergic dermatitis, allergic contact dermatitis, dermatitis herpetiformis, nummular dermatitis, seborrheic dermatitis, nonspecific dermatitis, primary irritant contact dermatitis, and atopic dermatitis; X-linked hyper-IgM syndrome, allergic intraocular inflammatory disease, urticaria such as chronic allergic urticaria and chronic idiopathic urticaria including chronic autoimmune urticaria; myositis, polymyositis / dermatomyositis; juvenile dermatosis Myositis, toxic epidermal necrolysis, scleroderma (including systemic sclerosis), multiple sclerosis (MS) such as systemic sclerosis, spinal-optic MS, primary progressive MS (PPMS), and relapsing-remitting MS (RRMS), progressive systemic sclerosis, atherosclerosis, arteriosclerosis, disseminated sclerosis, ataxic sclerosis, neuromyelitis optica (NMO), inflammatory bowel disease (IBD) (e.g., Crohn's disease, autoimmune-mediated gastrointestinal diseases, ulcerative colitis, colitis, ulcerative colitis (colitis ulcerosa), microscopic colitis, collagenous colitis, polypoid colitis, necrotizing enterocolitis, and transmural colitis, as well as autoimmune inflammatory bowel diseases), enteritis, pyoderma gangrenosum, erythema nodosum, primary sclerosing cholangitis, respiratory distress syndromes including adult or acute respiratory distress syndrome (ARDS), meningitis, inflammation of all or part of the uvea, iritis, choroiditis, autoimmune hematologic disorders, rheumatoid spondylitis, rheumatoid synovitis, hereditary angioedema, cranial nerve damage as seen in meningitis, herpes gestationis, pemphigoid of gestationis, scrotal pruritus (pruritis) scroti), autoimmune premature ovarian failure, sudden hearing loss due to autoimmune conditions, 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, non-granulomatous uveitis, phacoantigenic uveitis, posterior uveitis, or autoimmune uveitis, Glomerulonephritis (GN) with and without nephrotic syndrome, e.g., chronic or acute glomerulonephritis, such as primary GN, immune-mediated GN, membranous GN (membranous nephropathy), idiopathic membranous GN or idiopathic membranous nephropathy, membranous or membranoproliferative GN (MPGN), including types I and II, and rapidly progressive GN, proliferative nephritis, autoimmune polyendocrine dysfunction, balanitis circumscripta, balanitis including erythema annulare centrifugum, erythema dyschromicus perstans, erythema multiforme, granuloma annulare, lichen sclerosus and atrophicus, lichen simplex chronicus, lichen spinous, lichen planus, ichthyosis lamellar, epidermolytic keratosis, precancerous keratosis, pyoderma gangrenosum, allergic conditions and responses, allergic reactions, eczema including allergic or atopic eczema, asteatotic eczema, dyshidrotic eczema, and bullous palmoplantar eczema, asthma bronchiale, bronchial asthma asthma, and autoimmune asthma; conditions with T cell infiltration and chronic inflammatory responses, immune responses to foreign antigens such as fetal ABO blood group during pregnancy; chronic pulmonary inflammatory disease; autoimmune myocarditis, leukocyte adhesion deficiency, lupus nephritis, lupus encephalitis, lupus including pediatric lupus, non-renal lupus, extrarenal lupus, discoid lupus and discoid lupus erythematosus, depilatory lupus, systemic lupus erythematosus (SLE) such as cutaneous SLE or subacute cutaneous SLE, neonatal lupus syndrome (NLE), and disseminated lupus erythematosus; juvenile-onset (type 1) diabetes, including childhood insulin-dependent diabetes mellitus (IDDM), and adult-onset diabetes mellitus (type 2 diabetes), and autoimmune diabetes.Also contemplated are immune responses associated with acute and delayed hyp...

Claims

1. An engineered invariant natural killer T (iNKT) cell that expresses at least one invariant natural killer (iNKT) T cell receptor (TCR) and in which the genome of the cell has been modified to (1) express an exogenous suicide gene product and (2) eliminate surface expression of at least one HLA-I or HLA-II molecule; wherein the at least one iNKT TCR is expressed from an exogenous nucleic acid and / or from an endogenous invariant TCR gene under the transcriptional control of a recombinantly modified promoter region; the engineered invariant natural killer cells are a) selecting CD34+ cells from a plurality of hematopoietic stem or progenitor cells; b) introducing one or more nucleic acids encoding at least one human invariant natural killer (iNKT) T cell receptor (TCR); c) eliminating surface expression of one or more HLA-I and / or HLA-II molecules on the isolated human CD34+ cells; d) culturing the isolated CD34+ cells expressing the iNKT TCR to produce said invariant natural killer iNKT cells; 2. An engineered iNKT cell prepared by a method comprising:

2. The engineered iNKT cell described in claim 1, wherein the invariant TCR gene product is an alpha TCR gene product.

3. An engineered iNKT cell described in claim 1 or 2, wherein the invariant TCR gene product is a beta TCR gene product.

4. An engineered iNKT cell described in any one of claims 1 to 3, in which both an alpha TCR gene product and a beta TCR gene product are expressed.

5. An engineered iNKT cell described in any of claims 1 to 4, wherein at least one invariant TCR gene product is expressed from an exogenous nucleic acid.

6. An engineered iNKT cell described in any of claims 1 to 5, wherein the exogenous suicide gene product and / or exogenous nucleic acid has one or more codons optimized for expression in the cell.

7. The engineered iNKT cell of any one of claims 1 to 6, wherein the suicide gene product is herpes simplex virus thymidine kinase (HSV-TK), purine nucleoside phosphorylase (PNP), cytosine deaminase (CD), carboxypeptidase G2, cytochrome P450, linamarase, beta-lactamase, nitroreductase (NTR), carboxypeptidase A, or inducible caspase 9.

8. The engineered iNKT cell of any of claims 1 to 7, wherein the iNKT cell does not express surface HLA-I or surface HLA-II molecules by disrupting expression of genes encoding beta-2-microglobulin (B2M), major histocompatibility complex II transactivator (CIITA), and / or individual HLA-I and HLA-II molecules.

9. An engineered iNKT cell described in any one of claims 1 to 8, wherein the iNKT cell contains nucleic acid derived from a recombinant vector introduced into the cell.

10. An engineered iNKT cell according to any one of claims 1 to 9, wherein the cell has not been exposed to a medium containing animal serum.

11. The engineered iNKT cells of any one of claims 1 to 10, wherein the cells have been pre-frozen and are stable at room temperature for at least 1 hour.

12. An engineered iNKT cell described in any one of claims 1 to 11, wherein the suicide gene product is activated by a substrate.

13. An engineered iNKT cell described in any of claims 1 to 12, wherein the cell contains an exogenous nucleic acid encoding a polypeptide having a substrate that can be labeled for imaging.

14. A method for preparing the engineered invariant natural killer T (iNKT) cells of claim 1, comprising: a) selecting CD34+ cells from a plurality of hematopoietic stem or progenitor cells; b) introducing one or more nucleic acids encoding at least one human invariant natural killer (iNKT) T cell receptor (TCR); c) eliminating surface expression of one or more HLA-I and / or HLA-II molecules on the isolated human CD34+ cells; d) culturing the isolated CD34+ cells expressing the iNKT TCR to produce said invariant natural killer iNKT cells; wherein the method comprises culturing isolated CD34+ cells that express an iNKT TCR in a culture medium that is free of animal serum.

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