Operated Stem Cells and Their Use

Abstract

Compositions and methods are provided for a cell population comprising engineered stem cells comprising synthetic cytokine receptors for non-physiological ligands. The non-physiological ligands activate the synthetic cytokine receptors within the engineered stem cells to induce differentiation of the stem cells and expansion and / or activation of the resulting cytotoxic natural lymphocyte-like cells. TIFF2025524373000046.tif51159

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority to U.S. Provisional Application No. 63 / 351,144, entitled "Engineered Stem Cells and Uses Thereof", filed on June 10, 2022; U.S. Provisional Application No. 63 / 392,861, entitled "Engineered Stem Cells and Uses Thereof", filed on July 27, 2022; U.S. Provisional Application No. 63 / 411,065, entitled "Engineered Stem Cells and Uses Thereof", filed on September 28, 2022; U.S. Provisional Application No. 63 / 422,882, entitled "Engineered Stem Cells and Uses Thereof", filed on November 4, 2022; U.S. Provisional Application No. 63 / 447,337, entitled "Engineered Stem Cells and Uses Thereof", filed on February 21, 2023; and U.S. Provisional Application No. 63 / 451,536, entitled "Engineered Stem Cells and Uses Thereof", filed on March 10, 2023, and incorporates by reference the entire contents of each thereof.

[0002] Incorporation by Reference of Sequence Listing This application is filed with a sequence listing in electronic form. The sequence listing is provided as a file entitled 260132000340SeqList.xml created on June 9, 2023, and its size is 83,181 bytes. The electronic form information of the sequence listing is incorporated by reference in its entirety.

[0003] Field The present disclosure provides compositions and methods related to cell populations comprising engineered stem cells that include synthetic cytokine receptors for non - physiological ligands. The non - physiological ligands activate the synthetic cytokine receptors within the engineered stem cells to induce differentiation of the stem cells and expansion and / or activation of the resulting cytotoxic natural lymphocyte - like cells.

Background Art

[0004] Background Cytotoxic innate lymphoid cells (CIL) are a class of immune cells that can be used in immunotherapies, including cancer immunotherapy. One type of CIL is the natural killer (NK) cell, which is a type of cell that is generally identified as being positive for the cell surface protein CD56 (CD56+) and other markers and having cytotoxic activity.

[0005] CIL cells for use in immunotherapy can be obtained from primary sources such as peripheral blood or umbilical cord blood. Artificial sources of CIL cells include pluripotent stem cells, including induced pluripotent stem cells (iPSC) that are derived from somatic cells (generally fibroblasts or peripheral blood mononuclear cells [PBMC]) that have been induced to be capable of unlimited proliferation and differentiation into other cell types when subjected to appropriate differentiation conditions, and human embryonic stem cells (hESC). CIL cells can be derived from iPSCs by sequentially differentiating the iPSCs into hematopoietic progenitor cells (HPC), also called hematopoietic stem cells (HSC), the HPC into common lymphoid progenitor cells (CLP), and then the CLP into CIL cells called iPSC-derived cytotoxic innate lymphoid cells (iPSC-CIL). Generally, iPSC-CIL cells express CD56 and have cytotoxic activity, similar to NK cells, but iPSC-CIL cells can be different from NK cells in phenotype and other respects.

[0006] Methods are known for differentiating iPSCs into CD34+ HPC using either embryoid bodies (EB) or culture of single iPSCs using feeder cells. The CD34+ HPC can then be differentiated into CLP.

[0007] There is still a need in the art for compositions and methods related to engineered stem cells, methods of making such cells, methods of differentiating such cells into CIL, and methods of using them in immunotherapy.

Summary of the Invention

[0008] Summary of the Disclosure The present disclosure is based in part on the discovery that stem cells engineered to express a synthetic cytokine receptor improve or enhance differentiation into hematopoietic precursors and CLPs in response to a cognate non-physiological ligand of the receptor. Such precursors are then differentiated into engineered CIL cells. As demonstrated herein, stem cells are genetically engineered to express a synthetic cytokine receptor and, in some embodiments, genes are simultaneously disrupted to avoid immune rejection (e.g., beta-2-microglobulin) and / or to provide resistance to rapamycin using CRISPR.

[0009] According to the methods described herein, CIL cells can be produced in large quantities from engineered stem cells to have desired functional characteristics. Non-limiting advantages of certain embodiments include the ability to differentiate or supplement exogenous factors without using exogenous factors, e.g., SCF, TPO, BMP4, FGF, and / or VEGF, to differentiate the CIL cells described herein. Furthermore, CIL cells expressing a synthetic cytokine receptor provide the ability to expand and proliferate the CIL cells described herein without using exogenous factors, e.g., IL-2, IL-7, IL-15, and / or IL-21. The CIL cells and related compositions described herein can be used in immunotherapy using ex vivo expansion and proliferation.

[0010] Accordingly, in some aspects, the present disclosure provides engineered stem cells comprising synthetic cytokine receptors for non-physiological ligands, the cytokine receptors comprising a synthetic gamma chain polypeptide comprising a first dimerization domain, a first transmembrane domain, and an interleukin-2 receptor subunit gamma (IL-2RG) intracellular domain, and a synthetic beta chain polypeptide comprising a second dimerization domain, a second transmembrane domain, and an intracellular domain selected from an interleukin-2 receptor subunit beta (IL-2RB) intracellular domain, an interleukin-7 receptor subunit beta (IL-7RB) intracellular domain, and / or an interleukin-21 receptor subunit beta (IL-21RB) intracellular domain.

[0011] In some embodiments, the first dimerization domain and the second dimerization domain are extracellular domains. In some embodiments, the synthetic gamma chain polypeptide comprises, in order from N-terminus to C-terminus, the first dimerization domain, the first transmembrane domain, and the interleukin-2 receptor subunit gamma (IL-2RG) intracellular domain, and the synthetic beta chain polypeptide comprises, in order from N-terminus to C-terminus, the second dimerization domain, the second transmembrane domain, and the intracellular domain.

[0012] In some embodiments, the IL-2RG intracellular domain comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:1, or the polypeptide sequence set forth in SEQ ID NO:1. In some embodiments, the first transmembrane domain comprises the IL-2RG transmembrane domain. In some embodiments, the IL-2RG transmembrane domain comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:8 or 31, or the polypeptide sequence set forth in SEQ ID NO:8 or 31.

[0013] In some embodiments, the beta chain intracellular domain comprises the IL-2RB intracellular domain. In some embodiments, the IL-2RB intracellular domain comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:2, or the polypeptide sequence set forth in SEQ ID NO:2.

[0014] In some embodiments, the beta chain intracellular domain comprises the IL-7RB intracellular domain. In some embodiments, the IL-7RB intracellular domain comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:3, or the polypeptide sequence set forth in SEQ ID NO:3.

[0015] In some embodiments, the beta chain intracellular domain comprises the IL-21RB intracellular domain. In some embodiments, the IL-21RB intracellular domain comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:4, or the polypeptide sequence set forth in SEQ ID NO:4.

[0016] In some embodiments, the second transmembrane domain comprises a transmembrane domain derived from the same beta chain intracellular domain. In some embodiments, the second transmembrane domain is the transmembrane domain of IL-2RB that comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:35 or 36, or the polypeptide sequence set forth in SEQ ID NO:35 or 36.

[0017] In some embodiments, the synthetic gamma chain polypeptide contains the IL-2RG TM domain comprising the sequence set forth in SEQ ID NO:8 or 31 and the IL-2RG intracellular domain comprising the sequence set forth in SEQ ID NO:1, and the synthetic beta chain polypeptide contains the IL-2RB TM domain comprising the sequence set forth in SEQ ID NO:35 or 36 and the IL-2RB intracellular domain comprising the sequence set forth in SEQ ID NO:2.

[0018] In some embodiments, the first dimerization domain and the second dimerization domain are heterodimerization domains selected from the 12 kD FK506-binding protein (FKBP) and the FKBP12-rapamycin binding (FRB) domain. In some embodiments, the non-physiological ligand is rapamycin or a rapalog. In some embodiments, the FRB domain comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:6 or SEQ ID NO:7. In some embodiments, the FRB domain comprises the polypeptide sequence set forth in SEQ ID NO:6 or SEQ ID NO:7. In some embodiments, the FKBP domain comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:5, SEQ ID NO:49 or SEQ ID NO:30.

[0019] In some embodiments, the FKBP domain comprises the polypeptide sequence set forth in SEQ ID NO:5, SEQ ID NO:49 or SEQ ID NO:30. In some embodiments, the first dimerization domain and the second dimerization domain are heterodimerization domains selected from the 12 kD FK506-binding protein (FKBP) and the calcineurin domain. In some embodiments, the non-physiological ligand is FK506 or an analog thereof. In some embodiments, the FKBP domain comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:5, SEQ ID NO:49 or SEQ ID NO:30. In some embodiments, the FKBP domain comprises the polypeptide sequence set forth in SEQ ID NO:5, SEQ ID NO:49 or SEQ ID NO:30.

[0020] In some embodiments of any aspect, the synthetic gamma chain polypeptide has the amino acid sequence set forth in SEQ ID NO:28, or an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:28, and the synthetic beta chain polypeptide has the amino acid sequence set forth in SEQ ID NO:33, or an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:33. In some embodiments of any aspect, the synthetic gamma chain polypeptide has the amino acid sequence set forth in SEQ ID O:28, and the synthetic beta chain polypeptide has the amino acid sequence set forth in SEQ ID NO:33.

[0021] In some embodiments, the first dimerization domain and the second dimerization domain are homodimerization domains selected from: i) FK506-binding protein (FKBP) of size 12kD; ii) cyclophilin A (CypA); or iii) gyrase B (CyrB), and the non-physiological ligands are, respectively, i) FK1012, AP1510, AP1903 or AP20187 or analogs thereof; ii) cyclosporine-A (CsA) or analogs thereof; or iii) coumermycin or analogs thereof.

[0022] In some embodiments of any aspect, the stem cells are pluripotent stem cells. In some embodiments, the stem cells are induced pluripotent stem cells (iPSCs).

[0023] In some embodiments, the stem cells are resistant to rapamycin-mediated mTOR inhibition.

[0024] In some embodiments, the stem cells express a cytosolic polypeptide that binds to a non-physiological ligand. In some embodiments, the non-physiological ligand is rapamycin or a rapalog, and the stem cells express a cytosolic FRB domain or a variant thereof. In some embodiments, the cytosolic FRB domain comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:6 or SEQ ID NO:7. In some embodiments, the cytosolic FRB domain comprises a polypeptide sequence that is at least 98% identical to SEQ ID NO:6 or SEQ ID NO:7.

[0025] In some embodiments, the stem cells comprise a disrupted FKBP12 gene that reduces the expression of FKBP12. In some embodiments, the stem cells comprise a knockout of the FKBP12 gene.

[0026] In some embodiments, the stem cells comprise a nucleotide sequence encoding a synthetic cytokine receptor that is inserted into the genome of the stem cells. In some embodiments, the nucleotide sequence encoding the synthetic cytokine receptor is inserted into a non-target locus within the genome of the stem cells. In some embodiments, the nucleotide sequence encoding the synthetic cytokine receptor is inserted into an endogenous gene of the stem cells. In some embodiments, the insertion reduces the expression of the endogenous gene within the locus. In some embodiments, the insertion knocks out the endogenous gene within the locus. In some embodiments, the insertion is by homologous recombination repair.

[0027] In some embodiments, the endogenous gene is a housekeeping gene, a blood lineage-specific locus, or an immune-related gene. In some embodiments, the endogenous gene is a housekeeping gene, and the housekeeping gene is selected from eukaryotic translation elongation factor 1 alpha (EEF1A), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ubiquitin C (UBC), and actin beta (ACTB). In some embodiments, the endogenous gene is a blood lineage-specific locus, and the blood lineage-specific locus is selected from protein tyrosine phosphatase receptor type C (PTPRC), IL2RG, and IL2RB. In some embodiments, the immune-related gene is selected from the beta-2-microglobulin (B2M) gene, the T cell receptor alpha constant (TRAC) gene, and the signal regulatory protein alpha (SIRPA) gene. In some embodiments, the endogenous gene is B2M. In some embodiments, the stem cells comprise a B2M knockout. In some embodiments, the cell has a disruption of the gene encoding FKBP12. In some embodiments, the disruption is an FKBP12 knockout that inactivates the gene encoding FKBP12.

[0028] In some embodiments, the stem cells comprise a B2M knockout and an FKBP12 knockout.

[0029] In some embodiments, the stem cells comprise a chimeric antigen receptor (CAR). In some embodiments, the CAR is an anti-FITC CAR.

[0030] In some of any of the embodiments, binding of a non-physiological ligand to the synthetic cytokine receptor activates the synthetic cytokine receptor in the stem cell to induce differentiation of the engineered stem cells within the cell population.

[0031] Also provided herein is a cell population comprising any of the engineered stem cells provided.

[0032] Accordingly, in some aspects, the present disclosure provides a cell population comprising engineered stem cells comprising synthetic cytokine receptors for non-physiological ligands, wherein the cytokine receptor comprises a synthetic gamma chain polypeptide comprising a first dimerization domain, a first transmembrane domain, and an interleukin-2 receptor subunit gamma (IL-2RG) intracellular domain, and a synthetic beta chain polypeptide comprising a second dimerization domain, a second transmembrane domain, and an intracellular domain selected from an interleukin-2 receptor subunit beta (IL-2RB) intracellular domain, an interleukin-7 receptor subunit beta (IL-7RB) intracellular domain, and / or an interleukin-21 receptor subunit beta (IL-21RB) intracellular domain, and wherein the non-physiological ligand activates the synthetic cytokine receptor in the stem cell to induce differentiation of the stem cell.

[0033] In some embodiments, the beta chain intracellular domain comprises the IL-2RB intracellular domain. In some embodiments, the IL-2RB intracellular domain comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:2, or the polypeptide sequence set forth in SEQ ID NO:2.

[0034] In some embodiments, the beta chain intracellular domain comprises the IL-7RB intracellular domain. In some embodiments, the IL-7RB intracellular domain comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:3, or the polypeptide sequence set forth in SEQ ID NO:3.

[0035] In some embodiments, the beta chain intracellular domain comprises the IL-21RB intracellular domain. In some embodiments, the IL-21RB intracellular domain comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:4, or the polypeptide sequence set forth in SEQ ID NO:4.

[0036] In some or any of the foregoing aspects, the first dimerization domain and the second dimerization domain are extracellular domains, and the synthetic gamma chain polypeptide comprises, in order from the N-terminus to the C-terminus, the first dimerization domain, the first transmembrane domain, and the interleukin-2 receptor subunit gamma (IL-2RG) intracellular domain, and the synthetic beta chain polypeptide comprises, in order from the N-terminus to the C-terminus, the second dimerization domain, the second transmembrane domain, and the intracellular domain.

[0037] In some or any of the foregoing aspects, the first dimerization domain and the second dimerization domain are heterodimerization domains selected from the FK506-binding protein (FKBP) of size 12kD and the FKBP12-rapamycin binding (FRB) domain, and / or the non-physiological ligand is rapamycin or a rapalog. In some aspects, the FKBP domain comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:5. In some aspects, the FKBP domain comprises the polypeptide sequence set forth in SEQ ID NO:5. In some aspects, the FKBP domain comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:49. In some aspects, the FKBP domain comprises the polypeptide sequence set forth in SEQ ID NO:49. In some aspects, the FRB domain comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:6 or SEQ ID NO:7. In some aspects, the FRB domain comprises the polypeptide sequence set forth in SEQ ID NO:6 or SEQ ID NO:7.

[0038] In some or any of the foregoing aspects, the first dimerization domain and the second dimerization domain are heterodimerization domains selected from FK506-binding protein (FKBP) of size 12 kD and the calcineurin domain, and / or the non-physiological ligand is FK506 or an analog thereof. In some aspects, the FKBP domain comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:5. In some aspects, the FKBP domain comprises the polypeptide sequence set forth in SEQ ID NO:5. In some aspects, the FKBP domain comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:49. In some aspects, the FKBP domain comprises the polypeptide sequence set forth in SEQ ID NO:49.

[0039] In some or any of the foregoing aspects, the first dimerization domain and the second dimerization domain are i) FK506-binding protein (FKBP) of size 12 kD; ii) Cyclophilin A (CypA); or iii) Gyrase B (CyrB) selected homodimerization domains, The non-physiological ligands are, respectively, i) FK1012, AP1510, AP1903 or AP20187 or an analog thereof; ii) Cyclosporin-A (CsA) or an analog thereof; or iii) Coumermycin or an analog thereof is.

[0040] In some or any of the foregoing embodiments, the stem cells express a cytosolic polypeptide that binds to a non-physiological ligand. In some embodiments, the non-physiological ligand is rapamycin or a rapalog, and the stem cells express a cytosolic FRB domain or a variant thereof. In some embodiments, the cytosolic FRB domain comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:6 or SEQ ID NO:7. In some embodiments, the cytosolic FRB domain comprises a polypeptide sequence that is at least 98% identical to SEQ ID NO:6 or SEQ ID NO:7.

[0041] In some or any of the foregoing embodiments, the stem cells are induced pluripotent stem cells (iPSCs).

[0042] In some or any of the foregoing embodiments, the stem cells comprise a nucleotide sequence encoding a synthetic cytokine receptor. In some embodiments, the nucleotide sequence is inserted into an endogenous gene of the stem cells. In some embodiments, the endogenous gene is a housekeeping gene or a blood lineage-specific locus. In some embodiments, the housekeeping gene is selected from eukaryotic translation elongation factor 1 alpha (EEF1A), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ubiquitin C (UBC), and actin beta (ACTB). In some embodiments, the blood lineage-specific locus is selected from protein tyrosine phosphatase receptor type C (PTPRC), IL2RG, and IL2RB. In some embodiments, the nucleotide sequence is inserted into a disrupted gene of the stem cells. In some embodiments, the disrupted gene is selected from the disrupted beta-2-microglobulin (B2M) gene, the disrupted T cell receptor alpha constant (TRAC) gene, and the disrupted signal regulatory protein alpha (SIRPA) gene.

[0043] In some or any of the foregoing embodiments, the stem cells contain a disrupted B2M gene. In some embodiments, the stem cells involve a decrease in B2M expression. In some embodiments, the stem cells are knocked out for B2M.

[0044] In some or any of the foregoing embodiments, the stem cells are rapamycin-resistant. In some embodiments, the rapamycin-resistant stem cells contain a disrupted FKBP12 gene. In some embodiments, the stem cells involve a decrease in FKBP12 expression. In some embodiments, the stem cells are knocked out for FKBP12.

[0045] In some or any of the foregoing embodiments, the non-physiological ligand activates a synthetic cytokine receptor within the stem cells to induce differentiation of the stem cells into hematopoietic precursors. In some embodiments, the non-physiological ligand activates a synthetic cytokine receptor within the stem cells to induce differentiation of the stem cells into common lymphoid progenitors (CLPs) or common myeloid progenitors (CMPs).

[0046] In some or any of the foregoing embodiments, the stem cells contain a chimeric antigen receptor (CAR).

[0047] In some aspects, the present disclosure provides a method for generating cytotoxic innate lymphoid (iCIL) cells, including the step of contacting a cell population of any one of the provided embodiments with a non-physiological ligand for a first period sufficient to generate CLPs, and the step of contacting the CLPs with a differentiation medium for a second period sufficient to generate iCILs.

[0048] In some embodiments, the differentiation medium contains stem cell factor (SCF), FLT3L, IL-7, IL-12, IL-15, SR-1, and UM729. In some embodiments, the differentiation medium contains a non-physiological ligand.

[0049] In some embodiments, the first period is 1 to 15 days, and the second period is 1 to 15 days. In some embodiments, the method includes contacting iCIL with a pre-activation medium comprising IL-7, IL-12, IL-15, IL-18, and IL-21 for a third period sufficient to produce mature iCIL. In some embodiments, the pre-activation medium comprises a non-physiological ligand. In some embodiments, the third period is 1 to 10 days.

[0050] In some or any of the foregoing embodiments, mature iCIL expresses NKp46, NKG2D, LFA1, DNAM1, CD16, and CD56.

[0051] In some aspects, the present disclosure provides a method of genetically engineering stem cells to express a synthetic cytokine receptor, the method comprising contacting a population of stem cells with a recombinant vector comprising (i) a guide RNA (gRNA) targeting a target site within an endogenous gene, (ii) an RNA-guided endonuclease, and (iii) a nucleotide sequence encoding a synthetic cytokine receptor for a non-physiological ligand, thereby inserting the nucleotide sequence into the endogenous gene. The cytokine receptor comprises a synthetic gamma chain polypeptide comprising a first dimerization domain, a first transmembrane domain, and an intracellular domain of interleukin-2 receptor subunit gamma (IL-2RG), and a synthetic beta chain polypeptide comprising a second dimerization domain, a second transmembrane domain, and an intracellular domain selected from the intracellular domain of interleukin-2 receptor subunit beta (IL-2RB), the intracellular domain of interleukin-7 receptor subunit beta (IL-7RB), and / or the intracellular domain of interleukin-21 receptor subunit beta (IL-21RB).

[0052] In some embodiments or aspects, the nucleotide sequence is inserted via homologous recombination repair (HDR). In some embodiments, the recombinant vector contains 5' and 3' homologous arms adjacent to the nucleotide sequence encoding the synthetic cytokine receptor, the 3' homologous arm is homologous to the region upstream of the gRNA target site, and the 5' homologous arm is homologous to the region downstream of the gRNA target site. In some embodiments or aspects, the method comprises contacting, from 5' to 3', a cell with a vector comprising a nucleic acid comprising (a) a nucleotide sequence homologous to the region located upstream of the target site, (b) a nucleotide sequence encoding a synthetic cytokine receptor for a non-physiological ligand, and (c) a nucleotide sequence homologous to the region located downstream, wherein a double-strand break occurs at the target site within the endogenous gene and the nucleic acid is exchanged with the homologous nucleotide sequence of the endogenous gene.

[0053] In some embodiments or aspects, the nucleotide sequence is inserted via non-homologous end joining (NHEJ).

[0054] In some or any of the foregoing embodiments, the RNA-guided endonuclease is selected from Cas endonucleases, Mad endonucleases, and Cpf1 endonucleases. In some embodiments, the RNA-guided endonuclease is Cas9. In some embodiments, the RNA-guided endonuclease is Mad7.

[0055] In some or any of the foregoing embodiments, the method comprises disrupting a target gene and inserting a nucleotide sequence encoding a synthetic cytokine receptor into the disrupted target gene, and the step of disrupting the target gene comprises contacting a population of stem cells with (i) a gRNA targeting a target site within the target gene and (ii) an RNA-guided endonuclease. In some embodiments, the endogenous target gene is selected from B2M, TRAC, and SIRPA.

[0056] In some embodiments of any aspect, the endogenous gene is B2M. In some embodiments, the gRNA comprises the sequence set forth in SEQ ID NO:18.

[0057] In some embodiments of any aspect, the nucleotide sequence homologous to the region located upstream of the target site comprises a nucleic acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:22, and the nucleotide sequence homologous to the region located downstream comprises a nucleic acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:23.

[0058] In some embodiments of any aspect, the nucleotide sequence encoding the synthetic cytokine receptor comprises a first nucleic acid sequence encoding a gamma chain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:37, and a second nucleic acid sequence encoding a beta chain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:38. In some embodiments, the nucleotide sequence encoding the synthetic cytokine receptor comprises the first nucleic acid as set forth in SEQ ID NO:37 and the second nucleic acid as set forth in SEQ ID NO:38.

[0059] In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are separated by a cleavable linker or an IRES. In some embodiments, the cleavable linker is a Protein Quantification Reporter Linker (PQR). In some embodiments, the PQR linker has the sequence set forth in SEQ ID NO:42.

[0060] In some of any embodiment, the nucleotide sequence encoding the synthetic cytokine receptor for a non-physiological ligand is under the functional control of a heterologous promoter. In some embodiments, the heterologous promoter is an EF1α promoter or an MND promoter. In some embodiments, the promoter is a dual promoter under the functional control of two promoters where the synthetic cytokine receptor is. In some embodiments, the dual promoter is a dual EF1α promoter.

[0061] In some of any embodiment, the nucleotide sequence encoding the synthetic cytokine receptor comprises a polyadenylation sequence.

[0062] In some embodiments, the recombinant vector comprises the sequence set forth in SEQ ID NO:40, or a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:40. In some embodiments, the recombinant vector has the sequence set forth in SEQ ID NO:40.

[0063] In some or any of the foregoing aspects, the method includes the step of manipulating a population of stem cells to be resistant to rapamycin. In some aspects, the resistance to rapamycin is rapamycin-mediated mTOR inhibition. In some aspects, the population of stem cells resistant to rapamycin includes disrupting the FKBP12 gene within the stem cells. In some aspects, the population of stem cells resistant to rapamycin has a reduced expression of FKBP12. In some aspects, the population of stem cells resistant to rapamycin includes knocking out the FKBP12 gene.

[0064] In some aspects, the stem cells are manipulated by CRISPR-Cas and gRNA targeting the FKBP12 gene to disrupt FKBP12 within the cell. In some of any aspect, the method includes the step of further contacting the population of stem cells with a guide RNA (gRNA) targeting a target site within the FKBP12 gene. In some aspects, the RNA-guided endonuclease is selected from Cas endonuclease, Mad endonuclease and Cpf1 endonuclease. In some aspects, the RNA-guided endonuclease is Cas9. In some aspects, the RNA-guided endonuclease is Mad7. In some aspects, the step of further contacting is performed, optionally in combination with the same RNA-guided endonuclease, by a guide RNA (gRNA) targeting a target site within an endogenous gene simultaneously with the step of contacting in (i).

[0065] In some aspects, the gRNA includes one or more gRNAs selected from gRNAs including the sequences set forth in SEQ ID NO:19, SEQ ID NO:20 or SEQ ID NO:21. In some aspects, the one or more gRNAs are a pool of gRNAs including two or three gRNAs.

[0066] In some of any aspect, the method further includes the step of introducing a chimeric antigen receptor (CAR) into the population of stem cells. In some aspects, the CAR is an anti-FITC CAR.

[0067] In some embodiments of any aspect, the stem cells are pluripotent stem cells.

[0068] In some or any of the foregoing embodiments, the stem cells are iPSCs.

[0069] In some embodiments of any aspect, (a) culturing a cell population comprising any of the engineered iPSCs provided herein under conditions that form aggregates; (b) culturing the cells produced in (a) under conditions that induce mesoderm formation in a plurality of cells, wherein the start of the culturing step in (b) is day 0; and (c) culturing the cells produced in (b) under conditions that differentiate the cells into a population of hematopoietic progenitors (HP), wherein at least a portion of one or more of steps (a)-(c) is performed in the presence of a non-physiological ligand of a synthetic cytokine receptor, a method for generating hematopoietic progenitor (HP) cells is provided herein.

[0070] In some embodiments, it includes culturing a cell population comprising engineered iPSCs of any of the provided embodiments under conditions that differentiate the iPSCs into cytotoxic innate lymphoid cells (iCIL), wherein the non-physiological ligand of the synthetic cytokine receptor is added during at least a portion of the culturing. A method for generating cytotoxic innate lymphoid cell (iCIL) cells is provided herein. In some embodiments, the culturing step includes: (a) culturing a cell population comprising engineered iPSCs under conditions that form aggregates; (b) culturing the cells produced in (a) under conditions that induce mesoderm formation in a plurality of cells, wherein the start of the culturing in (b) is day 0; (c) culturing the cells produced in (b) under conditions that differentiate the cells into a population of hematopoietic progenitors (HP); and (d) culturing the cells produced in (c) under conditions that generate iCIL cells, wherein at least a portion of one or more of steps (a)-(d) is performed in the presence of a non-physiological ligand of a synthetic cytokine receptor.

[0071] Accordingly, in some embodiments, a method for generating cytotoxic innate lymphoid cells (iCIL) is provided herein, comprising: (a) culturing a cell population comprising engineered iPSCs of any of the provided embodiments under conditions that form aggregates; (b) culturing the cells produced in (a) under conditions that induce mesoderm formation in a plurality of cells, wherein the start of the culturing step in (b) is day 0; (c) culturing the cells produced in (b) under conditions that differentiate the cells into a population of hematopoietic progenitors (HP); and (d) culturing the cells produced in (c) under conditions that generate iCIL cells, wherein at least a portion of one or more of steps (a)-(d) is performed in the presence of a non-physiological ligand of a synthetic cytokine receptor.

[0072] In some embodiments, the culturing is performed in a vessel that has been treated to promote cell attachment and proliferation. In some embodiments, the vessel is Matrigel. In some embodiments, the culturing is performed in a non-adherent culture vessel. In some embodiments, the non-adherent culture vessel is an Aggrewell™ plate. In some embodiments of the method, the aggregate of (a) is an embryoid body (EB).

[0073] In some embodiments, the culturing is performed in suspension. In some embodiments, the culturing is performed in a culture vessel that has not been treated to promote cell attachment and proliferation. In some of any such embodiments, the culturing in step a) comprises: (i) performing a first incubation comprising culturing a cell population of engineered stem cells under conditions that form a first aggregate; (ii) contacting the aggregate with a dissociating agent to form a population of dissociated cells; and (iii) performing a second incubation comprising culturing the population of dissociated cells under conditions that form a second aggregate. In some embodiments, the incubation is performed in suspension.

[0074] In some of any aspects of the method, the culture in (b) is in a medium containing one or more of BMP4, FGF2, VEGF, and a Rock inhibitor. In some aspects, the Rock inhibitor is Y27632. In some aspects, the culture in (b) is in a medium containing BMP4, FGF2, VEGF, and Y27632. In some aspects, the culture in (b) is in a medium containing BMP4, FGF2, and VEGF. In some of any aspects, the culture in (b) is in a medium containing a non-physiological ligand. In some aspects, the culture in (b) is in a medium containing a non-physiological ligand and no additional growth factors. In some aspects, the culture in (b) lasts for 2 to 4 days. In some aspects, the culture in (b) lasts for 3 days or about 3 days.

[0075] In some of any aspects, the culture in (c) is in a medium containing one or more of BMP4, FGF2, VEGF, TPO, SCF, and LDL. In some of any aspects, the culture in (c) is in a medium containing one or more of BMP4, FGF2, VEGF, and LDL. In some of any aspects, the culture in (c) is in a medium containing BMP4 and FGF2. In some of any aspects, the culture with BMP4 and FGF2 in (c) lasts from day 3 to day 15. In some of any aspects, the culture in (c) contains a PI3K inhibitor. In some of any aspects, the PI3K inhibitor is LY2940002. In some of any aspects, the PI3K inhibitor is added during part of the culture in (c). In some of any aspects, the PI3K inhibitor is added from about day 6 to day 15.

[0076] In some of any aspects, the culture in (c) is in a medium without SCF and TPO. In some aspects, the culture in (c) is in a medium containing a non-physiological ligand. In some aspects, the culture in (c) is in a medium containing a non-physiological ligand and no arbitrary additional growth factors, cytokines, or both.

[0077] In some embodiments, the culture in (c) is for 3 to 15 days. In some embodiments, during at least a portion of the culture in (c), the medium contains an aryl hydrocarbon receptor (AHR) antagonist (e.g., StemRegenin-1), a pyrido-[4,5-b]-indole derivative (e.g., UM729), or both. In some embodiments, a portion of the culturing step is on days 9 to 15 or about days 9 to 15. In some embodiments, the AHR antagonist is StemRegenin 1 (SR1). In some embodiments, the pyrido-[4,5-b]-indole derivative is UM729. In some embodiments, SR1 and UM729 are added to the culture in (c) starting on days 6 to 9. In some embodiments, SR1 and UM729 are added to the culture in (c) starting on about day 6.

[0078] In some embodiments, the culture in (d) is in a medium containing one or more of FLT3L, IL-7, IL-12, IL-15, SR-1, and UM729. In some embodiments, the culture in (d) is in a medium containing a non-physiological ligand. In some embodiments, the culture in (d) is in a medium containing a non-physiological ligand and not containing any additional growth factors, cytokines, or both. In some embodiments, the culture in (d) is over a period of 15 to 40 days. In some embodiments, the culture in (d) is over days 15 and 30.

[0079] In some aspects, (a) culturing a cell population comprising engineered iPSCs according to any one of claims 1 to 48 under conditions that form aggregates; (b) culturing the cells produced in (a) in a medium comprising one or more selected from the group consisting of BMP4, VEGF, FGF2, and ROCKi to induce mesoderm formation in a plurality of cells, wherein the start of the culturing step in (b) is day 0; (c) culturing the cells produced in (b) in a medium comprising BMP4, FGF2, and LY2940002 to differentiate the cells into a population of hematopoietic progenitors (HP), wherein the start of the culturing step in (c) is day 3; and (d) culturing the cells produced in (d) in a medium comprising SCF and IL-15 to generate iCIL cells, wherein the start of the culturing step in (b) is day 15, wherein at least a portion of one or more of steps (a) to (d) is performed in the presence of a non-physiological ligand of a synthetic cytokine receptor. Provided herein is a method for generating cytotoxic innate lymphoid cell lineage (iCIL) cells. In some embodiments, SR1 and UM729 are added to the culturing in (c) starting on days 6 to 9. In some embodiments, SR1 and UM729 are added to the culturing in (c) starting on about day 6.

[0080] A method of generating cytotoxic innate lymphoid (iCIL) cells is also provided, which includes contacting a cell population comprising an engineered stem cell of any one of the provided modalities with a non-physiological ligand for a first period sufficient to generate CLP, and contacting the CLP with a differentiation medium for a second period sufficient to generate iCIL. In some embodiments, the differentiation medium comprises stem cell factor (SCF), FLT3L, IL-7, IL-12, IL-15, SR-1, and UM729. In some embodiments, the differentiation medium comprises a non-physiological ligand. In some embodiments, the first period is 1 to 15 days, and the second period is 1 to 15 days. In some embodiments, the method includes contacting the iCIL with a pre-activation medium comprising IL-7, IL-12, IL-15, IL-18, and IL-21 for a third period sufficient to generate mature iCIL. In some embodiments, the pre-activation medium comprises a non-physiological ligand. In some embodiments, the third period is 1 to 10 days. In some embodiments, the mature iCIL expresses NKp46, NKG2D, LFA1, DNAM1, CD16, and CD56.

[0081] In some aspects of any embodiment, the non - physiological ligand is rapamycin or a rapamycin analog. In some embodiments, the rapamycin analog is a rapalog. In some embodiments, the non - physiological ligand is added to the medium at a concentration of 5 nM to 200 nM, 5 nM to 150 nM, 5 nM to 100 nM, 5 nM to 50 nM, 5 nM to 20 nM, 5 nM to 10 nM, 10 nM to 200 nM, 10 nM to 150 nM, 10 nM to 100 nM, 10 nM to 50 nM, 10 nM to 20 nM, 20 nM to 200 nM, 20 nM to 150 nM, 20 nM to 100 nM, 20 nM to 50 nM, 50 nM to 200 nM, 50 nM to 150 nM, 50 nM to 100 nM, 100 nM to 200 nM, 100 nM to 150 nM, and 150 nM to 200 nM. In some embodiments, the non - physiological ligand is added to the medium at a concentration of 10 nM or about 10 nM. In some embodiments, the non - physiological ligand is added to the medium at a concentration of 100 nM or about 100 nM. In some embodiments, the non - physiological ligand is added to the medium at a concentration of 2.5 nM to 10 nM. In some embodiments, the non - physiological ligand is added to the medium at a concentration of 3 nM to 7 nM. In some embodiments, the non - physiological ligand is added to the medium at a concentration of 3.1 nM or about 3.1 nM.

[0082] In some aspects of any embodiment, hematopoietic progenitor (HP) cells produced by any of the methods provided herein are provided herein. In some embodiments, the HP cells comprise lower expression of HLF, HOXA9 and / or CD133 compared to CD34+ cord blood cells. In some embodiments, the expression of HLF, HOXA9 and / or CD133 in the HP cells is 1 / 8, 1 / 7, 1 / 6, 1 / 5, 1 / 4, 1 / 3, 1 / 2, or 1 / 1 compared to CD34+ cord blood cells. In some embodiments, the CD34+ cord blood cells comprise hematopoietic stem cells (HSCs).

[0083] In some aspects, provided herein are hematopoietic progenitor (HP) cells differentiated from pluripotent stem cells according to any of the methods provided herein, wherein the HP cells comprise a synthetic cytokine receptor.

[0084] In some aspects, provided herein is a population of hematopoietic progenitor (HP) cells produced by any of the methods provided herein. In some aspects, the population of HP cells comprises lower expression of HLF, HOXA9, and / or CD133 as compared to a population of CD34+ cord blood cells. In some aspects, the expression of HLF, HOXA9, and / or CD133 in the HP cells is 1 / 8, 1 / 7, 1 / 6, 1 / 5, 1 / 4, 1 / 3, 1 / 2, or 1 / 1 as compared to a population of CD34+ cord blood cells. In some aspects, the population of CD34+ cord blood cells comprises hematopoietic stem cells (HSCs).

[0085] Also provided herein are cytotoxic innate lymphoid (iCIL) cells produced by any of the methods provided herein.

[0086] Also provided herein are cytotoxic innate lymphoid (iCIL) cells differentiated from any of the engineered stem cells provided herein, wherein the iCIL cells comprise a synthetic cytokine receptor.

[0087] Also provided is a population of cytotoxic innate lymphoid (iCIL) cells produced by any of the methods provided herein.

[0088] Also provided are pharmaceutical compositions comprising any iCIL or population of iCILs of the aspects provided herein.

[0089] In some aspects, the disclosure provides a population of cells produced by the methods described herein.

[0090] In some aspects, the present disclosure provides a pharmaceutical composition comprising the cell populations described herein.

[0091] A method of expanding and proliferating cytotoxic innate lymphoid cells (iCIL), the method comprising contacting an iCIL or population of iCILs provided herein, or a pharmaceutical composition comprising the same, with a non-physiological ligand of a synthetic cytokine receptor, is also provided herein. A method of killing cancer cells or inhibiting the growth of cancer cells, the method comprising contacting cancer cells with a non-physiological ligand of a synthetic cytokine receptor together with an iCIL or population of iCILs provided herein, or a pharmaceutical composition comprising the same, is also provided herein. In some embodiments, the synthetic cytokine receptor has a first dimerization domain and a second dimerization domain that are heterodimerization domains selected from a 12 kD FK506-binding protein (FKBP) and an FKBP12-rapamycin binding (FRB) domain. In some embodiments, the non-physiological ligand is rapamycin or a rapamycin analog. In some embodiments, the rapamycin analog is a rapalog.

[0092] In some embodiments, such methods are performed in vitro or ex vivo. In some of any of the embodiments, the method is performed ex vivo on a subject and the non-physiological ligand is contacted with stem cells (e.g., iPSCs) from the subject.

[0093] In some embodiments, the non - physiological ligand is contacted at a concentration of 2.5 nM to 200 nM, 2.5 nM to 150 nM, 2.5 nM to 100 nM, 5 nM to 200 nM, 5 nM to 150 nM, 5 nM to 100 nM, 5 nM to 50 nM, 5 nM to 20 nM, 5 nM to 10 nM, 10 nM to 200 nM, 10 nM to 150 nM, 10 nM to 100 nM, 10 nM to 50 nM, 10 nM to 20 nM, 20 nM to 200 nM, 20 nM to 150 nM, 20 nM to 100 nM, 20 nM to 50 nM, 50 nM to 200 nM, 50 nM to 150 nM, 50 nM to 100 nM, 100 nM to 200 nM, 100 nM to 150 nM, and 150 nM to 200 nM. In some embodiments, the non - physiological ligand is contacted at a concentration of 10 nM or about 10 nM. In some embodiments, the non - physiological ligand is contacted at a concentration of 100 nM or about 100 nM.

[0094] In some of any embodiment, the method is performed in vivo on a subject and the non - physiological ligand is administered to the subject.

[0095] In some aspects, the present disclosure provides a method of treating cancer in a subject, comprising administering to the subject an effective amount of the cell population or pharmaceutical composition described herein. In some embodiments, the cell population is a population of iCIL provided herein. In some embodiments, the method comprises administering to the subject a non - physiological ligand in an amount effective to induce the expansion and proliferation of iCIL in the subject.

[0096] In some of any embodiment, the subject has not been subjected to lymphodepletion therapy prior to administration of iCIL, a population of iCIL, or a pharmaceutical composition containing such cells.

[0097] In some of any aspects, iCIL expresses a CAR that targets cancer cells in a subject. In some of any aspects, the CAR is an anti-FITC CAR, and to tag cancer cells in the subject, a FITC-ligand is administered to the subject, and the ligand specifically binds to a molecule expressed on the tumor. In some of any aspects, the FITC-ligand is FITC-folate.

[0098] In some of any aspects, the method includes administering to the subject a non-physiological ligand of a synthetic cytokine receptor.

[0099] In some of any aspects, the non-physiological ligand is rapamycin or a rapamycin analog. In some of any aspects, the rapamycin analog is a rapalog.

[0100] In some of any aspects, the non-physiological ligand is administered at a dose of 1 mg to 100 mg. In some aspects, the non-physiological ligand is administered at a dose of 10 to 100 mg. In some of any aspects, the non-physiological ligand is administered at a dose of 10 mg, 20 mg, 25 mg, 30 mg, 40 mg, 50 mg, or any value between any of the foregoing.

[0101] In some of any aspects, multiple doses of the non-physiological ligand are administered to the subject. In some of any aspects, after administering iCIL or a population or composition thereof to the subject, multiple doses are administered intermittently or at regular intervals. In some aspects, the doses are administered over a predetermined period. In some of any aspects, 2 to 8 doses of the non-physiological ligand are administered to the subject. In some of any aspects, a single dose of the non-physiological ligand is administered to the subject.

[0102] In some of any aspects, the iCIL population or a composition thereof is from 1×10 8 cells or about 1×10 8 iCIL cells to 100×10 9 cells or about 100×10 9administered at a dose of individual iCIL cells. In some of any embodiments, the iCIL population or its composition is 5×10 9 or more than or about 5×10 9 administered at a dose of individual iCIL cells. In some embodiments, the dose is 5×10 9 or about 5×10 9 from individual iCIL cells to 100×10 9 or about 100×10 9 individual iCIL cells.

[0103] In some aspects, the disclosure provides a kit comprising a cell population described herein and instructions for administering the cell population to a subject in need thereof. In some embodiments, the kit comprises a container containing a non - physiological ligand and instructions for administering the non - physiological ligand to the subject after administration of the cell population. In some embodiments, the subject has cancer.

[0104] In some aspects, a population of induced cytotoxic innate lymphoid (iCIL) cells is provided herein, where the iCIL are mature iCIL expressing CD56 and LFA1, at least 25% of the iCIL express a cytotoxic receptor, at most 75% of the iCIL express a dysfunctional receptor, and / or at least 25% of the iCIL are proliferative. In some embodiments, at least 25% of the iCIL express a cytotoxic receptor. In some embodiments, the cytotoxic receptor is one or more of NKp30, NKp46, and NKG2D. In some embodiments, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, or at least 60% of the iCIL express NKp30+. In some embodiments, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80% of the iCIL express NKp46. In some embodiments, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% of the iCIL express NKG2D. In some embodiments, at most 75% of the iCIL express a dysfunctional receptor. In some embodiments, the dysfunctional receptor is one or more of KLRG1, CD73, and CD38. In some embodiments, at most 45%, at most 40%, at most 35%, at most 30%, at most 25%, at most 20%, at most 15%, at most 10%, or at most 5% of the iCIL express KLRG1. In some embodiments, at most 10%, at most 9%, at most 8%, at most 7%, at most 6%, at most 5%, at most 4%, at most 3%, at most 2%, or at most 1% of the iCIL express CD73. In some embodiments, at most 75%, at most 65%, at most 55%, at most 45%, at most 35%, at most 25%, at most 15%, or at most 5% of the iCIL express CD38.

[0105] In some embodiments, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55% or at least 60% of the iCILs are proliferative. In some embodiments, the proliferative iCILs are CD56bright CD57-.

[0106] In some embodiments, the iCILs further comprise a synthetic cytokine receptor for a non-physiological ligand, the cytokine receptor comprising a synthetic gamma chain polypeptide comprising a first dimerization domain, a first transmembrane domain, and an interleukin-2 receptor subunit gamma (IL-2RG) intracellular domain, and a synthetic beta chain polypeptide comprising a second dimerization domain, a second transmembrane domain, and an intracellular domain selected from the interleukin-2 receptor subunit beta (IL-2RB) intracellular domain, the interleukin-7 receptor subunit beta (IL-7RB) intracellular domain, and / or the interleukin-21 receptor subunit beta (IL-21RB) intracellular domain. In some embodiments, the first dimerization domain and the second dimerization domain are extracellular domains.

[0107] In some embodiments, the synthetic gamma chain polypeptide comprises, in order from N-terminus to C-terminus, a first dimerization domain, a first transmembrane domain, and an interleukin-2 receptor subunit gamma (IL-2RG) intracellular domain, and the synthetic beta chain polypeptide comprises, in order from N-terminus to C-terminus, a second dimerization domain, a second transmembrane domain, and an intracellular domain.

[0108] In some embodiments where the IL-2RG intracellular domain comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:1 or the polypeptide sequence set forth in SEQ ID NO:1, in some embodiments, the first transmembrane domain comprises the IL-2RG transmembrane domain. In some embodiments, the IL-2RG transmembrane domain comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:8 or 31, or the polypeptide sequence set forth in SEQ ID NO:8 or 31. In some embodiments, the beta chain intracellular domain comprises the IL-2RB intracellular domain. In some embodiments, the IL-2RB intracellular domain comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:2 or the polypeptide sequence set forth in SEQ ID NO:2. In some embodiments, the beta chain intracellular domain comprises the IL-7RB intracellular domain. In some embodiments, the IL-7RB intracellular domain comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:3 or the polypeptide sequence set forth in SEQ ID NO:3. In some embodiments, the beta chain intracellular domain comprises the IL-21RB intracellular domain. In some embodiments, the IL-21RB intracellular domain comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:4 or the polypeptide sequence set forth in SEQ ID NO:4. In some embodiments, the second transmembrane domain comprises a transmembrane domain derived from the same beta chain intracellular domain. In some embodiments where the second transmembrane domain is the transmembrane domain of IL-2RB that comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:35 or 36, or the polypeptide sequence set forth in SEQ ID NO:35 or 36, in some embodiments, the synthetic gamma chain polypeptide contains the IL-2RG TM domain comprising the sequence set forth in SEQ ID NO:8 or 31 and the IL-2RG intracellular domain comprising the sequence set forth in SEQ ID NO:1, and The synthetic beta chain polypeptide contains the IL-2RB TM domain comprising the sequence set forth in SEQ ID NO:35 or 36 and the IL-2RB intracellular domain comprising the sequence set forth in SEQ ID NO:2.

[0109] The first dimerization domain and the second dimerization domain are heterodimerization domains selected from the 12 kD FK506-binding protein (FKBP) and the FKBP12-rapamycin binding (FRB) domain, and / or in some embodiments where the non-physiological ligand is rapamycin or a rapalog, the FRB domain comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:6 or SEQ ID NO:7. In some embodiments, the FRB domain comprises the polypeptide sequence set forth in SEQ ID NO:6 or SEQ ID NO:7. In some embodiments, the first dimerization domain and the second dimerization domain are heterodimerization domains selected from the 12 kD FK506-binding protein (FKBP) and the calcineurin domain, and / or the non-physiological ligand is FK506 or an analog thereof.

[0110] In some embodiments, the FKBP domain comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:5 or SEQ ID NO:30. In some embodiments, the FKBP domain comprises the polypeptide sequence set forth in SEQ ID NO:5 or SEQ ID NO:30. In some embodiments, the synthetic gamma chain polypeptide has the amino acid sequence set forth in SEQ ID NO:28, or an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:28, and the synthetic beta chain polypeptide has the amino acid sequence set forth in SEQ ID NO:33, or an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:33. In some embodiments, the synthetic gamma chain polypeptide has the amino acid sequence set forth in SEQ ID O:28, and the synthetic beta chain polypeptide has the amino acid sequence set forth in SEQ ID NO:33.

[0111] In some embodiments, the first dimerization domain and the second dimerization domain are homodimerization domains selected from: i) FK506-binding protein (FKBP) of size 12 kD; ii) cyclophilin A (CypA); or iii) gyrase B (CyrB), and the non-physiological ligands are, respectively, i) FK1012, AP1510, AP1903 or AP20187 or analogs thereof; ii) cyclosporin-A (CsA) or analogs thereof; or iii) coumermycin or analogs thereof. In some embodiments, the population comprises about 1×10 6 to about 1×10 12 individual iCILs, about 1×10 6 to about 1×10 10 individual iCILs, about 1×10 6 to about 1×10 8 individual iCILs, about 1×10 8 to about 1×10 12 individual iCILs, about 1×10 8 to about 1×10 10 individual iCILs, or about 1×10 10 to about 1×10 12 individual iCILs. In some embodiments, the volume of the population is about 1 mL to about 100 mL, about 1 mL to about 80 mL, about 1 mL to about 60 mL, about 1 mL to about 40 mL, about 1 mL to about 20 mL, about 1 mL to about 10 mL, about 10 mL to about 100 mL, about 10 mL to about 80 mL, about 10 mL to about 60 mL, about 10 mL to about 40 mL, about 10 mL to about 20 mL, about 20 mL to about 100 mL, about 20 mL to about 80 mL, about 20 mL to about 60 mL, about 20 mL to about 40 mL, about 40 mL to about 100 mL, about 40 mL to about 80 mL, about 40 mL to about 60 mL, about 60 mL to about 100 mL, about 60 mL to about 80 mL, or about 80 mL to about 100 mL.

[0112] In some aspects, pharmaceutical compositions are provided that include any population of iCILs of any one of the aspects provided herein. In some aspects, the pharmaceutical composition further includes a cryoprotectant. In some aspects, cryopreservation compositions are provided that include any population of iCILs of any one of the aspects provided herein. In some aspects, the composition is from about 1×10 6 to about 1×10 12 iCILs, from about 1×10 6 to about 1×10 10 iCILs, from about 1×10 6 to about 1×10 8 iCILs, from about 1×10 8 to about 1×10 12 iCILs, from about 1×10 8 to about 1×10 10 iCILs, or from about 1×10 10 to about 1×10 12 iCILs. In some aspects, the volume of the composition is from about 1 mL to about 100 mL, from about 1 mL to about 80 mL, from about 1 mL to about 60 mL, from about 1 mL to about 40 mL, from about 1 mL to about 20 mL, from about 1 mL to about 10 mL, from about 10 mL to about 100 mL, from about 10 mL to about 80 mL, from about 10 mL to about 60 mL, from about 10 mL to about 40 mL, from about 10 mL to about 20 mL, from about 20 mL to about 100 mL, from about 20 mL to about 80 mL, from about 20 mL to about 60 mL, from about 20 mL to about 40 mL, from about 40 mL to about 100 mL, from about 40 mL to about 80 mL, from about 40 mL to about 60 mL, from about 60 mL to about 100 mL, from about 60 mL to about 80 mL, or from about 80 mL to about 100 mL.

[0113] In some aspects, methods are provided for killing target cells or inhibiting the growth of target cells that include contacting the target cells with a population of iCILs of any one of the aspects provided herein or with a composition of any one of the aspects provided herein.

[0114] In some embodiments, the target cells are cancer cells. In some embodiments, the iCIL further comprises a synthetic cytokine receptor for a non-physiological ligand, and the method comprises contacting the target cells with the non-physiological ligand of the synthetic cytokine receptor. In some embodiments, the method is performed in vitro or ex vivo. In some embodiments, the non-physiological ligand is rapamycin or a rapamycin analog. In some embodiments, the rapamycin analog is a rapalog. In some embodiments, the non-physiological ligand is contacted at a concentration of 5 nM to 200 nM, 5 nM to 150 nM, 5 nM to 100 nM, 5 nM to 50 nM, 5 nM to 20 nM, 5 nM to 10 nM, 10 nM to 200 nM, 10 nM to 150 nM, 10 nM to 100 nM, 10 nM to 50 nM, 10 nM to 20 nM, 20 nM to 200 nM, 20 nM to 150 nM, 20 nM to 100 nM, 20 nM to 50 nM, 50 nM to 200 nM, 50 nM to 150 nM, 50 nM to 100 nM, 100 nM to 200 nM, 100 nM to 150 nM, or 150 nM to 200 nM. In some embodiments, the non-physiological ligand is contacted at a concentration of 10 nM or about 10 nM. In some embodiments, the non-physiological ligand is contacted at a concentration of 100 nM or about 100 nM. In some embodiments, the non-physiological ligand is added to the medium at a concentration of 2.5 nM to 10 nM. In some embodiments, the non-physiological ligand is added to the medium at a concentration of 3 nM to 7 nM. In some embodiments, the method is performed in vivo on a subject, and the population or composition of iCIL is administered to the subject. In some embodiments, the iCIL further comprises a synthetic cytokine receptor for a non-physiological ligand, and the method comprises administering the non-physiological ligand to the subject.

[0115] In some aspects, provided herein is a method of inducing natural killer (NK) cell-mediated cytotoxicity in a subject, comprising administering to the subject any population of iCILs of any one of the aspects provided herein or any effective amount of any composition of any one of the aspects provided herein. In some aspects, provided herein is a method of treating cancer in a subject, comprising administering to the subject any population of iCILs of any one of the aspects provided herein or an effective amount of any composition of any one of the aspects provided herein. In some embodiments, the subject has not been subjected to lymphodepletion therapy prior to administration of the population of iCILs or the composition thereof.

[0116] In some embodiments, the iCIL expresses a CAR that targets cancer cells in a subject. In some embodiments, the CAR is an anti-FITC CAR, and to tag cancer cells in the subject, a FITC-ligand has been administered to the subject, where the ligand specifically binds to a molecule expressed on the tumor. In some embodiments, the FITC-ligand is FITC-folate. In some embodiments, the iCIL further comprises a synthetic cytokine receptor for a non-physiological ligand, and the method comprises administering the non-physiological ligand of the synthetic cytokine receptor to the subject. In some embodiments, the non-physiological ligand is rapamycin or a rapamycin analog. In some embodiments, the rapamycin analog is a rapalog. In some embodiments, the non-physiological ligand is administered at a dose of 1 mg to 100 mg, optionally 10 - 100 mg, optionally 10 mg or about 10 mg, 20 mg or about 20 mg, 25 mg or about 25 mg, 30 mg or about 30 mg, 40 mg or about 40 mg, 50 mg or about 50 mg, or any value between any of the foregoing. In some embodiments, multiple doses of the non-physiological ligand are administered to the subject. In some embodiments, after administering a population of iCIL or a composition thereof to the subject, multiple doses are administered intermittently or at regular intervals, optionally over a predetermined period of time. In some embodiments, 2 - 8 doses of the non-physiological ligand are administered to the subject. In some embodiments, a single dose of the non-physiological ligand is administered to the subject.

[0117] In some embodiments, the population of iCIL or a composition thereof is administered at a dose of 1×10 8 cells or about 1×10 8 cells of iCIL, from 100×10 9 cells or about 100×10 9 cells of iCIL. In some embodiments, the population of iCIL or a composition thereof is administered at a dose of more than 5×10 9 cells or about more than 5×10 9 cells of iCIL, and optionally, the dose is from 5×10 9 cells or about 5×10 9 cells of iCIL to 100×109 one or about 100×10 9 iCIL cells. In some embodiments, multiple doses of iCIL cells are administered to a subject. In some embodiments, multiple doses of iCIL cells are administered intermittently or at regular intervals, optionally over a predetermined period of time.

[0118] In some embodiments, 2 to 8 doses of iCIL cells are administered to a subject. In some embodiments, a single dose of iCIL cells is administered to a subject.

[0119] In some aspects, a kit is provided herein that includes any population of iCILs of any one of the embodiments provided herein, or any one of the compositions provided herein, and instructions for administering the population of iCILs or the composition to a subject in need thereof. In some embodiments, the iCIL further includes a synthetic cytokine receptor for a non-physiological ligand, and the kit further includes a container containing the non-physiological ligand and instructions for administering the non-physiological ligand to the subject after administration of the population of iCILs or the composition.

[0120] In some embodiments, the subject has cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0121]

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Figure 51

Mode for Carrying Out the Invention

[0122] Detailed Description of the Invention Induced pluripotent stem cells (iPSCs) are a renewable, modifiable, and expandable source of materials for cell therapy manufacturing. However, there are significant challenges in current iPSC - based approaches to cell therapy. Specifically, current methods for differentiating iPSCs into therapeutic immune cell types require the presence of exogenous growth factors and, in some cases, feeder cells.

[0123] iPSCs can be generated by reprogramming adult cells into a cell state similar to embryonic stem cells. iPSCs are thought to be able to differentiate into any cell type found in the human body and have an unlimited expansion capacity, meaning that they can replicate and grow indefinitely and can supply an almost unlimited amount of starting material. Furthermore, iPSCs are suitable for precise multiplex genome editing and allow for the safe introduction of multiple gene modifications. Because of these properties, iPSCs provide a defined starting material derived from a single cell (clone) that allows for defined genomic integrity in the process intermediates and final cell products.

[0124] Current approaches to cell therapy manufacturing involve either autologous cell starting materials or allogeneic cell starting materials, and cell products for disease-targeted therapy are engineered from either autologous cell starting materials or allogeneic cell starting materials. In some embodiments, allogeneic cell therapy or “off-the-shelf” cell therapy has the potential to transform cell therapy from personalized medicine to a common treatment. However, current “off-the-shelf” cell therapies struggle to demonstrate in vivo engraftment and persistence of cells, the same as achieved by approved autologous cell therapy products. In some respects, this is due to allogeneic elements that can be recognized and rejected by the host immune system, or even the foreignness of engineered elements of autologous cells. To date, cell therapy has achieved cell engraftment by treating patients with a highly toxic chemotherapy regimen called lymphodepletion (LD) administered prior to the administration of cell therapy products. LD essentially removes the host immune system, provides many benefits to cell therapy products, first provides free “homeostatic cytokines” for ex vivo cell therapy products, and reduces the graft-versus-host response to foreign grafts by the host immune system. However, LD is a transient solution and the host immune system rapidly reconstitutes. Thus, multiple rounds of LD and cell infusion are required to sustain allogeneic cell exposure. Additionally, exogenous cytokines such as IL-2 are administered, but these cytokine therapies have short exposure times and high toxicity associated with their use. Finding better ways to enhance cell persistence is key to achieving durable tumor remission and has proven to be a challenge in the field of allogeneic cell therapy.

[0125] Allogeneic cells can be further classified into donor-derived cells or iPSC-derived cells. Donor-derived cells are generally supplied from the circulating blood or cord blood of healthy donors, and therapeutic cell types (e.g., natural killer cells or NK cells) are selected in a complex cell culture process that generally includes multiple cytokines, growth factors, genetic manipulation, and feeder cells, followed by collection, expansion, and production of multiple doses. Alternatively, in the case of iPSC-derived cells, which also require multiple complex cell culture conditions, these conditions must be implemented stepwise to drive the cells through the precursor stages necessary to ultimately obtain the intended final cell product (e.g., immune effector cells). For example, an effective method for the expansion of CIL cells for clinical-scale purposes is known to require exogenous cytokines including IL-2, IL-15, and / or IL-7, as well as antigen molecules, costimulatory molecules, and / or cell adhesion molecules. CIL cells are cytotoxic lymphocytes characterized by the ability to distinguish self from non-self by monitoring the expression of MHC class I molecules and the release of cytokines, and to directly kill non-self cells or infected target cells. It is known in the art that CIL cells are not a homogeneous population. Rather, there are many distinguishable subsets of CIL cells. In many studies, these exogenous factors have been supplemented during the ex vivo expansion of CIL cells and / or after injection of CIL cells into a subject. Effectively expanding CIL cells that rely on a large variety of exogenous factors using currently known methods often requires a complex and expensive manufacturing process. A further difficulty in the art involves modulating the activity of endogenous CIL cells in vivo, especially considering that cancer patients exhibit significantly reduced NK cell activity compared to healthy patients.

[0126] Furthermore, the gold standard of cell therapy is autologous chimeric antigen receptor (CAR) T cell therapy. In the decades of CAR T cell therapy attempts, starting from the "first-generation" CAR T cell therapy in the 1990s and leading to the first CAR T cell therapy approved by the FDA in 2017, the treatment of B cell malignancies has been successful, and long-term remission has been achieved in 30-40% of specific patient populations. Importantly, the effectiveness of CAR T cells requires lymphodepleting chemotherapy to eliminate niches for survival factors such as IL-15. Although CAR T cell therapy has revolutionized the treatment of malignancies (e.g., hematological malignancies), major limitations are hindering its widespread application. The allogeneic CAR T therapy field has shown promising early clinical results, but the sustained response profile has generally been poor compared to autologous CAR T cell therapy, despite the use of LD regimens of increasing intensity. This is likely due to limitations such as the product cell type, manufacturing process, and the anti-graft response against the therapeutic cells. Thus, despite the promising clinical effectiveness of CAR T cells in hematological malignancies, significant challenges remain, including patient access, complex manufacturing, and high costs. The engineered CIL cells and related methods provided offer an "off-the-shelf" cancer treatment to overcome these challenges.

[0127] Also, for example, iPSCs can be modified via CRISPR to express CARs to overcome issues associated with targeting, for example, the heterogeneous solid tumor microenvironment.

[0128] Overall, iPSC-based cell therapies are generally inefficient at generating the necessary intermediate progenitor cells, resulting in low initial yields of the therapeutic cell type (e.g., cytotoxic natural lymphocyte (CIL) cells), which then require feeder cell-facilitated expansion. In this feeder cell-facilitated expansion, the proliferative capacity of the final cell therapy product can be dramatically reduced. Thus, to achieve engraftment necessary for any therapeutic effect, in addition to repeated cycles of lymphodepleting chemotherapy, high cell numbers (about 1 billion cells) and repeated administrations are required.

[0129] Current methods for differentiating iPSCs into therapeutic effector cells such as natural killer (NK) cells require complex growth factors and feeder cells to achieve sufficient yields. Provided herein is differentiation enabled by synthetic receptors (ShRED), a directed differentiation and expansion process controlled by the rapamycin-activated cytokine receptor (RACR). RACR is activated upon addition of its synthetic ligand rapamycin, which induces JAK / STAT signaling, promoting the differentiation and expansion of cells into hematopoietic progenitors (HP) and then into immune effector cells called RACR-induced cytotoxic natural lymphocytes (RACR-iCIL). Furthermore, since rapamycin is a safe, effective, and approved therapeutic for immunosuppression, RACR can also be engaged in vivo by rapamycin administration to increase the persistence of RACR-iCIL while simultaneously protecting these cells from allogeneic rejection.

[0130] A platform for producing immune effector cells in the absence of exogenous cytokines and feeder cells is provided herein by genetically modifying iPSCs and iPSC-derived progenitor cells to express synthetic cytokine receptors. Synthetic small molecule ligands (e.g., rapamycin) activate the receptors to promote the differentiation and expansion of immune effector cells. The compositions and methods provided herein include CIL cells engineered to express synthetic cytokine receptors. Non-limiting advantages of the engineered CIL cells include excellent controllable expansion when administered to a subject, similar cytotoxic activity compared to native CIL cells, improved iPSC-derived cell manufacturing, and enhanced antitumor activity.

[0131] Improvement in cell manufacturing. In an aspect provided, the RACR manipulation platform provided herein improves iPSC-derived cell manufacturing by controlling cell production. By administering rapamycin and activating RACR, a more reproducible differentiation process and a uniform cell product are obtained. Since RACR activation eliminates the need to add expensive growth factors, cytokines and other raw materials, the RACR manipulation platform also reduces manufacturing costs. In certain embodiments, the methods disclosed herein can further enhance the expansion of the CIL cells described herein through their ability to expand without or with reduced exogenous factors, for example, without using IL-2, IL-15 and / or IL-7. In some embodiments, the methods disclosed herein can further enhance differentiation and / or expansion through the ability to generate the iCIL cells described herein by removing one or more exogenous factors as compared to conventional processes. For example, in some cases, at least seven reduced exogenous factors are required in the described processes. The RACR manipulation platform increases the yield of high-purity intermediate and final cell products. The RACR manipulation platform provided herein generated high-purity hematopoietic progenitors (HP), intermediate progenitor populations, and the resulting CILs that are highly pure and phenotypically mature. The RACR manipulation platform enhances the patient compatibility of the cells since the manufacturing process is completely free of feeder cells and heterologous cells. The RACR manipulation platform is also compatible with cells in suspension and promotes the scalability of cell production.

[0132] In another aspect provided, the RACR manipulation platform eliminates the need for additional physical processing of differentiated progenitor cells. In conventional processes for differentiating progenitor cells, residual cell aggregates must be removed prior to blood cell differentiation. Physical processing includes enzymatic digestion (e.g., collagenase or TrypLE™ enzyme) and filtration (e.g., cells are filtered to remove unwanted cell aggregates). In contrast, the RACR manipulation platform results in embryoid bodies that dissociate completely into pure HPs without the need for cell filtration.

[0133] Enhanced anti-tumor activity. In an aspect provided, the RACR manipulation platform provided herein improves the anti-tumor activity of iPSC-derived cells by increasing cell engraftment, persistence, and effector function. The RACR manipulation platform provided herein also improves the anti-tumor activity of iPSC-derived cells by inhibiting the host immune response via rapamycin administration, thereby further enabling engraftment of the cells (e.g., CIL). The RACR manipulation platform provided herein also improves the anti-tumor activity of iPSC-derived cells by removing the need for toxic LD by activating the RACR system to selectively support the expansion, growth, and survival of RACR cells.

[0134] In an additional aspect provided, the results demonstrate the surprising finding that RACR-engineered iPSC-derived CIL (iCIL) cells express low levels of CD38, which is a target of certain therapeutic antibodies such as daratumumab. A problem with many iPSC-derived cell therapies against specific cancer target antigens or tumor target antigens such as CD38 is that iPSC-derived cells can express CD38. Indeed, NK cell compositions have been reported to contain large populations of cells that express a high percentage (e.g., >90%) of CD38+ NK cells. Expression of CD38 in iPSC-derived cell therapies such as NK cell therapy can be problematic because anti-CD38 targeting antibodies (e.g., daratumumab) bind to CD38, thereby causing fratricide, which can lead to the elimination of the tumor and the cell therapy due to ADCC. In contrast, the findings herein demonstrate that the percentage of CD38+ cells is significantly lower on iCIL cells compared to conventional bdNK cells or iNK cells differentiated using feeder cells. These results support the utility of the combination therapy comprising the provided iCIL cells and anti-CD38 antibodies. This combination therapy will result in enhanced anti-tumor activity without the iCIL cells undergoing fratricide-related depletion.

[0135] In some aspects, the advantages of the RACR system for CIL include the ability to engineer unrestricted starting materials that are extremely efficient at generating immediate precursors and are characterized by minimizing the expansion requirements for the final cell type; the ability to efficiently edit cells; not requiring feeder cells, thereby minimizing complex raw materials; not requiring lymphocyte depletion in the subject to whom the RACR-engineered cells are administered; low or no cytokine release syndrome (CRS) or immune effector cell-associated neurotoxicity syndrome (ICAN); and including promotion of engraftment, expansion, and persistence by administration of rapamycin or a rapalog. In some embodiments, RACR-iCIL provides a cell source for allogeneic cell therapy that can be achieved while minimizing or eliminating low immune engineering requirements in some aspects.

[0136] Synthetic cytokine receptors are provided herein that can be applied to support the induction of cytotoxic innate lymphoid (CIL) cells. CIL cells can be derived from stem cells or progenitor cells, and such cells are referred to herein as "induced cytotoxic innate lymphoid" (iCIL) cells. iCIL cells share characteristic cell surface markers and functional attributes as described herein. As used herein, the term "induced cytotoxic innate lymphoid cell" or "iCIL" refers to CIL generated by inducing the differentiation of progenitor cells. As disclosed herein, iCIL can be generated and / or expanded by expressing a synthetic cytokine receptor in a stem cell or progenitor cell and activating the synthetic cytokine receptor with a non-physiological ligand. Such processes can involve the differentiation of progenitor cells engineered to express the synthetic cytokine receptor upon activation of the synthetic cytokine receptor. The process can also, or alternatively, involve the expansion of progenitor cells or CIL upon activation of the synthetic cytokine receptor.

[0137] In some aspects, the present disclosure provides stem cells (e.g., iPSCs) and CIL cells engineered to express a rapamycin-activated cytokine receptor (RACR), a synthetic cytokine receptor activated by a small molecule rapamycin or a rapalog. CIL cells that contain RACR and are activated by rapamycin or a rapalog are referred to herein as "RACR-iCIL" cells. Stem cells that contain RACR and are activated by rapamycin or a rapalog are referred to herein as "RACR-SC". RACR has been demonstrated to support the differentiation and / or expansion of RACR-SC and RACR-iCIL cells in a feeder-free manufacturing process. RACR-iCIL cells express multiple natural tumor-targeting receptors and can exhibit CAR-induced cell lysis activity when engineered to express a chimeric antigen receptor (CAR). Thus, RACR-iCIL cells provide an "off-the-shelf" allogeneic cell therapy.

[0138] The present disclosure relates in part to the surprising discovery that stem cells engineered to express a synthetic cytokine receptor differentiate into hematopoietic precursors, CLP, or CMP in response to the receptor's cognate non-physiological ligand. CIL cells differentiated from the engineered stem cells retain the synthetic cytokine receptor and expand in response to the receptor's cognate non-physiological ligand. The engineered CIL cells can be produced in large quantities with greater functional activity than CIL cells from other sources.

[0139] As shown in the figure of FIG. 1A, isolated CIL cells may be transduced with a vector containing at least one polynucleotide encoding a synthetic cytokine receptor. When the transduced CIL cells are contacted with a non-physiological ligand, the extracellular domain of the cytokine receptor dimerizes by interaction with the non-physiological ligand. This dimerization generates an expansion signal within the CIL cells, thereby producing a phenotypically enriched and functionally active population of engineered CIL cells.

[0140] As shown in the figure of FIG. 1B, stem cells or progenitor cells may be transfected with a vector comprising at least one polynucleotide encoding a synthetic cytokine receptor. When the transfected cells are contacted with a non-physiological ligand, the extracellular domain of the cytokine receptor dimerizes. Dimerization generates a differentiation signal within the stem cell or progenitor cell, thereby inducing sequential differentiation to become CIL cells.

[0141] CIL cells can be derived from iPSCs, common lymphoid progenitor cells (CLPs), or other stem cells or progenitor cells. Further provided herein is a method of differentiating engineered stem cells or progenitor cells into CIL cells by engineering stem cells or progenitor cells to express a synthetic cytokine receptor and contacting the stem cells or progenitor cells with a cognate non-physiological ligand for the cytokine receptor.

[0142] In certain embodiments, CIL cells generated ex vivo are provided herein. The engineered cells and related compositions described herein can be used in immunotherapy using ligand-controlled ex vivo expansion. Further provided herein is a method of expanding and proliferating CIL cells by contacting the cells with a cognate non-physiological ligand for the synthetic cytokine receptor. Additionally, the engineered CIL cells disclosed herein can be further engineered to express a chimeric antigen receptor (CAR), enabling targeting of the engineered CIL cells to cells that express an antigen recognized by the CAR or are labeled with an antigen recognized by the CAR.

[0143] In some embodiments, engineered CIL cells are provided, as well as methods provided for improved immunotherapy compared to existing strategies. Chimeric antigen receptor (CAR) T cell therapy has revolutionized the treatment of hematological malignancies, but significant limitations have hindered their widespread application.

[0144] In some embodiments, the involvement of the synthetic cytokine receptor can not only promote differentiation, but also increase cell proliferation and promote expansion proliferation through the involvement of the synthetic cytokine receptor on the provided engineered iCIL cells. In some aspects, the provided engineered iCIL cells and related methods can be used to increase in vivo cell proliferation and expansion proliferation of the engineered cell therapy by administering rapamycin to a patient following the cell therapy product. In some embodiments, rapamycin simultaneously expands and protects the cells. Expansion proliferation is achieved by JAK / STAT signal activation, and protection is achieved by rapamycin inhibition of the host-versus-graft response. In some embodiments, the need for lymphocyte depletion and exogenous cytokine administration is not required. In some embodiments, the provided methods of administration and treatment using the engineered iCIL cells can be performed without using lymphocyte depletion (e.g., without performing lymphocyte depletion therapy such as cyclophosphamide and / or fludarabine). In some embodiments, the provided methods of administration and treatment using the engineered iCIL cells can be performed without using exogenous cytokine administration (e.g., without administering IL-2 and / or IL-15).

[0145] In some embodiments, the provided method may also include administering a non - physiological ligand of the synthetic cytokine receptor (e.g., rapamycin or a rapalog) to the subject to expand or re - activate the engineered iCILs in the subject. Thus, in some embodiments, since it is possible to expand cells in vivo using a non - physiological ligand, there is no need to re - administer iCILs to the subject. However, due to the low - immunogenic engineering described herein that enables allogeneic cell therapy, re - administration of iCILs is also possible. Further, the provided method can be performed without using lymphodepletion, which not only promotes the expansion of the transplanted cells but also provides the additional advantage of promoting the host anti - tumor response. This is because without lymphodepletion, the host immune system remains and is not severely depleted. Thus, the immune response generated by iCIL cells (e.g., the release of cytokines and other pro - inflammatory factors) can stimulate the host's existing immune system against the tumor. Additionally, the exemplary non - physiological ligand rapamycin not only promotes the expansion of the transplanted cells through the involvement of the synthetic cytokine receptor, but the transient mTOR inhibition achieved through rapamycin can re - activate T cells and promote the apoptosis of inhibitory macrophages. Also, the non - physiological ligand rapamycin or analogs can suppress the host's anti - graft response, but this is only transient, and it is predicted that when the administration of the non - physiological ligand is stopped, the host anti - tumor response with enhanced normality will resume.

[0146] In some embodiments, the engineered cells herein are further modified to be resistant to the effects of rapamycin that inhibit or reduce cell growth and expansion. In some embodiments, cells can be made "rapamycin-resistant" by providing free cytosolic FRB to the cells to form a complex with rapamycin, thereby eliminating or reducing rapamycin-mediated growth inhibition of the source cells or iCILs. In other embodiments, cells can be made "rapamycin-resistant" by disrupting FKBP12 within the engineered cells, e.g., by inactivating or knocking it out. It is found herein that in some cases, overexpression of FRB may not result in free FRB that can completely quench rapamycin. Thus, in some cases, complete rapamycin resistance of cells can be provided by editing endogenous genes within the cells, such as by FKBP12 knockout.

[0147] Accordingly, the provided embodiments using rapamycin or analogs, e.g., synthetic cytokine receptor systems such as rapamycin-activated cytokine receptor (RACR) in which a rapalog may be involved, protect and expand cells with a single technique. Additionally, by further including genetic disruption, e.g., knockout of specific immune genes such as beta-2-microglobulin (B2M), "stealth" cells can be produced that have additional advantages for allogeneic cell therapy.

[0148] All publications referenced in this application, including patent documents, scientific papers, and databases, are hereby incorporated by reference in their entirety for all purposes as if each individual publication were individually incorporated by reference. If the definitions set forth herein are contrary to or otherwise inconsistent with the definitions set forth in patents, patent applications, published patent applications, and other publications incorporated herein by reference, the definitions set forth herein shall control over the definitions incorporated herein by reference.

[0149] The section headings used in this specification are for the sole purpose of organization and should not be construed as limiting the described subject matter. Those skilled in the art will recognize that several aspects are possible within the scope and spirit of the present disclosure. The following description is illustrative of the present disclosure and, of course, should in no way be construed as limiting the scope of the invention described herein.

[0150] I. Definitions All publications, patent applications, patents, and other references mentioned in this specification are hereby incorporated by reference in their entirety.

[0151] Unless otherwise indicated in context, the various features described in this specification may be used arbitrarily in combination with any feature or combination of features described herein, and each feature may be excluded or omitted from the combination.

[0152] As used in this specification, the singular forms "a", "an", and "the" include the plural forms unless the context clearly dictates otherwise. The phrase "and / or" indicates any and all possible combinations of one or more of the listed items.

[0153] As used in this specification, "subject" refers to the recipient of the manipulated CIL cells or other agent. The term includes mammals, such as primates, mice, rats, dogs, cats, cows, horses, goats, camels, sheep, or pigs, preferably humans.

[0154] As used in this specification, "treat", "treating", or "treatment" refers to any type of action or administration that confers a benefit to a subject having a disease or disorder, including improvement of the patient's condition (i.e., improvement, reduction, or amelioration of one or more symptoms and partial or complete response to treatment).

[0155] The term "effective amount" refers to an amount effective to bring about a desired biochemical, cellular, or physiological response. The term "therapeutically effective amount" refers to an amount of treatment, dosage, or dosing regimen effective to cause a desired therapeutic effect.

[0156] As used herein, "polynucleotide" refers to a biopolymer composed of two or more nucleotide monomers covalently linked via an ester bond between the phosphoryl group of one nucleotide and the hydroxyl group of the sugar component of the next nucleotide in the chain. DNA and RNA are non-limiting examples of polynucleotides.

[0157] As used herein, "polypeptide" refers to a polymer consisting of amino acid residues linked together by peptide bonds that form part (or all) of a protein.

[0158] It will be understood by those skilled in the art that a number of different polynucleotides and nucleic acids can encode the same polypeptide as a result of the degeneracy of the genetic code. Furthermore, it should be understood that those skilled in the art may make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides described herein so as to reflect the codon usage frequency of any particular host organism in which the polypeptide is to be expressed, using routine techniques.

[0159] Nucleic acids can include DNA or RNA. They can be single-stranded or double-stranded. They can also be polynucleotides that contain synthetic or modified nucleotides therein. Several different types of modifications to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, and the addition of acridine or polylysine chains at the 3' and / or 5' ends of the molecule. For the uses described herein, it should be understood that polynucleotides can be modified by any method available in the art. Such modifications can be made to enhance the in vivo activity or lifespan of the polynucleotide of interest.

[0160] The term "variant" means a polynucleotide or polypeptide that has at least one substitution, insertion, or deletion in its sequence as compared to a reference polynucleotide or reference polypeptide. A "functional variant" is a variant that retains one or more functions of the reference polynucleotide or reference polypeptide.

[0161] As used herein, the terms "sequence identity" or "identity" with respect to a polynucleotide or polypeptide sequence refer to the degree to which two optimally aligned polynucleotide or polypeptide sequences match at each position in the alignment over the full length of the reference sequence. The "percent identity" is the number of matching positions in the optimal alignment divided by the sum of the length of the reference sequence + the length of any gaps in the reference sequence in the alignment. The optimal alignment is the alignment that yields the highest percent identity. Alignment of sequences for determining percent identity can be achieved by various well-known methods including, for example, using a mathematical algorithm such as those within the BLAST suite or the Clustal Omega sequence analysis program. Unless otherwise specified, the term "sequence identity" in the claims refers to the sequence identity calculated by BLAST version 2.12.0 using default parameters. Also, unless otherwise specified, the alignment is an alignment of all or part of the polynucleotide or polypeptide sequence of interest over the full length of the reference sequence.

[0162] As used herein, "small molecule" refers to an organic compound of low molecular weight (<1000 daltons). Small molecules can bind to specific biological macromolecules and can have various biological functions or uses including, without limitation, cell signaling molecules, drugs, secondary metabolites, or various other modes of action.

[0163] The term "analog" with respect to a small molecule refers to a compound that has a similar structure and / or function to another compound but is different from it with respect to certain components. An analog may differ in one or more atoms, functional groups, or substructures that have been replaced by other atoms, groups, or substructures. An analog can have different physical, chemical, physicochemical, biochemical, or pharmacological properties despite a high structural and / or functional similarity.

[0164] The term "rapalog" refers to a group of analogs recognized in the art of rapamycin analogs that share structural and functional similarities with rapamycin. Certain rapalogs are known to share some, but not all, of the functional attributes of rapamycin. For example, some rapalogs promote dimerization but have substantially no immunosuppressive activity (e.g., AP21967, AP23102, or iRAP), making them suitable for use as non-physiological ligands.

[0165] An exemplary rapalog of the present disclosure is AP21967 TIFF2025524373000002.tif45128.

[0166] An exemplary rapalog of the present disclosure is AP23102 TIFF2025524373000003.tif45128.

[0167] An exemplary rapalog of the present disclosure is iRAP TIFF2025524373000004.tif45128.

[0168] The term "cell population" refers to a mixture of cells that are suspended in a solution, attached to a substrate, or stored in a container. The characteristics of the entire cell population can be tested using bulk measurements of a sample volume containing multiple cells. Flow cytometry may be used to reduce the problem of background fluorescence encountered in bulk cell population measurements.

[0169] As used herein, the term "cytotoxic innate lymphoid cell" or "CIL cell" is used to refer to a class of cytotoxic lymphocytes that constitute a major component of the innate immune system. In humans, cytotoxic innate lymphoid cells typically express the surface markers CD16 (FCyRIII) and CD56, and may also express CD127. They may express one or more of CD45, CD94, CD122, KIR, NKG2A, NKG2D, NKp30, NKp44, NKP46, NKp80. Cytotoxic innate lymphoid cells generally do not express CD3 or express CD3 at lower levels than CD3+ T cells. CIL cells are cytotoxic and contain small granules in the cytoplasm that contain special proteins, such as perforin, and proteases known as granzymes. CIL cells provide a rapid response to virus-infected cells and respond to transformed cells. When released in close proximity to the cells to be killed, perforin forms pores in the cell membrane of the target cells, through which granzymes and related molecules can enter, inducing apoptosis. CIL cells can act as effectors of lymphocyte cell populations in anti-tumor immunity and anti-infection immunity. In some embodiments, CIL cells are NK cells. In some embodiments, CIL cells are blood-derived NK cells (bdNK), iPSC-derived NK cells (iPSC-NK) or other cytotoxic innate lymphoid cells. For clarity, the term "CIL cell" excludes adaptive immune cells and their precursors, and excludes common lymphoid progenitor cells (CLP). However, CIL can be derived from CLP. In some embodiments, CIL is an induced cytotoxic innate lymphoid cell (iCIL) induced to differentiate into cytotoxic innate lymphoid cells from iPSC using the methods described herein. In some aspects, iCIL may exhibit one or more unique phenotypic or functional characteristics compared to conventional NK cells such as bdNK cells or iPSC-NK cells.

[0170] As used herein, the term "engineered" refers to a cell that has been stably transfected with a heterologous polynucleotide, or a cell that has been subjected to gene editing to introduce, delete, or modify a polynucleotide within the cell, or a cell that has been transiently transfected with a polynucleotide to cause a stable phenotypic change within the cell.

[0171] As used herein, the term "stem cell" is used to represent a cell having an undifferentiated phenotype that can differentiate, for example, into hematopoietic precursors, common lymphoid precursors, cytotoxic natural lymphoid cells, and / or NK cells.

[0172] As used herein, the term "pluripotency" means that a stem cell can form substantially all of the differentiated cell types of an organism, at least in culture. For example, embryonic stem cells are a type of pluripotent stem cell that can form cells from each of the three germ layers, namely, the ectoderm, mesoderm, and endoderm.

[0173] As used herein, the terms "induced pluripotent stem cell" and "iPSC" are used to refer to cells derived from somatic cells that have been reprogrammed into a pluripotent state and are capable of proliferation, selectable differentiation, and maturation. iPSCs are stem cells that are derived from differentiated adult, neonatal, or fetal cells that have been induced or changed, i.e., reprogrammed, to differentiate into cells that can differentiate into all three germ layers or cortical tissues, namely, mesoderm, endoderm, and ectoderm. The iPSCs produced do not refer to cells as found in nature.

[0174] As used herein, the term "hematopoietic stem cell" refers to a stem cell that can give rise to both mature myeloid cell types and mature lymphoid cell types, including natural killer cells, T cells, and B cells. Hematopoietic stem cells are typically characterized by CD34+.

[0175] The term "precursor" refers to a cell that has partially differentiated into the desired cell type. Precursor cells retain a certain degree of pluripotency and can differentiate into multiple cell types.

[0176] As used herein, the term "hematopoietic precursor cell" refers to a cell of an intermediate cell type that can differentiate into the hematopoietic lineage, and hematopoietic precursor cells can differentiate into either common myeloid progenitor cells or common lymphoid progenitor cells. Hematopoietic precursor cells are typically characterized by CD34+ and CD45+. CD38 is also considered a marker for hematopoietic precursor cells. CD45 is considered a hematopoietic lineage marker.

[0177] As used herein, the term "lymphoid progenitor cell" or "lymphoblast" or "common lymphoid precursor" refers to a cell that is a precursor to lymphoid lineage cells, such as CIL cells and NK cells. Lymphoid progenitor cells are the first stage of differentiation of hematopoietic stem cells following the lymphoid lineage of differentiation. As used herein, the term "lymphoid precursor" refers to a cell capable of hematopoietic engraftment into hematopoietic cell types. Lymphoid progenitor cells may be characterized by CD45+CD7+CD5+ / lo CD3-CD56-. Lymphoid progenitor cells may be characterized by CD45+CD5+ / lo CD7+.

[0178] As used herein, "differentiate" or "differentiated" is used to refer to the process and conditions by which an undifferentiated or immature (e.g., unspecialized) cell acquires the characteristics of a mature (specialized) cell and thereby acquires a particular morphology and function. Stem cells (unspecialized) are often exposed to various conditions (e.g., growth factors and morphogens) to induce a specific lineage commitment or differentiation of the stem cells.

[0179] As used herein, "expand" or "expansion" refers to an increase in the number and / or purity of cell types within a cell population by mitosis of cells having limited proliferative capacity, such as CIL cells.

[0180] As used herein, "activated," "activating," or "activation" refers to the stimulation of activating receptors on cytotoxic natural lymphocyte cells that results in cell division, cytokine secretion (e.g., IFNγ and / or TNFα), and / or release of cytolytic granules to regulate or assist an immune response.

[0181] II. Source Cells and Methods for Differentiating Them into Cytokine-Induced Lymphocyte (CIL) Cells Stem cells or progenitor cells containing a synthetic cytokine receptor are provided herein. In some embodiments, the synthetic cytokine receptor is any of those described in Section II.B. In some embodiments, the synthetic cytokine receptor contains a common gamma chain intracellular signaling domain (e.g., interleukin-2 receptor subunit gamma, IL-2RG), and an intracellular domain derived from interleukin-2 receptor subunit beta (IL-2RB), interleukin-7 receptor subunit beta (IL-7RB), or interleukin-21 receptor subunit beta (IL-21RB). In some embodiments, the synthetic cytokine receptor also contains an extracellular domain that can be bound by a non-physiological ligand (e.g., rapamycin or an analog). Thus, binding of a non-physiological ligand to the extracellular domain of the synthetic cytokine receptor activates cytokine receptor-mediated signaling, including JAK / STAT signaling, which is an important pathway for differentiating stem cells, e.g., iPSCs or other pluripotent stem cells, into downstream cell lines, e.g., CIL. Accordingly, in the presence of a non-physiological ligand (e.g., rapamycin), the synthetic cytokine receptor can be involved during cell differentiation, eliminating the need for endogenous receptors or exogenous growth factors. In some embodiments, this results in increased control and decreased variability of JAK / STAT signaling during cell differentiation, thereby enabling efficient generation of induced CIL (iCIL).

[0182] As described above, provided herein are stem cells or progenitor cells that can be differentiated into lymphoid cells using a synthetic cytokine receptor complex activated by a non-physiological ligand for use in medical treatment, and differentiated cells produced from such stem cells or progenitor cells. The differentiated cells can be, without limitation, iCIL cells. As a non-limiting illustration of the compositions and methods described herein, cytotoxic natural lymphoid cells can be produced from pluripotent stem cells, such as induced pluripotent stem cells engineered to express a synthetic cytokine receptor that can be activated by a non-physiological ligand (e.g., rapamycin) described herein to induce differentiation, in addition to or instead of exogenous cytokines. In some embodiments, the synthetic cytokine receptor is a rapamycin-activated cytokine receptor (RACR) that uses rapamycin or a rapalog to induce differentiation, in addition to or instead of exogenous cytokines. Advantages of embodiments can include the ability to generate effector cells expressing a synthetic cytokine receptor complex activated by a non-physiological ligand from a rich source of cells (e.g., induced pluripotent stem cells) such that expansion of effector cells in a patient can be controlled by administration or cessation of the non-physiological ligand. Other advantages of embodiments can include, without limitation, the ability to generate effector cells from source cells in a medium substantially free of cytokines conventionally used in the art for CIL cell differentiation, such as IL-2, IL-7, and / or IL-15.

[0183] A. Source Cells CIL cells can be generated from multiple sources, exemplary examples of which include iPSCs, PBMCs, or UCBs. In some embodiments, the CIL source cells are autologous cells. In some embodiments, the CIL source cells are allogeneic cells. In some embodiments, the CIL source cells are xenogeneic cells. For example, allogeneic cells can be used if the subject being treated with the compositions of the present disclosure has received high-dose chemotherapy or radiation therapy to destroy the subject's immune system.

[0184] As used herein, the term "peripheral blood cell" is used to refer to cells derived from circulating blood that include hematopoietic stem cells capable of proliferation, selectable differentiation, and maturation. Thus, peripheral blood NK cells may alternatively be referred to as differentiated blood-derived NK cells (bdNK).

[0185] In some embodiments, the lymphocytes used to generate engineered stem cells or CIL cells may be obtained from a donor or subject by various means well known in the art (for autologous therapies). For example, lymphocytes can be obtained by collecting peripheral blood from a patient, subjecting the blood to Ficoll density gradient centrifugation and / or leukapheresis, and then isolating a population of lymphocytes from the peripheral blood using an isolation kit. In one exemplary embodiment, the population of lymphocytes need not be a selected pure cell type and may contain other cell types such as T cells, monocytes, macrophages, natural killer cells, and B cells. In some embodiments, the cell population collected can contain at least about 90% of the selected cell type, at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the selected cell type.

[0186] In certain embodiments, CIL cells are generated from a stem cell source.

[0187] In some embodiments, the source cells include hematopoietic stem cells characterized as CD34+ and / or CD45+; common lymphoid progenitor cells characterized as CD45+CD7+CD56-; CIL progenitor cells characterized as CD45+CD5-CD7+; and / or CIL cells characterized as CD45+CD56+CD3-, and optionally CD5- and / or CD7+.

[0188] In some embodiments, the stem cells are pluripotent stem cells. Various sources of pluripotent stem cells, including embryonic stem (ES) cells and induced pluripotent stem cells (iPSCs), can be used in the method. Various sources of pluripotent stem cells, including embryonic stem (ES) cells and induced pluripotent stem cells (iPSCs), can be used in the method. In some embodiments, the pluripotent stem cells are induced pluripotent stem cells (iPSCs) artificially induced from non-pluripotent cells. In some aspects, the non-pluripotent cells are cells with lower self-renewal and differentiation abilities than pluripotent stem cells. iPSCs can be generated by a process known as reprogramming, in which non-pluripotent cells are effectively "dedifferentiated" into an embryonic stem cell-like state by being manipulated to express genes such as OCT4, SOX2, and KLF4. Takahashi and Yamanaka Cell (2006) 126:663-76.

[0189] In some embodiments, the source cells can be human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs). In immunotherapy, the source cells are allogeneic or autologous, which means they are derived from a donor or the subject, respectively.

[0190] In some embodiments, CIL cells can be generated from induced pluripotent stem cells (iPSCs). iPSCs are a type of pluripotent stem cell derived from adult somatic cells that have been genetically reprogrammed into an embryonic stem cell-like state by the forced expression of genes and factors important for maintaining the distinct characteristics of embryonic stem cells. iPSCs can be generated from tissues containing somatic cells, including but not limited to skin, dental tissue, peripheral blood, and urine. To generate iPSCs, somatic cells can be reprogrammed by methods including but not limited to transient expression of reprogramming factors, virus-free methods, adenoviruses, plasmids, minicircle vectors, episomal vectors, Sendai virus, synthetic mRNA, self-replicating RNA, retroviruses, lentiviruses, PhiC31 integrase, excisable transposons, CRISPR-based gene editing, or recombinant proteins.

[0191] In the cell therapy industry, the use of genetically engineered patient-derived cells to treat specific disease indications has demonstrated potential transformative power, but there remain many challenges associated with the use of patient-derived materials, including limited expansion capacity and scalability, manufacturing complexity, high costs, patient-to-patient variability, and patient access. In contrast, iPSCs are pluripotent stem cells, a type of cell that can theoretically differentiate into any other cell type, including natural killer (NK) cells applicable to cancer treatment. Using iPSCs makes it possible to provide scalable and simplified manufacturing of cell therapies that fight target cells (e.g., fight cancer), reducing costs and improving patient access to cell therapies. iPSCs have an unlimited expansion capacity, meaning they can replicate and proliferate indefinitely, potentially providing an almost unlimited supply of differentiated immune cells for treatments such as cancer therapy. iPSCs are also suitable for precise multiplex genome editing, allowing the introduction of multiple gene modifications to enhance their disease targeting ability and the safety of the immune cells they ultimately become. iPSCs can likewise be engineered to protect them from allogeneic rejection by the patient's own immune system and to improve their initial expansion and engraftment duration. Furthermore, while neither patient-derived blood materials nor donor-derived blood materials are consistent, iPSCs provide a consistent starting material derived from a single cell clone, which may enable genomic stability and integrity in the final cell product.

[0192] In some embodiments, the PSCs (e.g., iPSCs) are autologous to the subject being treated, i.e., the PSCs are derived from the same subject to whom the differentiated cells are administered. In some embodiments, non-pluripotent cells (e.g., fibroblasts) derived from the patient to be treated are reprogrammed to become iPSCs prior to differentiating into CILs as described herein. In some embodiments, fibroblasts can be reprogrammed to iPSCs by transforming the fibroblasts with genes (OCT4, SOX2, NANOG, LIN28, and KLF4) cloned into a plasmid (see, e.g., Yu, et al., Science DOI:10.1126 / science.1172482). In some embodiments, patient-derived non-pluripotent fibroblasts are reprogrammed to become iPSCs prior to differentiating into CILs, for example, by reprogramming the cells using a non-integrating Sendai virus (e.g., using the CTS™ CytoTune™-iPS 2.1 Sendai Reprogramming Kit). In some embodiments, the resulting differentiated cells are then administered to the patient from whom they are derived in an autologous cell therapy.

[0193] In some embodiments, the PSCs (e.g., iPSCs) are allogeneic to the subject being treated, i.e., the PSCs are derived from an individual different from the individual to whom the differentiated cells are administered. In some embodiments, non-pluripotent cells (e.g., fibroblasts) derived from another individual (e.g., an individual without the disease or condition being treated, e.g., a healthy subject) are reprogrammed to become iPSCs prior to differentiating into CILs. In some embodiments, reprogramming is at least partially accomplished by reprogramming the cells using a non-integrating Sendai virus (e.g., using the CTS™ CytoTune™-iPS 2.1 Sendai Reprogramming Kit). In some embodiments, the resulting differentiated cells are then administered to an individual that is not the same individual from which the differentiated cells are derived (e.g., allogeneic cell therapy or allogeneic cell transplantation). In such embodiments, the PSCs (e.g., allogeneic cells) described herein can be genetically engineered to be hypoimmunogenic. Methods for reducing immunogenicity are known and include removing polymorphic HLA-A / -B / -C and HLA class II molecule expression. Exemplary methods for reducing one or more HLA molecules include disrupting the beta-2-microglobulin (B2M) gene as described herein.

[0194] In some embodiments, the iPSCs are genetically edited using a lentivirus. In some embodiments, the iPSCs are genetically edited using CRISPR.

[0195] In some embodiments, the HSCs are genetically edited using lentivirus. In some embodiments, the HSCs are genetically edited using CRISPR. In some embodiments, the blood progenitor cells are genetically edited using lentivirus. In some embodiments, the blood progenitor cells are genetically edited using CRISPR. In some embodiments, the common lymphoid progenitor cells are genetically edited using lentivirus. In some embodiments, the common lymphoid progenitor cells are genetically edited using CRISPR. In some embodiments, the common lymphoid progenitor (CLP) cells are genetically edited using lentivirus. In some embodiments, the common lymphoid progenitor (CLP) cells are genetically edited using CRISPR. Exemplary methods for gene editing are described in Section III.

[0196] In some embodiments, the stem cells can be engineered to express a synthetic cytokine receptor.

[0197] In some embodiments, the iPSCs can be engineered to express a synthetic cytokine receptor.

[0198] In some embodiments, the hematopoietic stem cells can be engineered to express a synthetic cytokine receptor. In some embodiments, the blood progenitor (leukocyte) cells can be engineered to express a synthetic cytokine receptor. In some embodiments, the common lymphoid progenitor cells can be engineered to express a synthetic cytokine receptor. In some embodiments, the CIL cells can be engineered to express a synthetic cytokine receptor.

[0199] Methods of producing CIL cells can include an ex vivo culture process in which CIL cells differentiate from non-terminally differentiated cells. In some embodiments, the non-terminally differentiated cells are stem cells. In some embodiments, the non-terminally differentiated cells are iPSC cells. In some embodiments, the non-terminally differentiated cells are progenitor cells. In embodiments, the non-terminally differentiated cells (e.g., stem cells such as iPSCs) express a synthetic cytokine receptor. In one aspect, the present disclosure provides a method of producing CIL cells that includes providing a stem cell or progenitor cell and differentiating the cell into a CIL cell by controlled activation of a synthetic cytokine receptor or without using activation of the synthetic cytokine receptor. In some embodiments, the differentiation is performed by activation of the synthetic cytokine receptor without using additional cytokines (e.g., without using one or more of IL-2, IL-15, and IL-7). In some embodiments, the differentiation is performed by activation of the synthetic cytokine receptor with one or more additional cytokines. In some embodiments, the differentiation can also be performed using cytokines without using activation of the synthetic cytokine receptor.

[0200] In any of the provided embodiments, the synthetic cytokine receptor can be any of those described herein that can be activated by a non-physiological ligand (e.g., rapamycin). In some embodiments, the synthetic cytokine receptor is a rapamycin-activated cytokine receptor (RACR) that can be activated by rapamycin or a rapalog. In the provided embodiments, activation of the synthetic cytokine receptor induces differentiation in addition to or instead of an exogenous cytokine.

[0201] In some embodiments, the non-physiological ligand can induce differentiation in addition to or instead of an exogenous cytokine. In some embodiments, the non-physiological ligand can induce differentiation during one or more of mesoderm formation, hematopoietic specification, lymphoid progenitor differentiation, and CIL cell differentiation.

[0202] In some embodiments, the non - physiological ligand can be contacted with cells in the differentiation stage that require IL - 7 and / or IL - 15 signals.

[0203] In some embodiments, the non - physiological ligand can be contacted with cells in the expansion stage that require IL - 2 signals.

[0204] In some embodiments, by engineering cells to express a synthetic cytokine receptor and activating the receptor with a non - physiological ligand, the generation of CIL cells that can be differentiated and / or expanded without using exogenous factors becomes possible.

[0205] In some embodiments, by engineering iPSCs to express RACR and activating the receptor with rapamycin or a rapalog, the generation of CIL cells that can be differentiated without using exogenous factors, for example, without using IL - 15 and / or IL - 7, becomes possible.

[0206] In some embodiments, by engineering iPSCs to express RACR and activating the receptor with rapamycin or a rapalog, the generation of CIL cells that can be expanded without using exogenous factors, for example, without using IL - 2 and / or IL - 7, becomes possible.

[0207] Since it is well - known in the relevant art that the differentiation and / or expansion of CIL cells depends on the presence of various exogenous stimuli, as described herein, the differentiation and / or expansion of engineered CIL cells without using one, two, or all of IL - 2, IL - 15, and IL - 7 is surprising and unexpected.

[0208] In some embodiments, the engineered common lymphoid progenitor differentiates into CIL cells without using IL - 15 in the differentiation medium.

[0209] In some embodiments, the engineered common lymphoid progenitor differentiates into CIL cells without using IL - 7 in the differentiation medium.

[0210] In some embodiments, the engineered common lymphoid progenitor differentiates into CIL cells without using either IL-15 or IL-7 in the differentiation medium.

[0211] In some embodiments, the common lymphoid progenitor is engineered to express a synthetic cytokine receptor, such as RACR, and is differentiated into CIL cells without using either IL-15 or IL-7 in the differentiation medium using a rapalog.

[0212] B. Synthetic Cytokine Receptor Complex The synthetic cytokine receptors of the present disclosure include a synthetic gamma chain and a synthetic beta chain, each of which includes a dimerization domain. The dimerization domain dimerizes controllably in the presence of a non-physiological ligand, thereby activating the signaling of the synthetic cytokine receptor.

[0213] The synthetic gamma chain polypeptide includes a first dimerization domain, a first transmembrane domain, and an interleukin-2 receptor subunit gamma (IL-2RG) intracellular domain. The dimerization domain can be extracellular (N-terminal to the transmembrane domain) or intracellular (C-terminal to the transmembrane domain and N-terminal or C-terminal to the IL-2G intracellular domain).

[0214] The synthetic beta chain polypeptide includes a second dimerization domain, a second transmembrane domain, and an intracellular domain selected from the interleukin-2 receptor subunit beta (IL-2RB) intracellular domain, the interleukin-7 receptor subunit beta (IL-7RB) intracellular domain, or the interleukin-21 receptor subunit beta (IL-21RB) intracellular domain. The dimerization domain can be extracellular (N-terminal to the transmembrane domain) or intracellular (C-terminal to the transmembrane domain and N-terminal or C-terminal to the IL-2RB intracellular domain or the IL-7RB intracellular domain).

[0215] In some embodiments, the synthetic gamma chain polypeptide is encoded by a nucleic acid sequence encoding a signal peptide. In some embodiments, the synthetic beta chain polypeptide is encoded by a nucleic acid sequence encoding a signal peptide. One skilled in the art is readily familiar with signal peptides that can provide signals for transporting nascent proteins within cells. Any of a variety of signal peptides can be used.

[0216] In some embodiments, the signal peptide is SEQ ID NO:12: the CD8a signal sequence shown as TIFF2025524373000005.tif3128

[0217] In some embodiments, the signal peptide is SEQ ID NO:29: the signal sequence shown as TIFF2025524373000006.tif4128

[0218] In some embodiments, the non-physiological ligand activates a synthetic cytokine receptor within cytotoxic natural lymphocyte lineage cells to induce expansion and / or activation of engineered cytotoxic natural lymphocyte lineage cells. In some embodiments, the non-physiological ligand is rapamycin or a rapalog, such a synthetic cytokine receptor called the rapamycin-activated cytokine receptor (RACR).

[0219] In some embodiments, the non-physiological ligand activates the synthetic cytokine receptor within the CIL cell to induce the expansion and proliferation of the CIL cell. In some embodiments, the activation of the synthetic cytokine receptor results in at least about 10-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 1000-fold, at least about 1500-fold, at least about 2000-fold, at least about 2500-fold, at least about 3000-fold, at least about 3500-fold, or at least about 4000-fold more CIL cells compared to non-induced cells. In some embodiments, the activation of the synthetic cytokine receptor results in at least about 5000-fold, at least about 6000-fold, at least about 7000-fold, at least about 8000-fold, at least about 9000-fold, at least about 10,000-fold, at least about 50,000-fold, at least about 100,000-fold, at least about 250,000-fold, at least about 500,000-fold, at least about 750,000-fold, or at least about 1,000,000-fold more CIL cells compared to non-induced cells.

[0220] In some embodiments, the CIL cells increase by about 10-fold to about 100-fold, about 50-fold to about 200-fold, about 100-fold to about 300-fold, about 200-fold to about 400-fold, about 300-fold to about 500-fold, about 400-fold to about 1000-fold, about 500-fold to about 1500-fold, about 1000-fold to about 2000-fold, about 1500-fold to about 2500-fold, about 2000-fold to about 3000-fold, about 2500-fold to about 3500-fold, about 3000-fold to about 4000-fold, or any value between these ranges. In some embodiments, the CIL cells increase by about 4000-fold to about 6000-fold, about 5000-fold to about 7000-fold, about 6000-fold to about 8000-fold, about 7000-fold to about 9000-fold, about 8000-fold to about 10000-fold, about 9000-fold to about 50,000-fold, about 10000-fold to about 100,000-fold, about 50,000-fold to about 250,000-fold, about 100,000-fold to about 500,000-fold, about 250,000-fold to about 750,000-fold, about 500,000-fold to about 1,000,000-fold, or any value between these ranges.

[0221] In some embodiments, the non - physiological ligand activates a synthetic cytokine receptor within the stem cell to induce differentiation. In some embodiments, the non - physiological ligand is rapamycin or a rapalog, such a synthetic cytokine receptor called the rapamycin - activated cytokine receptor (RACR).

[0222] In some embodiments, the non - physiological ligand activates a synthetic cytokine receptor within the stem cell to induce the expansion and proliferation of hematopoietic precursors or CLPs differentiated from the stem cell. In some embodiments, the activation of the synthetic cytokine receptor results in at least about 10 - fold, at least about 50 - fold, at least about 100 - fold, at least about 200 - fold, at least about 300 - fold, at least about 400 - fold, at least about 500 - fold, at least about 1000 - fold, at least about 1500 - fold, at least about 2000 - fold, at least about 2500 - fold, at least about 3000 - fold, at least about 3500 - fold, or at least about 4000 - fold the number of hematopoietic precursors or CLPs compared to unmanipulated cells.

[0223] In some embodiments, the hematopoietic precursors or CLPs increase by about 10 - fold to about 100 - fold, about 50 - fold to about 200 - fold, about 100 - fold to about 300 - fold, about 200 - fold to about 400 - fold, about 300 - fold to about 500 - fold, about 400 - fold to about 1000 - fold, about 500 - fold to about 1500 - fold, about 1000 - fold to about 2000 - fold, about 1500 - fold to about 2500 - fold, about 2000 - fold to about 3000 - fold, about 2500 - fold to about 3500 - fold, about 3000 - fold to about 4000 - fold, or any value between these ranges.

[0224] 1. Intracellular domain In some embodiments, the intracellular signaling domain of the first transmembrane receptor protein comprises the interleukin-2 receptor subunit gamma (IL2Rγ) domain. In some embodiments, the IL2Rγ domain comprises the sequence set forth in SEQ ID NO:1. In some embodiments, the IL2Rγ common gamma chain intracellular domain has at least 80% amino acid identity, at least 85% amino acid identity, at least 90% amino acid identity, at least 95% amino acid identity, or 100% amino acid identity to SEQ ID NO:1.

[0225] The sequence of the IL2RG common gamma chain intracellular domain is set forth in SEQ ID NO:1: as described in TIFF2025524373000007.tif11159.

[0226] In some embodiments, the synthetic cytokine receptor comprises a first transmembrane receptor protein comprising an IL-2RG intracellular domain, a first dimerization domain, a second transmembrane receptor protein comprising an IL-2RB intracellular domain, and a second dimerization domain.

[0227] In some embodiments, the synthetic beta chain comprises the interleukin-2 receptor subunit beta (IL2RB) intracellular domain. IL2RB is also known as IL15RB or CD122. Thus, when referred to herein, IL2RB can also mean IL15RB. That is, the term is used interchangeably in the present disclosure. In some embodiments, the IL2RB intracellular domain comprises the sequence set forth in SEQ ID NO:2. In some embodiments, the IL2RB intracellular domain has at least 80% amino acid identity, at least 85% amino acid identity, at least 90% amino acid identity, at least 95% amino acid identity, or 100% amino acid identity to SEQ ID NO:2.

[0228] The sequence of the IL2RB intracellular domain is set forth in SEQ ID NO:2: It is described in TIFF2025524373000008.tif33159.

[0229] In some embodiments, the synthetic cytokine receptor comprises a first transmembrane receptor protein comprising an IL-2RG intracellular domain, a first dimerization domain, a second transmembrane receptor protein comprising an IL-7RB intracellular domain, and a second dimerization domain.

[0230] In some embodiments, the synthetic beta chain comprises an interleukin-7 receptor subunit beta (IL7RB) intracellular domain. In some embodiments, the IL7RB intracellular domain comprises the sequence set forth in SEQ ID NO:3. In some embodiments, the IL7RB intracellular domain has at least 80% amino acid identity, at least 85% amino acid identity, at least 90% amino acid identity, at least 95% amino acid identity, or 100% amino acid identity to SEQ ID NO:3.

[0231] The sequence of the IL7RB intracellular domain is SEQ ID NO:3: It is described in TIFF2025524373000009.tif26159.

[0232] In some embodiments, the synthetic cytokine receptor comprises a first transmembrane receptor protein comprising an IL-2RG intracellular domain, a first dimerization domain, a second transmembrane receptor protein comprising an IL-21RB intracellular domain, and a second dimerization domain.

[0233] In some embodiments, the synthetic beta chain comprises the intracellular domain of the interleukin-21 receptor subunit beta (IL21RB). In some embodiments, the IL21RB intracellular domain comprises the sequence set forth in SEQ ID NO:4. In some embodiments, the IL21RB intracellular domain has at least 80% amino acid identity, at least 85% amino acid identity, at least 90% amino acid identity, at least 95% amino acid identity, or 100% amino acid identity to SEQ ID NO:4.

[0234] The sequence of the IL21RB intracellular domain is set forth in SEQ ID NO:4: as described in TIFF2025524373000010.tif33159.

[0235] 2. Dimerization domain The dimerization domain can be a heterodimerization domain including, but not limited to, a 12 kD FK506-binding protein (FKBP) that dimerizes in the presence of rapamycin or a rapalog, and an FKBP12-rapamycin binding (FRB) domain. In some embodiments, the FRB domain can include a polypeptide sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:6 or SEQ ID NO:7. In some embodiments, the FKBP domain can include a polypeptide sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:5. In some embodiments, the FKBP domain can include a polypeptide sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:49. In some embodiments, the FKBP domain can include a polypeptide sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:30.

[0236] In some embodiments, the sequence of an exemplary FKBP domain is set forth in SEQ ID NO:5: as described in TIFF2025524373000011.tif11157.

[0237] In some embodiments, the sequence of an exemplary FKBP domain is set forth in SEQ ID NO:49: as described in TIFF2025524373000012.tif11157.

[0238] In some embodiments, the sequence of an exemplary FKBP domain is set forth in SEQ ID NO:30: It is described in TIFF2025524373000013.tif at 11157.

[0239] In some embodiments, the sequence of the exemplary FRB domain is SEQ ID NO:6: It is described in TIFF2025524373000014.tif at 11158.

[0240] In some embodiments, the sequence of the variant FRB domain (FRB mutant domain) is SEQ ID NO:7: It is described in TIFF2025524373000015.tif at 11158.

[0241] In some embodiments, the first dimerization domain is described in SEQ ID NO:5, and the second dimerization domain is described in SEQ ID NO:6.

[0242] In some embodiments, the first dimerization domain is described in SEQ ID NO:49, and the second dimerization domain is described in SEQ ID NO:6.

[0243] In some embodiments, the first dimerization domain is described in SEQ ID NO:30, and the second dimerization domain is described in SEQ ID NO:6.

[0244] In some embodiments, the first dimerization domain is described in SEQ ID NO:5, and the second dimerization domain is described in SEQ ID NO:7.

[0245] In some embodiments, the first dimerization domain is described in SEQ ID NO:49, and the second dimerization domain is described in SEQ ID NO:7.

[0246] In some embodiments, the first dimerization domain is described in SEQ ID NO:30, and the second dimerization domain is described in SEQ ID NO:7.

[0247] Alternatively, the first dimerization domain and the second dimerization domain can be a 12 kD FK506-binding protein (FKBP) and a calcineurin domain, which are known in the art to dimerize in the presence of FK506 or an analog thereof.

[0248] In some embodiments, the dimerization domain is (i) a FK506-binding protein (FKBP) of size 12 kD; (ii) cyclophilin A (CypA); or (iii) gyroase B (CyrB) a homodimerization domain selected from, and the corresponding non-physiological ligands are, respectively (i) FK1012, AP1510, AP1903 or AP20187; (ii) cyclosporine-A (CsA); or (iii) coumermycin or an analog thereof is.

[0249] In some embodiments, the first and second dimerization domains of the transmembrane receptor protein are the FKBP domain and the cyclophilin domain.

[0250] In some embodiments, the first and second dimerization domains of the transmembrane receptor protein are the FKBP domain and the bacterial dihydrofolate reductase (DHFR) domain.

[0251] In some embodiments, the first and second dimerization domains of the transmembrane receptor protein are the calcineurin domain and the cyclophilin domain.

[0252] In some embodiments, the first and second dimerization domains of the transmembrane receptor protein are PYR1-like 1 (PYL1) and abscisic acid-insensitive 1 (ABI1).

[0253] 3. Transmembrane Domain The transmembrane domain is the sequence of the synthetic cytokine receptor that spans the membrane. The transmembrane domain may include a hydrophobic alpha helix. In some embodiments, the transmembrane domain is derived from a human protein.

[0254] The sequence of the transmembrane (TM) domain is shown as SEQ ID NO:8: TIFF2025524373000016.tif4128.

[0255] The sequence of the TM domain is shown as SEQ ID NO:9: TIFF2025524373000017.tif4128.

[0256] The sequence of the TM domain is shown as SEQ ID NO:10: TIFF2025524373000018.tif4128.

[0257] The sequence of the TM domain is shown as SEQ ID NO:11: TIFF2025524373000019.tif4128.

[0258] The sequence of the TM domain is shown as SEQ ID NO:36: TIFF2025524373000020.tif3128.

[0259] In some embodiments, the TM domain and the intracellular signaling domain are derived from the same cytokine receptor. In some embodiments, the synthetic gamma chain polypeptide contains the IL-2RG TM domain and the IL-2RG intracellular domain. In some embodiments, the synthetic beta chain polypeptide contains the IL-2RB TM domain and the IL-2RB intracellular domain. In some embodiments, the synthetic beta chain polypeptide contains the IL-7RB TM domain and the IL-7RB intracellular domain. In some embodiments, the synthetic beta chain polypeptide contains the IL-21RB TM domain and the IL-21RB intracellular domain.

[0260] In some embodiments, one or more additional contiguous amino acids of an extracellular domain that are immediately adjacent to the TM domain of the cytokine receptor can also be included as part of the polypeptide sequence of the chain of the synthetic cytokine receptor. In some embodiments, 1 to 20 contiguous amino acids of the extracellular domain adjacent to the TM domain of the cytokine receptor are included as part of the polypeptide sequence of the chain of the synthetic cytokine receptor. The portion of the extracellular domain can be a contiguous sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids that is immediately adjacent (e.g., N-terminal to the TM sequence) to the TM sequence.

[0261] In some embodiments, the synthetic gamma chain polypeptide contains an IL-2RG TM domain comprising the sequence set forth in SEQ ID NO:8 and an IL-2RG intracellular domain comprising the sequence set forth in SEQ ID NO:1. In some embodiments, the synthetic gamma chain polypeptide contains an IL-2RG TM domain comprising the sequence set forth in SEQ ID NO:31 and an IL-2RG intracellular domain comprising the sequence set forth in SEQ ID NO:1.

[0262] In some embodiments, the synthetic beta chain polypeptide contains an IL-2RB TM domain comprising the sequence set forth in SEQ ID NO:36 and an IL-2RB intracellular domain comprising the sequence set forth in SEQ ID NO:2. In some embodiments, the synthetic beta chain polypeptide contains an IL-2RB TM domain comprising the sequence set forth in SEQ ID NO:35 and an IL-2RB intracellular domain comprising the sequence set forth in SEQ ID NO:2.

[0263] In some embodiments, the synthetic cytokine receptor is composed of a synthetic gamma chain polypeptide containing an FKBP12 dimerization domain and an IL-2RG intracellular domain, and a synthetic beta chain polypeptide containing an FRB dimerization domain and an IL-2RB intracellular domain. In some embodiments, the synthetic gamma chain polypeptide has the amino acid sequence set forth in SEQ ID NO:28, or an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:28. In some embodiments, the synthetic beta chain polypeptide has the amino acid sequence set forth in SEQ ID NO:33, or an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:33.

[0264] In some embodiments, the synthetic cytokine receptor is composed of the synthetic gamma chain polypeptide set forth in SEQ ID NO:28 and the synthetic beta chain polypeptide set forth in SEQ ID NO:33.

[0265] In some embodiments, the synthetic cytokine receptor can be bound by a non-physiological ligand rapamycin or a rapamycin analog. In some embodiments, the synthetic cytokine receptor is responsive to a non-physiological ligand rapamycin or a rapamycin analog, and the binding of the non-physiological ligand to the dimerization domain of the synthetic cytokine receptor induces intracellular cytokine receptor-mediated signal transduction via, for example, the JAK / STAT pathway.

[0266] 4. Non-Physiological Ligand In various aspects of the compositions and methods of the present disclosure, the system includes a non-physiological ligand. Exemplary small molecules useful as ligands include, without limitation, rapamycin, fluorescein, fluorescein isothiocyanate (FITC), 4-[(6-methylpyrazin-2-yl)oxy]benzoic acid (aMPOB), folate, rhodamine, acetazolamide, and CA9 ligand.

[0267] In some aspects, the synthetic cytokine receptor is activated by a ligand. In some aspects, the ligand is a non-physiological ligand.

[0268] In some aspects, the non-physiological ligand is a rapalog.

[0269] In some aspects, the non-physiological ligand is rapamycin.

[0270] In some aspects, the non-physiological ligand is AP21967.

[0271] In some aspects, the non-physiological ligand is FK506.

[0272] In some aspects, the non-physiological ligand is FK1012. In some aspects, the non-physiological ligand is AP1510. In some aspects, the non-physiological ligand is AP1903. In some aspects, the non-physiological ligand is AP20187. In some aspects, the non-physiological ligand is cyclosporin-A (CsA). In some aspects, the non-physiological ligand is coumermycin.

[0273] In some embodiments, a synthetic cytokine receptor complex activated by folate, fluorescein, aMPOB, acetazolamide, a CA9 ligand, tacrolimus, rapamycin, a rapalog (rapamycin analog), a CD28 ligand, a poly(his) tag, a Strep-tag, a FLAG-tag, a VS-tag, a Myc-tag, an HA-tag, an NE-tag, biotin, digoxigenin, dinitrophenol, or a derivative thereof.

[0274] In some embodiments, the non-physiological ligand can be an inorganic or organic compound having a molecular weight of less than 1000 Daltons.

[0275] In some embodiments, the ligand can be rapamycin or a rapamycin analog (rapalog). In some embodiments, the rapalog includes a variant of rapamycin having one or more of the following modifications to rapamycin, namely, demethylation, elimination or substitution of the methoxy groups at C7, C42 and / or C29; elimination, derivatization or substitution of the hydroxy groups at C13, C43 and / or C28; reduction, elimination or derivatization of the ketones at C14, C24 and / or C30; substitution of the six-membered pipecolate ring by a five-membered prolyl ring; and alternative substitutions on the cyclohexyl ring, or substitution of the cyclohexyl ring by a substituted cyclopentyl ring.

[0276] Thus, in some embodiments, the rapalog is everolimus, sirolimus, pimecrolimus, deforolimus, tacrolimus, temsirolimus, umirolimus, zotarolimus, temsirolimus (CCI-779), C20-methylallyl rapamycin, C16-(S)-3-methylindole rapamycin, C16-(S)-3-methylindole rapamycin (C16-iRap), AP21967 (A / C Heterodimerizer, Takara Bio (registered trademark)), mycophenolate sodium, benidipine hydrochloride, rapamine, AP23573 (deforolimus), AP1903 (limuside), or a metabolite, derivative and / or combination thereof.

[0277] In some embodiments, the ligand includes FK1012 (a semi-synthetic dimer of FK506), tacrolimus (FK506), FKCsA (a complex of FK506 and cyclosporine), rapamycin, coumamycin, gibberellin, a HaXS dimerizer (a chemical dimerizer of HaloTag and SNAP-tag), TMP-HTag (a trimethoprim haloenzyme protein dimerizer), or ABT-737 or a functional derivative thereof.

[0278] In some embodiments, the non-physiological ligand is present or provided in an amount of 0 nM to 1000 nM, such as 0.05 nM, 0.1 nM, 0.5 nM, 1.0 nM, 5.0 nM, 10.0 nM, 15.0 nM, 20.0 nM, 25.0 nM, 30.0 nM, 35.0 nM, 40.0 nM, 45.0 nM, 50.0 nM, 55.0 nM, 60.0 nM, 65.0 nM, 70.0 nM, 75.0 nM, 80.0 nM, 90.0 nM, 95.0 nM, 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM or 1000 nM, or an amount within a range defined by any two of the above amounts.

[0279] In some embodiments, the non-physiological ligand is AP21967 and is present or provided at 10 nM. In some embodiments, the non-physiological ligand is AP21967 and is present or provided at 20 nM. In some embodiments, the non-physiological ligand is AP21967 and is present or provided at 50 nM. In some embodiments, the non-physiological ligand is AP21967 and is present or provided at 100 nM.

[0280] In some embodiments, the non - physiological ligand is rapamycin and is present or provided at 1 nM. In some embodiments, the non - physiological ligand is rapamycin and is present or provided at 10 nM. In some embodiments, the non - physiological ligand is rapamycin and is present or provided at 20 nM. In some embodiments, the non - physiological ligand is rapamycin and is present or provided at 50 nM.

[0281] In some embodiments, the non - physiological ligand is a rapalog and is present or provided at 1 nM. In some embodiments, the non - physiological ligand is a rapalog and is present or provided at 10 nM. In some embodiments, the non - physiological ligand is a rapalog and is present or provided at 20 nM. In some embodiments, the non - physiological ligand is a rapalog and is present or provided at 50 nM. In some embodiments, the non - physiological ligand is a rapalog and is present or provided at 100 nM.

[0282] In some embodiments, the non - physiological ligand is present or provided at 1 nM.

[0283] In some embodiments, the non - physiological ligand is present or provided at 10 nM.

[0284] In some embodiments, the non - physiological ligand is present or provided at 100 nM.

[0285] In some embodiments, the non - physiological ligand is present or provided at 1000 nM.

[0286] C. Cytosolic FRB In some embodiments, the engineered cells, such as stem cells or iCIL cells, can be contacted with the free cytosolic FRB. As described in more detail elsewhere herein, rapamycin normally binds to FBP12, and then the FKBP12-rapamycin complex binds to the FRB subunit of mTOR, blocking mTOR signaling. Thus, contacting cells with rapamycin can, in some cases, inhibit or reduce cell growth and expansion. In some embodiments, the cells can be made “rapamycin-resistant” by providing the free cytosolic FRB to the cells to form a complex therewith, thereby eliminating or reducing rapamycin-mediated growth inhibition of the source cells or iCIL.

[0287] In some embodiments, soluble FRB can be microinjected into stem cells or NK cells to eliminate or reduce rapamycin-mediated growth inhibition. In some embodiments, stem cells or NK cells can be transduced with a vector containing soluble FRB to eliminate or reduce rapamycin-mediated growth inhibition. In some embodiments, soluble FRB can be added to the cell culture medium to eliminate or reduce rapamycin-mediated growth inhibition.

[0288] In embodiments where soluble FRB is microinjected into stem cells or NK cells, the soluble FRB is injected at a concentration of 4 mg / mL, 4.5 mg / mL, 5 mg / mL, 5.5 mg / mL, or 6 mg / mL. In embodiments where soluble FRB is microinjected into stem cells or NK cells, the soluble FRB is injected at a concentration of 1 μM.

[0289] In some embodiments, a nucleic acid molecule encoding FRB is introduced into a cell, such as by introducing a vector construct encoding FRB. In some embodiments, the construct is designed to insert a nucleic acid encoding FRB into an endogenous locus within the cell. Methods of gene insertion or knock-in, including any of the methods described in Section III, are known. In some embodiments, insertion of the FRB-encoding construct is by homologous recombination repair, such as by using the CRISPR-Cas system. In some embodiments, an engineered cell expressing FRB at an endogenous locus is capable of expressing free cytosolic FRB within the cell.

[0290] The FRB domain is a domain of about 100 amino acids derived from the mTOR protein kinase. The FRB domain can be expressed in the cytosol as a freely diffusible soluble protein. Advantageously, the FRB domain reduces the inhibitory effect of rapamycin on mTOR in engineered cells, promotes consistent activation of the engineered cells, and confers a growth advantage over natural cells to the cells.

[0291] In some embodiments, the synthetic cytokine receptor complex comprises a cytosolic polypeptide that binds a ligand, or a complex that includes a ligand.

[0292] In some embodiments, the cytosolic polypeptide comprises an FRB domain. In some embodiments, the cytosolic polypeptide comprises an FRB domain and the ligand is rapamycin. The cytosolic FRB domain can comprise a polypeptide sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:6 or SEQ ID NO:7. The FRB domain can be a naked FRB domain consisting essentially of a polypeptide having a polypeptide sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:6 or SEQ ID NO:7.

[0293] In some embodiments, the cell is contacted with an FRB domain protein having the sequence set forth in SEQ ID NO:6.

[0294] In some embodiments, the cell is contacted with an FRB domain protein having the sequence set forth in SEQ ID NO:7.

[0295] Advantageously, cytosolic FRB confers resistance to the immunosuppressive effects of non-physiological ligands (e.g., rapamycin or rapalogs).

[0296] D. Engineered stem cells In some embodiments, the disclosure provides engineered stem cells that transiently or stably express a synthetic cytokine receptor complex. In some embodiments, the disclosure provides engineered stem cells that stably express a synthetic cytokine receptor complex.

[0297] In some embodiments, the engineered stem cells comprise a genome comprising a nucleotide sequence encoding a synthetic cytokine receptor complex as described in Section II.B. In some embodiments, the genome further comprises a disrupted B2M locus, TRAC locus, and / or SIRPA locus. In some embodiments, the genome further comprises a disrupted FKBP12 locus. In some embodiments, the locus of a gene is disrupted by a gene editing technique such as the CRISPR-Cas system. In some embodiments, the disrupted locus inactivates a gene in the cell. In some embodiments, the disrupted locus involves knockout of a gene in the cell. In some embodiments, the disrupted locus comprises an indel in an endogenous gene, or a deletion of a continuous stretch of genomic DNA of an endogenous gene. In some embodiments, the indel is a frameshift mutation, or a deletion of a continuous stretch of genomic DNA of a gene. In some embodiments, the indel is present in both alleles of the gene (indel / indel). Methods of gene editing and genetic engineering, including the methods described in Section III, are known. Any of such methods can be used to generate engineered stem cells further comprising the synthetic cytokine receptor complex described herein.

[0298] In some embodiments, the engineered stem cells comprise a genome that includes (i) a nucleotide sequence encoding a synthetic cytokine receptor complex as described in Section II.B, and (ii) a disrupted B2M locus. In some embodiments, the engineered stem cells comprise a genome that includes (i) a nucleotide sequence encoding a synthetic cytokine receptor complex as described in Section II.B, (ii) a disrupted B2M locus, and (iii) a disrupted FKBP12 locus. In some embodiments, the engineered stem cells comprise a genome that includes (i) a nucleotide sequence encoding a synthetic cytokine receptor complex, (ii) a disrupted TRAC locus, and (iii) a disrupted FKBP12 locus. In some embodiments, the engineered stem cells comprise a genome that includes (i) a nucleotide sequence encoding a synthetic cytokine receptor complex, (ii) a disrupted SIRPA locus, and (iii) a disrupted FKBP12 locus. In some embodiments, the engineered stem cells comprise a genome that includes (i) a nucleotide sequence encoding a synthetic cytokine receptor complex, (ii) a disrupted B2M locus, (iii) a disrupted TRAC locus, and (iv) a disrupted FKBP12 locus. In some embodiments, the engineered stem cells comprise a genome that includes (i) a nucleotide sequence encoding a synthetic cytokine receptor complex, (ii) a disrupted B2M locus, (iii) a disrupted SIRPA locus, and (iv) a disrupted FKBP12 locus. In some embodiments, the engineered stem cells comprise a genome that includes (i) a nucleotide sequence encoding a synthetic cytokine receptor complex, (ii) a disrupted SIRPA locus, (iii) a disrupted TRAC locus, and (iv) a disrupted FKBP12 locus.

[0299] Optionally, the engineered stem cells further comprise a polynucleotide encoding a chimeric antigen receptor (CAR), thereby generating engineered stem cells that express the CAR. Exemplary CARs and methods for engineering cells with CARs are described in Sections III and IV.

[0300] In some cases, the engineered stem cells further contain a polynucleotide encoding FRB, thereby generating engineered stem cells that express cytosolic FRB. FRB can have the sequences described in Section II.C. Methods of manipulating cells using exogenous FRB, including any of those described in Section III, are known.

[0301] In some embodiments, the engineered stem cells are iPSCs. In some embodiments, the engineered iPSCs are sequentially differentiated into hematopoietic progenitor cells (HPCs), the HPCs into common lymphoid progenitor cells (CLPs), and then the CLPs into CIL cells called "iCIL" cells. In a variant, CIL cells can be derived from HPCs by sequentially differentiating HPCs into CLPs and then CLPs into iCIL cells. In a further variant, CIL cells can be induced by differentiating CLPs into iCIL cells.

[0302] E. Differentiation of cells Methods are provided herein for differentiating pluripotent stem cells, such as iPSCs engineered with synthetic cytokine receptors, into CIL cells. In some embodiments, iPSC differentiation is by way of a pathway that includes differentiation into hematopoietic precursors (HPs) and common lymphoid progenitors (CLPs). In some embodiments, differentiation is at least partially guided by signaling derived from the synthetic cytokine receptor. In embodiments of the provided methods, engagement of the synthetic cytokine receptor (e.g., RACR) by a cognate non-physiological ligand (e.g., rapamycin) can deliver cytokine signals that induce the JAK / STAT pathway and promote differentiation to the cells. In some aspects, the need for additional growth factors or cytokines to promote differentiation in one or more different steps of the process is reduced or eliminated, thereby providing directed and consistent differentiation.

[0303] In some embodiments, the directed differentiation of stem cells, such as pluripotent stem cells (e.g., iPSCs), into hematopoietic precursors (HPs) and common lymphoid progenitors (CLPs) can be achieved by the involvement of synthetic cytokine receptors with non-physiological ligands. In some embodiments, the involvement of the provided synthetic cytokine receptor (e.g., RACR) with a non-physiological ligand (e.g., rapamycin or analogs) promotes differentiation by inducing STAT5 signaling via the JAK / STAT pathway, thereby eliminating or reducing the need for other growth factors or cytokines that can provide similar STAT5 signaling. The observations herein demonstrate that the involvement of an engineered synthetic cytokine receptor (e.g., RACR) on iPSCs with a non-physiological ligand (e.g., rapamycin) is sufficient to drive iPSCs to CLPs. This result was surprising as conventional methods for cell differentiation incorporate a complex milieu of media additives to achieve differentiation to CLPs or HPs.

[0304] iPSC-derived cell therapies are promising, but the current process of inducing immune cells from iPSCs is complex, prone to variation, and costly. Current protocols for inducing downstream cell types from iPSCs use a sequential process of "coaxing" iPSCs down the differentiation pathway by the provision of external factors that engage endogenous receptors. In some embodiments, this process can require long culture times, expensive protein materials, and can be highly prone to variation due to the dependence on the constantly changing expression patterns of endogenous genes and endogenous receptors. The provided embodiments in which iPSCs are engineered with synthetic cytokine receptors promote differentiation through highly controlled synthetic receptors, which have the potential to reduce the variability of cell differentiation and reduce the manufacturing costs of these cells by replacing expensive growth factors and cytokines with small molecule engagers (e.g., rapamycin).

[0305] In some embodiments, synthetic cytokine receptors engineered to engage stem cells such as iPSCs (e.g., RACR) provide an opportunity to direct cells via cytokine receptor signals that mimic normal signaling during cell differentiation. In particular, synthetic cytokine receptors induce JAK / STAT signaling, a downstream signaling pathway essential for hematopoiesis. Current protocols use multiple growth factors such as thrombopoietin (TPO), stem cell factor (SCF), bone morphogenetic protein (BMP4), and fibroblast activating protein (FGF2) to induce hematopoiesis in iPSCs, all of which induce JAK / STAT signaling in combination with other signaling pathways that help promote hematopoiesis. Thus, replacing or supplementing these endogenous receptor pathways with non-physiological stimulation of synthetic cytokine receptors, such as via rapamycin or analogs, to promote JAK / STAT signaling has the potential to improve the process of hematopoiesis and reduce the need for multiple exogenous protein signals. After blood cells appear during differentiation, blood precursors can further differentiate into lymphoid precursors, including CLP. In some aspects, common gamma chain cytokines such as IL-7, IL-15, IL-2, and IL-21 are typically used in the process of differentiating CD34+(HP) precursors into immune cells. However, because synthetic cytokine receptors are engineered from common gamma chain cytokine signaling, the provided embodiments and methods can also be performed with reduced or no additional cytokine support, thus replacing the need for these cytokine mixtures during cell differentiation and reducing the cost, variability, and complexity of cell production.

[0306] Furthermore, the generation of HPs and CLPs from iPSCs using conventional methods is generally inefficient and a current impediment in the manufacture of effector immunotherapy, and in some cases, achieves only a yield of about 1× of HPs from iPSCs (1 HP per 1 iPSC). In some embodiments, the provided method can result in an increased yield of greater than 10-fold (10×) or about greater than 10-fold, greater than 20-fold or about greater than 20-fold, greater than 40-fold or about greater than 40-fold, greater than 50-fold or about greater than 50-fold, greater than 60-fold or about greater than 60-fold, greater than 70-fold or about greater than 70-fold, greater than 80-fold or about greater than 80-fold, greater than 90-fold or about greater than 90-fold, greater than 100-fold or about greater than 100-fold, greater than 150-fold or about greater than 150-fold, greater than 200-fold or about greater than 200-fold, greater than 250-fold or about greater than 250-fold, greater than 300-fold or about greater than 300-fold of HPs from iPSCs. In some embodiments, the provided method can result in an increased yield of greater than 350-fold or about greater than 350-fold, greater than 400-fold or about greater than 400-fold, greater than 450-fold or about greater than 450-fold, greater than 500-fold or about greater than 500-fold, greater than 550-fold or about greater than 550-fold, greater than 600-fold or about greater than 600-fold, greater than 650-fold or about greater than 650-fold, greater than 1,000-fold or about greater than 1,000-fold, greater than 1,500-fold or about greater than 1,500-fold, greater than 2,000-fold or about greater than 2,000-fold, greater than 2,50-fold or about greater than 2,500-fold, greater than 3,000-fold or about greater than 3,000-fold, greater than 3,500-fold or about greater than 3,500-fold of HPs from iPSCs. In some embodiments, the provided method can result in an increased yield of greater than 630-fold or about greater than 630-fold of HPs from iPSCs. In some embodiments, the provided method can result in an increased yield of greater than 3,000-fold or about greater than 3,000-fold of HPs from iPSCs. This result represents a substantial improvement over other methods for differentiating iPSCs into HPs. Furthermore, the generation of iCILs from HPs using the provided embodiments results in extremely potent iCILs, as demonstrated by the killing of tumor target cells.

[0307] In some embodiments, the provided method is 1×10 6 -fold (1,000,000×) or about 1×10 6 -fold (1,000,000×) or greater, 1.5×106 more than or about 1.5×10 6 more than 2×10 6 more than or about 2×10 6 more than 2.5×10 6 more than or about 2.5×10 6 more than 3×10 6 more than or about 3×10 6 more than 3.5×10 6 more than or about 3.5×10 6 more than 4×10 6 more than or about 4×10 6 more than 4.5×10 6 more than or about 4.5×10 6 more than 5×10 6 more than or about 5×10 6 more than 5.5×10 6 more than or about 5.5×10 6 more than 6×10 6 more than or about 6×10 6 more than 6.5×10 6 more than or about 6.5×10 6 more than 7×10 6 more than or about 7×10 6 more than 7.5×10 6 more than or about 7.5×10 6 more than 8×10 6 more than or about 8×10 6 more than 8.5×10 6 more than or about 8.5×10 6 more than 9×10 6 more than or about 9×10 6 more than 9.5×10 6 more than or about 9.5×10 6 more than 10×10 6 more than or about 10×10 6 can result in an increased yield. In some embodiments, the provided method can result in an increased yield of 9×10 6 more than or about 9×10 6 times from iPSCs to iCILs.

[0308] In some embodiments, the cells selected to undergo differentiation are, for example, pluripotent stem cells (PSCs) engineered using synthetic cytokine receptors, such as iPSCs, as described in Section II.B. In some embodiments, the cells selected to undergo differentiation are, for example, pluripotent stem cells (PSCs) further disrupted in the gene encoding B2M, such as iPSCs, to reduce expression or knockout the gene encoding B2M. In some embodiments, the engineered synthetic cytokine receptor is integrated into the disrupted B2M locus by, for example, HDR or other methods. In some embodiments, the cells selected to undergo differentiation are, for example, pluripotent stem cells (PSCs) further disrupted in the gene encoding FBP12, such as iPSCs, to reduce expression or knockout the gene encoding FBP12. In some embodiments, the cells selected to undergo differentiation are pluripotent stem cells (PSCs), and for example, iPSCs are any of the engineered cells described in Section I.D.

[0309] In some embodiments, the provided method includes culturing engineered PSCs (e.g., iPSCs) by incubation with a non-physiological ligand of a synthetic cytokine receptor (e.g., rapamycin or an analog) under conditions that cause the stem cells to differentiate into iCIL cells or precursors thereof, such as HPs or CLPs. In some embodiments, the method can include a further incubation in which various molecules are added to the culture medium. In some embodiments, the method can include an exchange of the medium to replenish or add any one or more molecules to the culture medium.

[0310] In some embodiments, the method includes a first incubation with a non-physiological ligand of a synthetic cytokine receptor (e.g., rapamycin or an analog) under conditions that induce differentiation into hematopoietic progenitor (HP) cells. In some embodiments, one or more specific additional molecules (e.g., small molecules) can be added to further promote or induce differentiation into hematopoietic progenitor (HP) cells.

[0311] In some embodiments, one or more of the above-described steps of producing HP cells can include adding a non-physiological ligand of a synthetic cytokine receptor (e.g., rapamycin or an analog) to the culture medium to induce differentiation. In some embodiments, the non-physiological ligand is rapamycin or a rapamycin analog. In some embodiments, the rapamycin analog is a rapalog. In some embodiments, the non-physiological ligand (e.g., rapamycin or a rapamycin analog) is added to the medium at a concentration of 2.5 nM and 200 nM, 2.5 nM and 150 nM, 2.5 nM and 100 nM, 2.5 nM and 50 nM, 2.5 nM and 20 nM, 2.5 nM and 10 nM. In some embodiments, the non-physiological ligand (e.g., rapamycin or a rapamycin analog) is added to the medium at a concentration of 5 nM to 200 nM, 5 nM to 150 nM, 5 nM to 100 nM, 5 nM to 50 nM, 5 nM to 20 nM, 5 nM to 10 nM, 10 nM to 200 nM, 10 nM to 150 nM, 10 nM to 100 nM, 10 nM to 50 nM, 10 nM to 20 nM, 20 nM to 200 nM, 20 nM to 150 nM, 20 nM to 100 nM, 20 nM to 50 nM, 50 nM to 200 nM, 50 nM to 150 nM, 50 nM to 100 nM, 100 nM to 200 nM, 100 nM to 150 nM, and 150 nM to 200 nM.

[0312] In some embodiments, the non-physiological ligand (e.g., rapamycin or a rapamycin analog) is added to the medium at a concentration of 10 nM or about 10 nM. In some embodiments, the non-physiological ligand (e.g., rapamycin or a rapamycin analog) is added to the medium at a concentration of 100 nM or about 100 nM. In some embodiments, rapamycin is added to the medium at a concentration of 100 nM or about 100 nM. In some embodiments, a rapalog is added to the medium at a concentration of 100 nM or about 100 nM.

[0313] In some embodiments, surprisingly, it has been found that low concentrations of non - physiological ligands (e.g., rapamycin or rapamycin analogs) can support differentiation and / or expansion proliferation. In some embodiments, the non - physiological ligand (e.g., rapamycin or rapamycin analog) is added to the medium at a concentration of 10 nM or less than 10 nM. In some embodiments, the non - physiological ligand (e.g., rapamycin or rapamycin analog) is added to the medium at a concentration of 2.5 nM to 10 nM, such as 3 nM to 7 nM. In some embodiments, the non - physiological ligand (e.g., rapamycin or rapamycin analog) is added to the medium at a concentration of 3 nM or about 3 nM, 4 nM or about 4 nM, 5 nM or about 5 nM, 6 nM or about 6 nM, 7 nM or about 7 nM, 8 nM or about 8 nM, 9 nM or about 9 nM, or 10 nM or about 10 nM, or any value between any of the foregoing. In some embodiments, the non - physiological ligand (e.g., rapamycin or rapamycin analog) is added to the medium at a concentration of 3.1 nM or about 3.1 nM. In some embodiments, the non - physiological ligand (e.g., rapamycin or rapamycin analog) is added to the medium at a concentration of 6.2 nM or about 6.2 nM. In some embodiments, rapamycin is added to the medium at a concentration of 3.1 nM or about 3.1 nM. In some embodiments, rapamycin is added to the medium at a concentration of 6.2 nM or about 6.2 nM. In some embodiments, a rapalog is added to the medium at a concentration of 3.1 nM or about 3.1 nM. In some embodiments, a rapalog is added to the medium at a concentration of 6.2 nM or about 6.2 nM.

[0314] In some embodiments, the provided method includes culturing engineered stem cells, engineered using, for example, a synthetic cytokine receptor, with a non - physiological ligand for a first period sufficient to generate CLP, and contacting the CLP with a differentiation medium for a second period sufficient to generate iCIL.

[0315] In some embodiments, conditions in addition to or other than activation by a synthetic cytokine receptor can be used in methods for differentiating engineered stem cells into CILs.

[0316] In some embodiments, the provided stem cells engineered using a synthetic cytokine receptor, e.g., iPSCs, may instead or alternatively be differentiated via any other method known to differentiate CILs. In some embodiments, in addition to non-physiological ligand engagement of the synthetic cytokine receptor, one or more growth factors or cytokines conventionally used in relation to the differentiation of CILs can be used in the provided methods.

[0317] Various differentiation protocols for CIL cells are known in the art.

[0318] In some embodiments, the stem cells are adapted to feeder-free culture. As used herein, a "feeder-free" (FF) environment refers to an environment such as a culture condition, cell culture, or culture medium that is essentially free of feeder cells or stromal cells and / or has not been preconditioned by the culture of feeder cells. A "preconditioned" medium refers to a medium taken after feeder cells have been cultured in the medium for a period of time, e.g., at least one day. The preconditioned medium contains many mediator substances including growth factors and cytokines secreted by the feeder cells cultured in the medium.

[0319] Generally, techniques for differentiating cells involve directly or indirectly modulating specific cell pathways using methods based on polynucleotides, polypeptides, and / or small molecules. The developmental potency of cells can be modulated, for example, by contacting the cells with one or more modulators. In some embodiments, the cells are cultured in the presence of one or more agents (e.g., small molecules, proteins, peptides, etc.) that induce cell differentiation. In some embodiments, one or more differentiating agents are introduced into the cells during in vitro culture. The cells can be maintained in a culture medium containing one or more agents for a period of time sufficient for the cells to achieve the desired differentiated phenotype.

[0320] In some embodiments, the culture platform includes one or more of nutrients, extracts, growth factors, hormones, cytokines, and media additives. Exemplary nutrients and extracts can include, for example, DMEM / F-12 (Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12), a basal medium widely used to support the growth of many different mammalian cells; KOSR (Knockout Serum Replacement); L-glut; NEAA (non-essential amino acids). Media additives can include, without limitation, MTG, ITS, (ME, antioxidants (e.g., ascorbic acid).

[0321] In some embodiments, the differentiation medium contains supplements such as serum, extracts, growth factors, hormones, cytokines, and the like.

[0322] In some embodiments, the culture medium of the present invention comprises one or more of the following cytokines or growth factors, namely, epidermal growth factor (EGF), acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), leukemia inhibitory factor (LIF), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), insulin-like growth factor 2 (IGF-2), keratinocyte growth factor (KGF), nerve growth factor (NGF), platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-β), bone morphogenetic protein (BMP4), vascular endothelial growth factor (VEGF), transferrin, various interleukins (such as IL-1 to IL-18), various colony-stimulating factors (such as granulocyte / macrophage colony-stimulating factor (GM-CSF)), various interferons (such as IFNγ), and other cytokines such as stem cell factor (SCF) and erythropoietin (EPO).

[0323] These cytokines may be commercially available and can be either natural or recombinant. In some other embodiments, the culture medium of the present disclosure comprises one or more of bone morphogenetic protein (BMP4), insulin-like growth factor-1 (IGF-1), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), hematopoietic growth factors (e.g., SCF, GMCSF, GCSF, EPO, IL3, TPO, EPO), Fms-related tyrosine kinase 3 ligand (FLT3L), and one or more cytokines from leukemia inhibitory factor (LIF), IL3, IL6, IL7, IL11, IL15. In some embodiments, the growth factors, mitogens, and cytokines are at concentrations specific to stages and / or cell types that are empirically determined or derived by established cytokine techniques. Examples of exogenous cell culture medium additives and supplements and cell selection kit components are provided in International Publication No. WO 2020 / 124256, the entire disclosure of which is incorporated herein by reference.

[0324] In some other embodiments, the culture medium of the present disclosure comprises Roswell Park Memorial Institute (RPMI) medium, cRPMI1640 medium, fetal bovine serum (FBS), Glutamax, penicillin, streptomycin, Rosuvastatin, BX795, protamine sulfate, brefeldin A, monensin, UM729, IL-2, IL-15, IL-21, IL-18, IL-7, or any combination thereof. In some other embodiments, the culture medium of the present disclosure comprises AIM V medium, fetal bovine serum (FBS), Glutamax, penicillin, streptomycin, Rosuvastatin, BX795, protamine sulfate, brefeldin A, monensin, UM729, IL-2, IL-15, IL-21, IL-18, IL-7, or any combination thereof. In some other embodiments, the culture medium of the present disclosure comprises AIM V medium STEMdiff APEL 2 Medium (STEMCELL Technologies).

[0325] Examples of methods for differentiating stem cells (e.g., iPSCs) into pluripotent hematopoietic progenitor cells are provided in U.S. Patent No. 9,624,470, U.S. Patent Application No. 2020 / 0080059, and Mesquitta et al., Sci. Rep. 9:6622 (2019), the entire disclosures of which are incorporated herein by reference.

[0326] In some embodiments, the method for producing CIL cells of the present disclosure comprises forming an embryoid body (EB) comprising aggregates of stem cells; differentiating the cells into hematopoietic stem cells in a first differentiation medium; differentiating the cells into lymphoid progenitor cells in a second differentiation medium; and / or differentiating the cells into differentiated CIL cells in a third differentiation medium. In some embodiments, the methods provided herein result in EBs that completely dissociate into pure hematopoietic precursors or hematopoietic stem cells without the need for additional purification steps.

[0327] In some embodiments, the method for generating cytotoxic innate lymphoid system (iCIL) cells comprises culturing a cell population comprising engineered iPSCs described herein under conditions that cause the iPSCs to differentiate into cytotoxic innate lymphoid system (iCIL), wherein a non-physiological ligand of a synthetic cytokine receptor is added during at least a portion of the culturing.

[0328] In some embodiments, one or more of the above steps for producing CIL cells can include adding a non-physiological ligand of a synthetic cytokine receptor (e.g., rapamycin or analogs thereof) to the culture medium to induce differentiation.

[0329] In some embodiments, the non-physiological ligand is rapamycin or a rapamycin analog. In some embodiments, the rapamycin analog is a rapalog. In some embodiments, the non-physiological ligand (e.g., rapamycin or a rapamycin analog) is added to the medium at a concentration of 2.5 nM and 200 nM, 2.5 nM and 1,50 nM, 2.5 nM and 100 nM, 2.5 nM and 50 nM, 2.5 nM and 20 nM, 2.5 nM and 10 nM. In some embodiments, the non-physiological ligand (e.g., rapamycin or a rapamycin analog) is added to the medium at a concentration of 5 nM to 200 nM, 5 nM to 150 nM, 5 nM to 100 nM, 5 nM to 50 nM, 5 nM to 20 nM, 5 nM to 10 nM, 10 nM to 200 nM, 10 nM to 150 nM, 10 nM to 100 nM, 10 nM to 50 nM, 10 nM to 20 nM, 20 nM to 200 nM, 20 nM to 150 nM, 20 nM to 100 nM, 20 nM to 50 nM, 50 nM to 200 nM, 50 nM to 150 nM, 50 nM to 100 nM, 100 nM to 200 nM, 100 nM to 150 nM, and 150 nM to 200 nM.

[0330] In some embodiments, a non - physiological ligand (e.g., rapamycin or a rapamycin analog) is added to the medium at a concentration of 10 nM or about 10 nM. In some embodiments, a non - physiological ligand (e.g., rapamycin or a rapamycin analog) is added to the medium at a concentration of 100 nM or about 100 nM. In some embodiments, rapamycin is added to the medium at a concentration of 100 nM or about 100 nM. In some embodiments, a rapalog is added to the medium at a concentration of 100 nM or about 100 nM.

[0331] In some embodiments, surprisingly, it has been found that low concentrations of non - physiological ligands (e.g., rapamycin or rapamycin analogs) can support differentiation and / or expansion proliferation. In some embodiments, the non - physiological ligand (e.g., rapamycin or rapamycin analog) is added to the medium at a concentration of 10 nM or less than 10 nM. In some embodiments, the non - physiological ligand (e.g., rapamycin or rapamycin analog) is added to the medium at a concentration of 2.5 nM to 10 nM, for example, 3 nM to 7 nM. In some embodiments, the non - physiological ligand (e.g., rapamycin or rapamycin analog) is added to the medium at a concentration of 3 nM or about 3 nM, 4 nM or about 4 nM, 5 nM or about 5 nM, 6 nM or about 6 nM, 7 nM or about 7 nM, 8 nM or about 8 nM, 9 nM or about 9 nM, or 10 nM or about 10 nM, or any value between any of the foregoing. In some embodiments, the non - physiological ligand (e.g., rapamycin or rapamycin analog) is added to the medium at a concentration of 3.1 nM or about 3.1 nM. In some embodiments, the non - physiological ligand (e.g., rapamycin or rapamycin analog) is added to the medium at a concentration of 6.2 nM or about 6.2 nM. In some embodiments, rapamycin is added to the medium at a concentration of 3.1 nM or about 3.1 nM. In some embodiments, rapamycin is added to the medium at a concentration of 6.2 nM or about 6.2 nM. In some embodiments, a rapalog is added to the medium at a concentration of 3.1 nM or about 3.1 nM. In some embodiments, a rapalog is added to the medium at a concentration of 6.2 nM or about 6.2 nM.

[0332] In some embodiments, a method of generating cytotoxic innate lymphoid-like (iCIL) cells comprises: (a) culturing a cell population comprising the engineered iPSCs described herein under conditions that form aggregates; (b) culturing the cells produced in (a) under conditions that induce mesoderm formation in a plurality of the cells, wherein the start of the culturing step in (b) is day 0; (c) culturing the cells produced in (b) under conditions that differentiate the cells into a population of hematopoietic progenitors (HPs); and (d) culturing the cells produced in (c) under conditions that generate iCIL cells, wherein at least a portion of one or more of steps (a)-(d) is performed in the presence of a non-physiological ligand of a synthetic cytokine receptor.

[0333] In some embodiments, to facilitate aggregate formation, the engineered iPSCs are transferred to a suitable container. The container can be a 2D or 3D container. Examples of suitable 2D containers for culturing the source cells include any Petri dish or culture dish regularly used in the laboratory for culturing cells. The culture container can be coated with a suitable culture medium, such as an extracellular medium for attachment and / or differentiation of the cultured cells. In some embodiments, the container is treated to promote cell adhesion and proliferation. An example of a medium suitable for use in the methods of the invention is MATRIGEL™ Membrane Matrix (BD Biosciences, Franklin Lakes, N.J.).

[0334] In some embodiments, the container is suitable for three-dimensional (3D) culture. Without being bound to a particular theory or mechanism, 3D culture is thought to be more effective than two-dimensional (2D) culture in providing a scaffold for cell differentiation. Suitable 3D culture systems can include, for example, hanging drop 3D culture, such as hanging drop plates, 3D microwell culture, such as ultra-low attachment multiwell plates, 3D culture on a hydrophobic surface, rotary culture, static 3D suspension culture, or bioreactors. For example, hanging drop plates such as the PERFECTA3D hanging drop plate available from Biospherix, Parish, N.Y. are commercially available. Ultra-low attachment multiwell plates (sometimes also referred to as non-adhesive culture vessels), such as the AGGREWELL™ ultra-low attachment multiwell plate available from Stemcell Technologies, Vancouver, Canada, are also commercially available.

[0335] In some embodiments, the container is not treated to promote cell attachment and growth. In some embodiments, the container is a standard tissue culture plate but is not treated to promote cell attachment and growth. In some embodiments, the cells do not attach or substantially do not attach during culture. In some embodiments, the culturing step is in suspension.

[0336] In some embodiments, the container is a multiwell plate. The multiwell plate can be a 96-well plate, a 24-well plate, or a 6-well plate.

[0337] In some embodiments, the vessel is a bioreactor. In some embodiments, the bioreactor is used in the process of iCIL production and proliferation after EB development. The bioreactor enables optimization of cell culture conditions to achieve optimal hydrogen production yields and process robustness. Environmental conditions that can be adjusted or monitored within the bioreactor include gas composition (e.g., air, oxygen, nitrogen, carbon dioxide), gas flow rate, temperature, pH, dissolved oxygen level, and agitation / circulation rate within the cell culture. For cell culture for the differentiation and expansion of cultured iCIL cells, any type of bioreactor known in the art can be used, including, without limitation, stirred tank bioreactors, pneumatic bioreactors (e.g., bubble column bioreactors or airlift bioreactors), membrane bioreactors, hollow fiber bioreactors, wave bioreactors, vertical wheel bioreactors, gas-permeable rapid expansion growth (G-Rex) bioreactors, or disposable bioreactors. In some embodiments, the bioreactor is a gas-permeable rapid expansion growth (G-Rex) bioreactor. In some embodiments, the bioreactor is a vertical wheel bioreactor. In some embodiments, the bioreactor is a stirred tank bioreactor. In some embodiments, the vertical wheel bioreactor is a PBS bioreactor. In some embodiments, the stirred tank bioreactor is a Sartorius Ambr250 stirred tank bioreactor.

[0338] The expansion and proliferation of iCIL cells can be scaled up or down to any desired volume suitable for various purposes. For example, for high-throughput to medium-throughput screening of various culture conditions, the process can be scaled up or down to be carried out in a micro-bioreactor (e.g., about 15 mL to about 500 mL), or a bench-top scale bioreactor, e.g., in the range of about 0.5 L to about 15 L. Alternatively, the process can be scaled up to a pilot scale bioreactor (e.g., in the range of about 15 L to about 15,000 L), or a manufacturing scale bioreactor (e.g., about 15,000 L to about 75,000 L or more).

[0339] In some embodiments, pluripotent aggregates may be formed in a bioreactor by culturing the engineered iPSCs in suspension within the bioreactor. In some embodiments, the iPSCs may spontaneously aggregate directly into spheroids within the bioreactor. In some embodiments, the aggregates or spheroids formed directly within the bioreactor may be approximately the same size as spheroids formed in other 3D culture systems, including, for example, ultra-low attachment micro-well plates. In one embodiment, the method may include forming spheroids directly from the source cells within the bioreactor without forming embryoid bodies (EBs) by culturing the source cells in suspension in a xeno-free medium within the bioreactor. The spheroids may contain undifferentiated iPSCs, such as engineered iPSCs.

[0340] In some embodiments, the aggregates in (a) are embryoid bodies (EBs).

[0341] In some embodiments, the differentiation of cells to generate CIL cells requires a change in the culture system, such as a stimulant in the culture medium or a change in the physical state of the cells. Conventional strategies utilize the formation of embryoid bodies as a common important intermediate to initiate lineage-specific differentiation. An embryoid body is an aggregate of stem cells that is induced to differentiate by changes in environmental stimuli (e.g., exposure and / or removal of specific molecules / chemical factors; and / or exposure to a three-dimensional structure / interaction with a three-dimensional structure). The formation of embryoid bodies induces cells to differentiate towards mesoderm specification.

[0342] Hematopoietic cells can be generated from embryoid bodies derived from pluripotent cells. Pluripotent cells may be able to form embryoid bodies or aggregates as part of the differentiation process. The formation of "embryoid bodies" (EBs) or clusters of proliferating cells to induce differentiation generally involves the in vitro aggregation of human pluripotent stem cells into EBs, enabling the human pluripotent stem cells to differentiate spontaneously and randomly into multiple tissue types representing endodermal, ectodermal, and mesodermal origins. Without specific culture conditions, it may take about two weeks for EBs to differentiate into any of the three germ layers, and the differentiation process occurs in a random pattern. In the provided embodiments, specific growth factors or cytokines can be added to the culture conditions to induce or promote the differentiation of EBs towards the hematopoietic lineage formed through the mesodermal lineage. EB cells differentiated into the hematopoietic lineage can be identified by one or more major hematopoietic lineage markers, such as any one or more of CD34, CD43, CD45, CD41, C235, and CD90.

[0343] In some embodiments, a single-cell suspension of engineered iPSCs is cultured in a suitable container to form EBs.

[0344] In some embodiments, pluripotent stem cell aggregates are transferred to a differentiation medium that provides an inductive cue towards a selected lineage (e.g., the lymphoid lineage). In some embodiments, once EBs are formed, they are dissociated and then cultured in the medium to induce mesodermal specificity of the cells.

[0345] In some embodiments, the suspended aggregates can be generated by culturing in an untreated container or under conditions for suspension culture. For example, the container is a container that has not been treated to promote cell adhesion and proliferation. In some embodiments, the step (a) of culturing a cell population containing engineered iPSCs under conditions for forming aggregates includes: (i) performing a first incubation that includes culturing a cell population of engineered stem cells under conditions for forming a first aggregate; (ii) contacting the aggregate with a dissociating agent to form a population of dissociated cells; and (iii) performing a second incubation that includes culturing the population of dissociated cells under conditions for forming a second aggregate. Methods for dissociating cells are known to those skilled in the art. Any of a variety of methods can be used. In some embodiments, the dissociation is by Gentle Cell Dissociation Reagent (GDCR; Stem Cell Technologies). In some embodiments, the dissociation is by EDTA.

[0346] In some aspects of the present invention, the above induction of embryoid bodies and differentiation into the hematopoietic progenitor cell platform can be performed under serum-free conditions. Examples of commercially available serum-free media suitable for cell attachment and / or induction include mTeSR™1, STEMdiff APEL 2 Medium or TeSR™2 manufactured by Stem Cell Technologies (Vancouver, Canada), Primate ES / iPS Cell Medium manufactured by ReproCELL (Boston, Mass.), StemPro®-34 manufactured by Invitrogen (Carlsbad, Calif.), StemPro® hESC SFM manufactured by Invitrogen, and X-VIVO™ manufactured by Lonza (Basel, Switzerland).

[0347] In some embodiments, the medium for the mesoderm specification step comprises one or more of bone morphogenetic protein 4 (BMP4), vascular endothelial growth factor (VEGF), stem cell factor (SCF), thrombopoietin (TPO), Fms-related tyrosine kinase 3 ligand (FLT3L), basic fibroblast growth factor (bFGF also known as FGF2). In some embodiments, the medium for the mesoderm specification step comprises one or more of bone morphogenetic protein 4 (BMP4), vascular endothelial growth factor (VEGF), stem cell factor (SCF), thrombopoietin (TPO), Fms-related tyrosine kinase 3 ligand (FLT3L), basic fibroblast growth factor (bFGF also known as FGF2) and / or a ROCK inhibitor. In some embodiments, the medium for the mesoderm specification step comprises one or more of bone morphogenetic protein 4 (BMP4), vascular endothelial growth factor (VEGF), stem cell factor (SCF), basic fibroblast growth factor (bFGF also known as FGF2) and / or a ROCK inhibitor. In some embodiments, the method comprises differentiating mesoderm-specified cells into hematopoietic stem cells in a first differentiation medium.

[0348] In some embodiments, the hematopoietic stem cells in the first differentiation medium comprise one or more of bone morphogenetic protein 4 (BMP4), fibroblast growth factor 2 (FGF2), vascular endothelial growth factor (VEGF), stem cell factor (SCF), Fms-related tyrosine kinase 3 ligand (FLT3L), thrombopoietin (TPO), interleukin-6 (IL-6), interleukin-3 (IL-3), a TGF-β inhibitor, a PI3K inhibitor or any combination thereof.

[0349] In some embodiments, the TGF-β inhibitor is GW788388. In some embodiments, the PI3K inhibitor is LY294002. In some embodiments, the ROCK inhibitor is Y27632.

[0350] In some embodiments, the stem cells differentiate into the mesodermal lineage in a differentiation medium containing BMP4, FGF2, and VEGF. In some embodiments, the stem cells differentiate into the mesodermal lineage in a differentiation medium containing BMP4, FGF2, VEGF, and a ROCK inhibitor. In some embodiments, the stem cells differentiate into the mesodermal lineage in a differentiation medium containing BMP4, FGF2, VEGF, and Y27632.

[0351] In some embodiments, the culture in (b) is in a medium containing one or more of BMP4, FGF2, VEGF, and a Rock inhibitor. In some embodiments, the Rock inhibitor is Y27632. In some embodiments, the culture in (b) is in a medium containing BMP4, FGF2, VEGF, and Y27632. In some embodiments, the culture in (b) is in a medium containing BMP4, FGF2, and VEGF. In some embodiments, the culture in (b) is in a medium containing a non-physiological ligand. In some embodiments, the culture in (b) is in a medium containing a non-physiological ligand and no additional growth factors.

[0352] In some embodiments, the culturing in (b) lasts for 2 to 4 days. In some embodiments, the cells are cultured in the differentiation medium for 2 to 4 days. In some embodiments, the culturing step lasts for 2 days or about 2 days, 3 days or about 3 days, or 4 days or about 4 days. In some embodiments, the culturing step lasts for 3 days or about 3 days. In some embodiments, the concentration of BMP4 in the medium is about 0.5 ng / mL to 2.5 ng / mL, 0.5 ng / mL to 5 ng / mL, 0.5 ng / mL to 10 ng / mL, 0.5 ng / mL to 15 ng / mL, 0.5 ng / mL to 20 ng / mL, 0.5 ng / mL to 30 ng / mL, 0.5 ng / mL to 50 ng / mL, 2.5 ng / mL to 5 ng / mL, 2.5 ng / mL to 10 ng / mL, 2.5 ng / mL to 15 ng / mL, 2.5 ng / mL to 20 ng / mL, 2.5 ng / mL to 30 ng / mL, 2.5 ng / mL to 50 ng / mL, 5 ng / mL to 10 ng / mL, 5 ng / mL to 15 ng / mL, 5 ng / mL to 20 ng / mL, 5 ng / mL to 30 ng / mL, 5 ng / mL to 50 ng / mL, 10 ng / mL to 15 ng / mL, 10 ng / mL to 20 ng / mL, 10 ng / mL to 30 ng / mL, 10 ng / mL to 50 ng / mL (including the values at both ends). In some embodiments, the concentration of BMP4 in the medium is at least about 0.5 ng / mL, 2.5 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 30 ng / mL or 50 ng / mL, and in some embodiments, the concentration of BMP4 in the medium is about 10 ng / mL.

[0353] In some embodiments, the concentration of FGF2 in the medium is from about 0.5 ng / mL to 2.5 ng / mL, 0.5 ng / mL to 5 ng / mL, 0.5 ng / mL to 10 ng / mL, 0.5 ng / mL to 15 ng / mL, 0.5 ng / mL to 20 ng / mL, 0.5 ng / mL to 30 ng / mL, 0.5 ng / mL to 50 ng / mL, 2.5 ng / mL to 5 ng / mL, 2.5 ng / mL to 10 ng / mL, 2.5 ng / mL to 15 ng / mL, 2.5 ng / mL to 20 ng / mL, 2.5 ng / mL to 30 ng / mL, 2.5 ng / mL to 50 ng / mL, 5 ng / mL to 10 ng / mL, 5 ng / mL to 15 ng / mL, 5 ng / mL to 20 ng / mL, 5 ng / mL to 30 ng / mL, 5 ng / mL to 50 ng / mL, 10 ng / mL to 15 ng / mL, 10 ng / mL to 20 ng / mL, 10 ng / mL to 30 ng / mL, 10 ng / mL to 50 ng / mL (including the values at both ends). In some embodiments, the concentration of FGF2 in the medium is at least about 0.5 ng / mL, 2.5 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 30 ng / mL or 50 ng / mL, and in some embodiments, the concentration of FGF2 in the medium is about 10 ng / mL.

[0354] In some embodiments, the concentration of VEGF in the medium is about 0.5 ng / mL to 2.5 ng / mL, 0.5 ng / mL to 5 ng / mL, 0.5 ng / mL to 10 ng / mL, 0.5 ng / mL to 15 ng / mL, 0.5 ng / mL to 20 ng / mL, 0.5 ng / mL to 30 ng / mL, 0.5 ng / mL to 50 ng / mL, 0.5 ng / mL to 100 ng / mL, 2.5 ng / mL to 5 ng / mL, 2.5 ng / mL to 10 ng / mL, 2.5 ng / mL to 15 ng / mL, 2.5 ng / mL to 20 ng / mL, 2.5 ng / mL to 30 ng / mL, 2.5 ng / mL to 50 ng / mL, 2.5 ng / mL to 100 ng / mL, 5 ng / mL to 10 ng / mL, 5 ng / mL to 15 ng / mL, 5 ng / mL to 20 ng / mL, 5 ng / mL to 30 ng / mL, 5 ng / mL to 50 ng / mL, 5 ng / mL to 100 ng / mL, 10 ng / mL to 15 ng / mL, 10 ng / mL to 20 ng / mL, 10 ng / mL to 30 ng / mL, 10 ng / mL to 50 ng / mL, 10 ng / mL to 100 ng / mL (including the values at both ends). In some embodiments, the concentration of VEGF in the medium is at least about 0.5 ng / mL, 2.5 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 30 ng / mL, 50 ng / mL or 100 ng / mL, and in some embodiments, the concentration of VEGF in the medium is about 50 ng / mL.

[0355] In some embodiments, the concentration of Y27632 in the medium is from about 0.5 μM to 2.5 μM, 0.5 μM to 5 μM, 0.5 μM to 10 μM, 0.5 μM to 15 μM, 0.5 μM to 20 μM, 0.5 μM to 30 μM, 0.5 μM to 50 μM, 2.5 μM to 5 μM, 2.5 μM to 10 μM, 2.5 μM to 15 μM, 2.5 μM to 20 μM, 2.5 μM to 30 μM, 2.5 μM to 50 μM, 5 μM to 10 μM, 5 μM to 15 μM, 5 μM to 20 μM, 5 μM to 30 μM, 5 μM to 50 μM, 10 μM to 15 μM, 10 μM to 20 μM, 10 μM to 30 μM, 10 μM to 50 μM (including the values at both ends). In some embodiments, the concentration of Y27632 in the medium is at least about 0.5 μM, 2.5 μM, 5 μM, 10 μM, 15 μM, 20 μM, 30 μM or 50 μM, and in some embodiments, the concentration of Y27632 in the medium is about 10 μM.

[0356] In some embodiments, mesoderm cells differentiate into hematopoietic stem cells in a differentiation medium containing BMP4, FGF2, VEGF and SCF. In some embodiments, mesoderm cells differentiate into hematopoietic stem cells in a differentiation medium containing BMP4, FGF2, VEGF, SCF, TPO and LDL. In some embodiments, mesoderm cells differentiate into hematopoietic stem cells in a differentiation medium containing BMP4, FGF2 and VEGF. In some embodiments, the differentiation medium contains a non-physiological ligand of a synthetic cytokine receptor (e.g., rapamycin or an analog).

[0357] In some embodiments, hematopoietic stem cells differentiate into lymphoid progenitor cells in a differentiation medium containing BMP4, FGF2, VEGF and SCF. In some embodiments, hematopoietic stem cells differentiate into lymphoid progenitor cells in a differentiation medium containing BMP4, FGF2 and VEGF. In some embodiments, hematopoietic stem cells differentiate into lymphoid progenitor cells in a differentiation medium containing BMP4, FGF2, VEGF, SCF, TPO and LDL. In some embodiments, the differentiation medium contains a non-physiological ligand of a synthetic cytokine receptor (e.g., rapamycin or an analog).

[0358] In some embodiments, the culture in (c) is in a medium containing one or more of BMP4, FGF2, VEGF, TPO, SCF, and LDL. In some embodiments, the culture in (c) is in a medium containing one or more of BMP4, FGF2, VEGF, and LDL. In some embodiments, the culture in (c) is in a medium that does not contain SCF and TPO. In some of any embodiment, the culture in (c) is in a medium containing one or more of BMP4, FGF2, and a PI3K inhibitor. In some embodiments, the PI3K inhibitor is LY2940002. In some of any embodiment, the culture in (c) is in a medium that does not contain LDL, VEGF, SCF, and / or TPO. In some embodiments, the culture in (c) is in a medium containing a non-physiological ligand. In some embodiments, the culture in (c) is in a medium containing a non-physiological ligand that does not contain any additional growth factors, cytokines, or both.

[0359] In some embodiments, the culture in (c) is for 3 days to 15 days. In some embodiments, during at least a portion of the culture in (c), the medium contains an aryl hydrocarbon receptor (AHR) antagonist (e.g., StemRegenin-1), a pyrimido-[4,5-b]-indole derivative (e.g., UM729), or both. In some embodiments, a portion of the culturing step is on days 9 to 15 or about days 9 to 15. In some embodiments, a portion of the culturing step is on days 6 to 15 or about days 6 to 15. In some embodiments, the culture in (c) is on day 6. In some embodiments, the culture in (c) is on day 9.

[0360] In some embodiments, the concentration of BMP4 in the medium is from about 0.5 ng / mL to 2.5 ng / mL, 0.5 ng / mL to 5 ng / mL, 0.5 ng / mL to 10 ng / mL, 0.5 ng / mL to 15 ng / mL, 0.5 ng / mL to 20 ng / mL, 0.5 ng / mL to 30 ng / mL, 0.5 ng / mL to 50 ng / mL, 2.5 ng / mL to 5 ng / mL, 2.5 ng / mL to 10 ng / mL, 2.5 ng / mL to 15 ng / mL, 2.5 ng / mL to 20 ng / mL, 2.5 ng / mL to 30 ng / mL, 2.5 ng / mL to 50 ng / mL, 5 ng / mL to 10 ng / mL, 5 ng / mL to 15 ng / mL, 5 ng / mL to 20 ng / mL, 5 ng / mL to 30 ng / mL, 5 ng / mL to 50 ng / mL, 10 ng / mL to 15 ng / mL, 10 ng / mL to 20 ng / mL, 10 ng / mL to 30 ng / mL, 10 ng / mL to 50 ng / mL (including the values at both ends). In some embodiments, the concentration of BMP4 in the medium is at least about 0.5 ng / mL, 2.5 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 30 ng / mL or 50 ng / mL, and in some embodiments, the concentration of BMP4 in the medium is about 10 ng / mL.

[0361] In some embodiments, the concentration of FGF2 in the medium is about 0.5 ng / mL to 2.5 ng / mL, 0.5 ng / mL to 5 ng / mL, 0.5 ng / mL to 10 ng / mL, 0.5 ng / mL to 15 ng / mL, 0.5 ng / mL to 20 ng / mL, 0.5 ng / mL to 30 ng / mL, 0.5 ng / mL to 50 ng / mL, 2.5 ng / mL to 5 ng / mL, 2.5 ng / mL to 10 ng / mL, 2.5 ng / mL to 15 ng / mL, 2.5 ng / mL to 20 ng / mL, 2.5 ng / mL to 30 ng / mL, 2.5 ng / mL to 50 ng / mL, 5 ng / mL to 10 ng / mL, 5 ng / mL to 15 ng / mL, 5 ng / mL to 20 ng / mL, 5 ng / mL to 30 ng / mL, 5 ng / mL to 50 ng / mL, 10 ng / mL to 15 ng / mL, 10 ng / mL to 20 ng / mL, 10 ng / mL to 30 ng / mL, 10 ng / mL to 50 ng / mL (including the values at both ends). In some embodiments, the concentration of FGF2 in the medium is at least about 0.5 ng / mL, 2.5 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 30 ng / mL or 50 ng / mL, and in some embodiments, the concentration of FGF2 in the medium is about 10 ng / mL.

[0362] In some embodiments, the concentration of SCF in the medium is from about 0.5 ng / mL to 2.5 ng / mL, 0.5 ng / mL to 5 ng / mL, 0.5 ng / mL to 10 ng / mL, 0.5 ng / mL to 15 ng / mL, 0.5 ng / mL to 20 ng / mL, 0.5 ng / mL to 30 ng / mL, 0.5 ng / mL to 50 ng / mL, 0.5 ng / mL to 100 ng / mL, 2.5 ng / mL to 5 ng / mL, 2.5 ng / mL to 10 ng / mL, 2.5 ng / mL to 15 ng / mL, 2.5 ng / mL to 20 ng / mL, 2.5 ng / mL to 30 ng / mL, 2.5 ng / mL to 50 ng / mL, 2.5 ng / mL to 100 ng / mL, 5 ng / mL to 10 ng / mL, 5 ng / mL to 15 ng / mL, 5 ng / mL to 20 ng / mL, 5 ng / mL to 30 ng / mL, 5 ng / mL to 50 ng / mL, 5 ng / mL to 100 ng / mL, 10 ng / mL to 15 ng / mL, 10 ng / mL to 20 ng / mL, 10 ng / mL to 30 ng / mL, 10 ng / mL to 50 ng / mL, 10 ng / mL to 100 ng / mL (including the values at both ends). In some embodiments, the concentration of SCF in the medium is at least about 0.5 ng / mL, 2.5 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 30 ng / mL, 50 ng / mL or 100 ng / mL, and in some embodiments, the concentration of SCF in the medium is about 50 ng / mL.

[0363] In some embodiments, the concentration of UM729 in the medium is from about 0.5 μM to 1 μM, 0.5 μM to 5 μM, 0.5 μM to 10 μM, 0.5 μM to 15 μM, 0.5 μM to 20 μM, 0.5 μM to 30 μM, 0.5 μM to 50 μM, 1 μM to 5 μM, 1 μM to 10 μM, 1 μM to 15 μM, 1 μM to 20 μM, 1 μM to 30 μM, 1 μM to 50 μM, 5 μM to 10 μM, 5 μM to 15 μM, 5 μM to 20 μM, 5 μM to 30 μM, 5 μM to 50 μM, 10 μM to 15 μM, 10 μM to 20 μM, 10 μM to 30 μM, 10 μM to 50 μM (including the values at both ends). In some embodiments, the concentration of UM729 in the medium is at least about 0.5 μM, 1 μM, 5 μM, 10 μM, 15 μM, 20 μM, 30 μM or 50 μM, and in some embodiments, the concentration of UM729 in the medium is about 1 μM.

[0364] In some embodiments, the concentration of SR1 in the medium is from about 0.5 μM to 1 μM or less than about 0.5 μM to 1 μM, from about 0.5 μM to 5 μM or less than about 0.5 μM to 5 μM, from about 0.5 μM to 10 μM or less than about 0.5 μM to 10 μM, from about 0.5 μM to 15 μM or less than about 0.5 μM to 15 μM, from about 0.5 μM to 20 μM or less than about 0.5 μM to 20 μM, from about 0.5 μM to 30 μM or less than about 0.5 μM to 30 μM, from about 0.5 μM to 50 μM or less than about 0.5 μM to 50 μM, from about 1 μM to 5 μM or less than about 1 μM to 5 μM, from about 1 μM to 10 μM or less than about 1 μM to 10 μM, from about 1 μM to 15 μM or less than about 1 μM to 15 μM, from about 1 μM to 20 μM or less than about 1 μM to 20 μM, from about 1 μM to 30 μM or less than about 1 μM to 30 μM, from about 1 μM to 50 μM or less than about 1 μM to 50 μM, from about 5 μM to 10 μM or less than about 5 μM to 10 μM, from about 5 μM to 15 μM or less than about 5 μM to 15 μM, from about 5 μM to 20 μM or less than about 5 μM to 20 μM, from about 5 μM to 30 μM or less than about 5 μM to 30 μM, from about 5 μM to 50 μM or less than about 5 μM to 50 μM, from about 10 μM to 15 μM or less than about 10 μM to 15 μM, from about 10 μM to 20 μM or less than about 10 μM to 20 μM, from about 10 μM to 30 μM or less than about 10 μM to 30 μM, from about 10 μM to 50 μM or less than about 10 μM to 50 μM (including both end values). In some embodiments, the concentration of SR1 in the medium is at least about 0.5 μM, 1 μM, 5 μM, 10 μM, 15 μM, 20 μM, 30 μM or 50 μM, and in some embodiments, the concentration of SR1 in the medium is about 1 μM

[0365] In some embodiments, lymphoid progenitor cells differentiate into cytotoxic natural lymphoid cells in a differentiation medium containing a non - physiological ligand, SCF, FLT3L, and UM729. In some embodiments, lymphoid progenitor cells differentiate into cytotoxic natural lymphoid cells in a differentiation medium containing a non - physiological ligand, SCF, and UM729. In some embodiments, the non - physiological ligand is a rapalog. In some embodiments, the non - physiological ligand is rapamycin.

[0366] In some embodiments, the step of differentiating mesoderm-specified cells into hematopoietic stem cells in the first differentiation medium is performed for a period sufficient for the mesoderm-specified cells to become CD34+ hematopoietic stem cells.

[0367] In some embodiments, the method includes the step of differentiating hematopoietic stem cells into lymphoid progenitor cells in a second differentiation medium.

[0368] In some embodiments, the lymphoid progenitor cell differentiation medium comprises one or more of bone morphogenetic protein 4 (BMP4), fibroblast growth factor 2 (FGF2), vascular endothelial growth factor (VEGF), stem cell factor (SCF), Fms-related tyrosine kinase 3 ligand (FLT3L), thrombopoietin (TPO), interleukin-6 (IL-6), interleukin-3 (IL-3), a TGF-β inhibitor, a PI3K inhibitor, or any combination thereof.

[0369] In some embodiments, the step of differentiating hematopoietic stem cells into lymphoid progenitor cells in the second differentiation medium is performed for a period sufficient for the hematopoietic stem cells to become Lin-CD34+CD38- / lo CD45RA+CD90-lymphoid progenitor cells.

[0370] In some embodiments, the first differentiation medium further comprises a non-physiological ligand of the present disclosure and / or the second differentiation medium further comprises a non-physiological ligand of the present disclosure.

[0371] In some embodiments, the first differentiation medium and / or the second differentiation medium substantially does not contain at least one cytokine (e.g., IL-2, IL-15, and / or IL-7).

[0372] In some embodiments, the differentiation medium comprises one or more of stem cell factor (SCF), interleukin-7 (IL-7), interleukin-15 (IL-15), Fms-related tyrosine kinase 3 ligand (FLT3L), pyrimido-indole derivatives, or any combination thereof. The pyrimido-indole derivative is UM 729 (pyrimido-[4,5-b]-indole derivative).

[0373] In some embodiments, the culturing in (d) is in a medium comprising one or more of FLT3L, IL-7, IL-12, IL-15, SR-1, and UM729. In some embodiments, the culturing in (d) is in a medium comprising one or more of IL-15, SCR, SR-1, and UM729. In some embodiments, the culturing in (d) is in a medium that does not contain FLT3L, IL-7 and / or IL-12. In some embodiments, the culturing in (d) is in a medium comprising a non-physiological ligand. In some embodiments, the culturing in (d) is in a medium comprising a non-physiological ligand that does not contain any additional growth factors, cytokines, or both. In some embodiments, the culturing in (d) lasts for a period from day 15 to day 40. In some embodiments, the culturing in (d) lasts from day 15 to day 35. In some embodiments, the culturing in (d) lasts from day 15 to day 30.

[0374] In some embodiments, differentiating lymphoid progenitor cells into CIL cells in a differentiation medium is carried out for a period sufficient for the lymphoid progenitor cells to become CD3−CD56+CD45+CD94+CD122+ / IL-2Rβ+CD127 / IL-7Rα−FcγRIII / CD16+KIR+NKG2A+NKG2D+NKp30+NKp44+NKP46+NKp80+. In some embodiments, differentiating lymphoid progenitor cells into CIL cells in a differentiation medium is carried out for a period sufficient for the lymphoid progenitor cells to become CD45+CD56+. In some embodiments, differentiating lymphoid progenitor cells into CIL cells in a differentiation medium is carried out for a period sufficient for the lymphoid progenitor cells to become CD45+CD5−CD7+CD56+.

[0375] In some embodiments, differentiating lymphoid progenitor cells into CIL cells in a differentiation medium is carried out over about 8 to about 18 days. In some embodiments, differentiating lymphoid progenitor cells into CIL cells in a differentiation medium is carried out over about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19 or about 20 days.

[0376] In some embodiments, the CIL cell differentiation medium is substantially free of interleukin-15 (IL-15). In some embodiments, the CIL cell differentiation medium is substantially free of interleukin-7 (IL-7). In some embodiments, the CIL cell differentiation medium is substantially free of interleukin-2 (IL-2). In some embodiments, the CIL cell differentiation medium is substantially free of IL-15, IL-7 and / or IL-2.

[0377] In some embodiments, after differentiation, the CIL cells are expanded and proliferated in a medium containing a non-physiological ligand and CD2 / NKp46 stimulation. In some embodiments, the CIL cell expansion and proliferation medium contains activation beads comprising conjugated anti-CD2 antibody and anti-NKp46 antibody that stimulate and activate the CIL cells.

[0378] In some embodiments, after differentiation, the RACR-iCIL cells are expanded and proliferated in a medium containing a non-physiological ligand, membrane-bound IL-21 (mbIL21), and 41BBL K562 feeder cells.

[0379] F. Induced cytotoxic innate lymphoid cell line (iCIL) and expansion and proliferation of iCIL cells In some embodiments, CIL cells can be induced from iPSCs by sequentially differentiating iPSCs into hematopoietic progenitor cells (HPCs), HPCs into common lymphoid progenitor cells (CLPs), and then CLPs into CIL cells called "iCIL" cells. In a variant, CIL cells can be induced from HPCs by sequentially differentiating HPCs into CLPs and then CLPs into iCIL cells. In a further variant, CIL cells can be induced by differentiating CLPs into iCIL cells. Manipulating the cells to express a synthetic cytokine receptor can be done at the iPSC, HPC, CLP, or iCIL cell stage of the differentiation process.

[0380] In some embodiments, iCIL cells are characterized by being CD3-CD56+CD45+CD94+CD122+ / IL-2Rβ+CD127 / IL-7Rα-FcγRIII / CD16+KIR+NKG2A+NKG2D+NKp30+NKp44+NKP46+NKp80+. In some embodiments, iCIL cells are characterized by being CD3-CD56+CD45+ cells. In some embodiments, iCIL cells are characterized by being CD3-CD56+ cells. In some embodiments, iCIL cells comprise one or more cell markers selected from the group consisting of CD56+, CD45+, CD94+, CD122+ / IL-2Rβ+, FcγRIII / CD16+, KIR+, NKG2A+, NKG2D+, NKp30+, NKp44+, NKP46+, NKp80+, or any combination thereof. iCIL cells can be CD45+; CD45+CD5-; or CD45+CD5-CD56+.

[0381] In some embodiments, the iCIL cells are CD45+CD7+CD56 + / lo characterized by being.

[0382] In some embodiments, after transducing the iCIL cells with a CAR, the cells are cultured under conditions that promote activation of the cells.

[0383] The culture conditions can be such that the cells can be administered to a patient without considering the reactivity to the components of the culture medium. For example, the culture conditions may omit bovine serum products such as bovine serum albumin. In an exemplary aspect, activation can be achieved by introducing a known activator into the culture medium. In one aspect, a population of iCIL cells can be cultured for about 1 to about 4 days under conditions that promote activation. In one embodiment, an appropriate activation level can be determined by cell size, growth rate, or an activation marker determined by flow cytometry. In some embodiments, any of the culture methods disclosed herein may be used to promote activation of the iCIL cells.

[0384] In some embodiments, provided herein are methods of making and / or expanding a population of engineered cells comprising a synthetic cytokine receptor for a non-physiological ligand. In some embodiments, the method comprises a. optionally, providing engineered cells comprising a synthetic cytokine receptor by introducing a polynucleotide encoding the synthetic cytokine receptor into a source cell, and b. incubating the engineered cells in a medium comprising the non-physiological ligand comprising.

[0385] In this aspect, the cytokine receptor comprises a synthetic gamma chain polypeptide comprising a first dimerization domain, a first transmembrane domain, and an intracellular domain of interleukin-2 receptor subunit gamma (IL-2RG), and a synthetic beta chain polypeptide comprising a second dimerization domain, a second transmembrane domain, and an intracellular domain selected from the intracellular domain of interleukin-2 receptor subunit beta (IL-2RB), the intracellular domain of interleukin-7 receptor subunit beta (IL-7RB), and / or the intracellular domain of interleukin-21 receptor subunit beta (IL-21RB). The non-physiological ligand activates the synthetic cytokine receptor in the engineered cell to induce expansion and / or activation of the engineered cell.

[0386] In some aspects, the engineered CIL cells are expanded without using IL-2 in the expansion medium.

[0387] In some aspects, CIL cells are engineered to express a synthetic cytokine receptor such as RACR and are expanded in an expansion medium using a rapalog without using a recombinant cytokine in the expansion medium.

[0388] In some aspects, the CIL cell expansion step is performed in a cell culture medium substantially free of recombinant cytokine.

[0389] In some aspects, the expansion step is performed in feeder-free cell culture.

[0390] In some aspects, the CIL cell expansion step is performed in a culture vessel not coated with a recombinant ligand for CIL cell expansion.

[0391] In some aspects, provided is a method of administering an effective amount of a non-physiological ligand to a subject, wherein the non-physiological ligand differentiates iPSC cells into iCIL cells according to any of the foregoing aspects.

[0392] In some embodiments, provided is a method of administering an effective amount of a non - physiological ligand to a subject, wherein the non - physiological ligand differentiates common lymphoid progenitor cells into CIL cells according to any of the foregoing embodiments.

[0393] In some embodiments, provided is a method of differentiating and expanding iCIL cells by culturing them in a suitable container. The container can be a 2D container or a 3D container. Examples of suitable 2D containers for culturing source cells include any Petri dish or culture dish that is regularly used in the laboratory for culturing cells. The culture container can be coated with a suitable culture medium, such as an extracellular medium for cell attachment and / or differentiation. In some embodiments, the container is treated to promote cell adhesion and growth. Examples of media suitable for use in the methods of the present invention include MATRIGEL™ membrane matrix (BD Biosciences, Franklin Lakes, N.J.).

[0394] In some embodiments, the container is suitable for three - dimensional (3D) culture. Without being bound by a particular theory or mechanism, 3D culture is thought to be more effective than two - dimensional (2D) culture in providing a scaffold for cell differentiation. Suitable 3D culture systems can include, for example, hanging - drop 3D culture, such as hanging - drop plates, 3D microwell culture, such as ultra - low attachment multi - well plates, 3D culture on a hydrophobic surface, rotating culture, static 3D suspension culture, or bioreactors. For example, hanging - drop plates such as the PERFECTA3D hanging - drop plate available from Biospherix, Parish, N.Y. are commercially available. Also commercially available are ultra - low attachment multi - well plates (sometimes also referred to as non - adherent culture containers), such as the AGGREWELL™ ultra - low attachment multi - well plate available from Stemcell Technologies, Vancouver, Canada.

[0395] In some embodiments, the container is not treated to promote cell adhesion and growth. In some embodiments, the container is a standard tissue culture plate but is not treated to promote cell adhesion and growth. In some embodiments, the cells do not adhere or substantially do not adhere during culture. In some embodiments, the culturing step is in suspension.

[0396] In some embodiments, the container is a multi-well plate. The multi-well plate can be a 96-well plate, a 24-well plate, or a 6-well plate.

[0397] In some embodiments, the container is a bioreactor. In some embodiments, the bioreactor is used in the process of iCIL production and growth after EB development. The bioreactor enables optimization of cell culture conditions to achieve an optimal hydrogen production yield and process robustness. Environmental conditions that can be adjusted or monitored within the bioreactor include gas composition (e.g., air, oxygen, nitrogen, carbon dioxide), gas flow rate, temperature, pH, dissolved oxygen level, and agitation / circulation rate within the cell culture. For cell culture for the differentiation and expansion of cultured iCIL cells, any type of bioreactor known in the art can be used, including, without limitation, a stirred tank bioreactor, a pneumatic bioreactor (e.g., a bubble column bioreactor or an airlift bioreactor), a membrane bioreactor, a hollow fiber bioreactor, a wave bioreactor, a vertical wheel bioreactor, a gas-permeable rapid expansion growth (G-Rex) bioreactor, or a disposable bioreactor. In some embodiments, the bioreactor is a gas-permeable rapid expansion growth (G-Rex) bioreactor. In some embodiments, the bioreactor is a vertical wheel bioreactor. In some embodiments, the bioreactor is a stirred tank bioreactor. In some embodiments, the vertical wheel bioreactor is a PBS bioreactor. In some embodiments, the stirred tank bioreactor is a Sartorius Ambr250 stirred tank bioreactor.

[0398] The expansion and proliferation of iCIL cells can be scaled up or down to any desired volume suitable for various purposes. For example, for high-throughput to medium-throughput screening of various culture conditions, the process can be scaled up or down to be carried out in a micro-bioreactor (e.g., about 15 mL to about 500 mL), or a bench-top scale bioreactor, e.g., in the range of about 0.5 L to about 15 L. Alternatively, the process can be scaled up to a pilot scale bioreactor (e.g., in the range of about 15 L to about 15,000 L), or a manufacturing scale bioreactor (e.g., about 15,000 L to about 75,000 L or more).

[0399] In some embodiments, one or more containers can be used for iCIL expansion and proliferation. In some embodiments, the cells can be cultured in the container from day 0 to day 3, day 0 to day 10, day 0 to day 15, day 0 to day 20, day 0 to day 25, day 0 to day 30, day 0 to day 35, day 0 to day 40, day 0 to day 50, day 0 to day 60, day 0 to day 100, day 3 to day 10, day 3 to day 15, day 3 to day 20, day 3 to day 25, day 3 to day 30, day 3 to day 35, day 3 to day 40, day 3 to day 50, day 3 to day 60, day 3 to day 100, day 10 to day 20, day 10 to day 25, day 10 to day 30, day 10 to day 35, day 10 to day 40, day 10 to day 50, day 10 to day 60, day 10 to day 100, day 15 to day 20, day 15 to day 25, day 15 to day 30, day 15 to day 35, day 15 to day 40, day 15 to day 50, day 15 to day 60, day 15 to day 100, day 20 to day 25, day 20 to day 30, day 20 to day 35, day 20 to day 40, day 20 to day 50, day 20 to day 60, day 20 to day 100, day 25 to day 30, day 25 to day 35, day 25 to day 40, day 25 to day 50, day 25 to day 60, day 25 to day 100, day 30 to day 35, day 30 to day 40, day 30 to day 50, day 30 to day 60, day 30 to day 100, day 35 to day 40, day 35 to day 50, day 35 to day 60, day 35 to day 100, day 40 to day 50, day 40 to day 60, day 40 to day 100 (including the values at both ends). In some embodiments, the cells can be cultured in the container until day 0 to day 35. In some embodiments, the cells can be cultured in the container until day 3 to day 35. In some embodiments, the container is a bioreactor. In some embodiments, one or more bioreactors can be used for iCIL expansion and proliferation. In some embodiments, one or more bioreactors are different types of bioreactors. In some embodiments, one or more bioreactors are bioreactors of different sizes.

[0400] G. Characteristics of Engineered Stem Cells and iCIL Cells In some embodiments, the CIL cells are CD3−CD5−, CD16+, CD56+, CD57+, NKp30+, NKp46+, NKG2A+, and / or NKG2D+.

[0401] In some embodiments, the population of engineered cells is 40% - 60% CD16+, 50% - 70% CD16+, 60% - 80% CD16+, 70% - 90% CD16+, 80% - 100% CD16+, or any percentage within a range defined by any two of the foregoing values.

[0402] In some embodiments, the population of engineered cells is 60% - 80% CD56+, 65% - 85% CD56+, 70% - 90% CD56+, 75% - 95% CD56+, 80% - 99% CD56+, or any percentage within a range defined by any two of the foregoing values.

[0403] In some embodiments, the population of engineered CIL cells is CD56lo. In some embodiments, the population of engineered CIL cells is CD56high.

[0404] In some embodiments, the population of engineered CIL cells is 60% - 80% CD16+CD56+, 65% - 85% CD16+CD56+, 70% - 90% CD16+CD56+, 75% - 95% CD16+CD56+, 80% - 99% CD16+CD56+, or any percentage within a range defined by any two of the foregoing values.

[0405] In some embodiments, the population of engineered CIL cells is at least 40% CD16+, at least 50% CD16+, at least 60% CD16+, at least 70% CD16+, at least 80% CD16+, at least 90% CD16+, or 100% CD16+.

[0406] In some embodiments, the population of engineered CIL cells is at least 80% CD56+, at least 85% CD56+, at least 90% CD56+, at least 95% CD56+, or 100% CD56+.

[0407] In some embodiments, the population of engineered CIL cells is at least 40% CD16+CD56+, at least 50% CD16+CD56+, at least 60% CD16+CD56+, at least 70% CD16+CD56+, at least 80% CD16+CD56+, at least 90% CD16+CD56+, or 100% CD16+CD56+.

[0408] In some embodiments, the CIL cells are characterized by being CD45+CD56+.

[0409] In some embodiments, the population of engineered CIL cells is 40% - 60% CD45+, 50% - 70% CD45+, 60% - 80% CD45+, 70% - 90% CD45+, 80% - 100% CD45+, or any percentage within a range defined by any two of the foregoing values.

[0410] In some embodiments, the population of engineered CIL cells is 60% - 80% CD45+CD56+, 65% - 85% CD45+CD56+, 70% - 90% CD45+CD56+, 75% - 95% CD45+CD56+, 80% - 99% CD45+CD56+, or any percentage within a range defined by any two of the foregoing values.

[0411] In some embodiments, the population of engineered CIL cells is at least 40% CD45+, at least 50% CD45+, at least 60% CD45+, at least 70% CD45+, at least 80% CD45+, at least 90% CD45+, or 100% CD45+.

[0412] In some embodiments, the population of engineered CIL cells is at least 40% CD45+CD56+, at least 50% CD45+CD56+, at least 60% CD45+CD56+, at least 70% CD45+CD56+, at least 80% CD45+CD56+, at least 90% CD45+CD56+, or 100% CD45+CD56+.

[0413] K562 cells are a sensitive target for in vitro cytotoxic natural lymphocyte - like cell cytotoxicity assays. In such assays, CIL cells are co - incubated with K562 target tumor cells, which are known to be sensitive to CIL cell - mediated cytotoxicity, at various ratios. The target cells (K562) are pre - labeled with a fluorescent dye to enable discrimination from effector cells (CIL cells). After the incubation period, killed target cells are identified by nucleic acid staining that specifically penetrates dead cells. The percentage of death is calculated by comparing the total number of viable cells in each experimental assay well to non - effector control wells. As used herein, the term "activity" refers to a measure of the cytotoxic ability of CIL cells against target cells.

[0414] In some embodiments, engineered CIL cells secrete CD107a in response to an antigen recognized by the engineered CIL cells.

[0415] Stimulation of natural CIL cell receptors causes secretion of CD107a by CIL cells. Since CD107a expression correlates with both cytokine secretion and CIL cell - mediated lysis of target cells, as used herein, CD107a secretion is a marker of CIL cell functional activity.

[0416] In some embodiments, engineered CIL cells secrete interferon - gamma (IFNγ) and / or tumor necrosis factor - alpha (TNF - α) in response to an antigen recognized by the engineered CIL cells.

[0417] Activation of CIL cells results in the secretion of IFNγ and TNF - α, which synergistically enhance the cytotoxicity of CIL cells. In engineered CIL cells, the expression of IFNγ and / or TNF - α is a marker of CIL cell activity.

[0418] In some embodiments, the engineered CIL cells can be fresh or can be frozen. In some embodiments, the engineered CIL cells are fresh. In some embodiments, the engineered CIL cells are frozen. In some embodiments, when the frozen CIL cells are thawed, they retain viability and cytotoxic function compared to fresh CIL cells. In some embodiments, the frozen / thawed CIL cells function as fresh CIL cells and control tumors.

[0419] In some embodiments, the cells can be frozen by a cryopreservation method. In some embodiments, the iCILs are subjected to cryopreservation after being differentiated according to the provided method. In some embodiments, the iCILs are subjected to cryopreservation after those operations. In some embodiments, the engineered iCILs produced according to the provided method are subjected to cryopreservation. In some embodiments, the method includes the step of cryopreserving the cells in the presence of a cryoprotectant, thereby generating a cryopreserved composition. In some aspects, any of various known cryopreservation solutions and parameters can be used. In some embodiments, the cryoprotectant is DMSO. In some embodiments, the cells are frozen, e.g., cryopreserved, in a solution having a final concentration of 1% - 15%, 6% - 12%, 5% - 10%, or 6% - 8% DMSO. In some embodiments, the cryopreservation medium is 5% - 10% or about 5% - about 10% DMSO (v / v). In some embodiments, the cryopreservation medium contains one or more additional excipients such as plasmalyte A or human serum albumin (HSA). In some embodiments, the solution for cryopreservation may also contain human serum albumin (HSA). In certain embodiments, the cells are frozen, e.g., cryopreserved, in a solution having a final concentration of 0.1% - 5%, 0.25% - 4%, 0.5% - 2%, or 1% - 2% HSA. In some embodiments, the cryopreservation medium contains a commercially available cryopreservation solution (CryoStor™ CS10 or CS5). CryoStor™ CS10 is a cryopreservation medium containing 10% dimethyl sulfoxide (DMSO). CryoStor™ CS5 is a cryopreservation medium containing 5% dimethyl sulfoxide (DMSO). In some embodiments, the cells are then generally frozen to -80°C or about -80°C at a rate of 1°C per minute or about 1°C per minute and stored in the vapor phase of a liquid nitrogen storage tank. In some aspects, the engineered iCILs are thawed before use, e.g., in connection with the therapeutic methods described herein. In some embodiments, after thawing the cells, the method includes the step of washing the cryopreserved composition under conditions to reduce or remove the cryoprotectant.

[0420] III. Gene Editing and Manipulation In some embodiments, pluripotent stem cells (e.g., iPSCs) or iCILs can be modified by gene editing. In some embodiments, pluripotent stem cells (e.g., iPSCs) or iCILs can be modified by genetic manipulation, for example, by introducing an exogenous nucleic acid encoding a transgene such as a chimeric antigen receptor (CAR). In some embodiments, the gene-edited iPSCs described can be used as a source cell for differentiation into iCILs.

[0421] Genome editing generally refers to the process of editing or altering the nucleotide sequence of a genome, preferably in an accurate, desirable, and / or predetermined manner. Examples of genome editing compositions, systems, and methods described herein use site-specific nucleases to cleave DNA at precise target locations within the genome, thereby creating a double-strand break (DSB) in the DNA. Such cleavage can be repaired by endogenous DNA repair pathways such as homologous recombination repair (HDR) and / or non-homologous end joining (NHEJ) repair (see, e.g., Cox et al., (2015) Nature Medicine 21(2):121-31).

[0422] In some embodiments, the cells described herein (e.g., stem cells, CIL) are genetically modified. In some embodiments, the modification involves using a DNA targeting protein and a nuclease or an RNA-guided nuclease to knock out one or more endogenous genes, and / or to knock in one or more exogenous genes of interest. In some embodiments, the gene of interest is knocked in at a specific locus of interest. In some embodiments, the gene of interest is a synthetic cytokine receptor complex. In some embodiments, the synthetic cytokine receptor complex is activated by rapamycin. In some embodiments, the synthetic cytokine receptor complex is a rapamycin-activated cytokine receptor (RACR). In some embodiments, the RACR is knocked in at the locus of interest. In some embodiments, the gene of interest is a chimeric antigen receptor.

[0423] In some embodiments, the modification comprises contacting the cell with a DNA targeting protein and a nuclease or an RNA-guided nuclease. In some embodiments, the DNA targeting protein and the nuclease or the RNA-guided nuclease comprise a zinc finger protein (ZFP), a clustered regularly interspaced short palindromic nucleic acid (CRISPR), or a transcription activator-like effector nuclease (TALEN). In some embodiments, CRISPR-Cas9 is used. In some embodiments, CRISPR-Mad7 is used.

[0424] Rejection of cell therapies (e.g., CAR T cells) is at least caused by a mismatch of human leukocyte antigen (HLA) between the donor and the recipient. One solution identified in recent years is to disrupt the expression of genes involved in this rejection, such as T cell receptor alpha constant (TRAC), beta-2-microglobulin (B2M), and signal regulatory protein alpha (SIRPA). Thus, in some embodiments, the cells described herein (e.g., iPSCs, CILs) are genetically engineered to knockout the B2M locus, the TRAC locus, and / or the SIRPA locus. In some embodiments, the cells described herein are genetically engineered to knockout the B2M locus. In some embodiments, the cells described herein are genetically engineered to knockout the TRAC locus. In some embodiments, the cells described herein are genetically engineered to knockout the SIRPA locus.

[0425] In some embodiments, the cells described herein are genetically engineered to be rapamycin resistant. Rapamycin is a small molecule drug that inhibits the mTOR pathway, an essential pathway for cell growth and expansion. Thus, when cells are contacted with rapamycin, in some cases, cell growth and expansion can be inhibited or reduced. In some embodiments, in the cells provided, the endogenous genes involved in rapamycin function are disrupted to eliminate or reduce rapamycin-mediated growth inhibition of source cells or CILs using the methods provided, whereby such cells become "rapamycin resistant". Reference to "rapamycin-resistant" cells is understood to refer to the ability of the endogenous mTOR pathway of the cells not to be affected by the presence of rapamycin or rapamycin analogs. However, it is further understood that "rapamycin-resistant" cells can still be responsive to rapamycin via pathways not involving mTOR, for example, due to the activation of the synthetic RACR described herein.

[0426] In some embodiments, the cells are genetically engineered to disrupt genes associated with rapamycin recognition. In some embodiments, the cells are genetically engineered to disrupt the mTOR gene. In some embodiments, the mTOR gene is FKBP-12 (also known as FKBP-1A, FKBP1, FKBP12, PKC12, PKCI2, PPIASE). FKBP12 is an essential binder of rapamycin and is required for its function. In some embodiments, the cells are genetically engineered to disrupt the FKBP12 gene. In some embodiments, the cells are genetically engineered to knockout the FKB12 gene to induce rapamycin resistance. In some embodiments, disruption of the endogenous FKBP12 gene in donor stem cells (e.g., iPSCs) is by genetic knockout using the CRISPR-Cas system. In normal cells without genetic disruption of FKBP12, FKBP12 is the major binder of rapamycin, and then the FKBP12-rapamycin complex binds to the FRB subunit of mTOR, blocking mTOR signaling. Disrupting the expression of the FKBP12 gene, such as by FKBP12 knockout, has demonstrated success in rapamycin inhibitory activity from the results herein, because rapamycin does not first complex with FKBP1A and has no function. Thus, genetic disruption of FKBP12, such as by gene knockout, highly tolerizes stem cells (e.g., iPSCs) to rapamycin-mediated mTOR inhibition and enables robust proliferation of stem cells (e.g., iPSCs) even in the presence of high doses of rapamycin. In some embodiments, the ability to render cells resistant to rapamycin growth inhibition allows for the involvement of rapamycin in RACR during cell differentiation without deleterious effects. Furthermore, FKBP12 knockout avoids competition between FKBP12 and RACR for binding to rapamycin. Thus, in some embodiments, the ability to render cells resistant to rapamycin growth by FKBP12 knockout also allows for the activation of RACR-containing cells in vivo and suppresses potential allogeneic anti-graft responses through mTOR inhibition of the host immune system.

[0427] In some embodiments, the cells described herein are genetically engineered to contain a nucleotide sequence encoding a synthetic cytokine receptor in an endogenous gene. In some embodiments, the synthetic cytokine receptor is engineered to integrate into the gene such that expression of the endogenous gene is not disrupted. In some embodiments, the cells described herein are genetically engineered to contain a nucleotide sequence encoding a synthetic cytokine receptor complex in a disrupted gene, e.g., a gene that has been inactivated or knocked out intracellularly.

[0428] In some embodiments, the cells described herein are genetically engineered to contain a nucleotide sequence encoding a synthetic cytokine receptor in a target endogenous gene. In some embodiments, the synthetic cytokine receptor is engineered to integrate into a safe harbor locus. In some embodiments, the target endogenous gene is a housekeeping gene. In some embodiments, the housekeeping gene is eukaryotic translation elongation factor 1 alpha (EEF1A), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ubiquitin C (UBC), or actin beta (ACTB). In some embodiments, the target endogenous gene is B2M, TRAC or SIRP alpha.

[0429] In some embodiments, the gene of interest inserted into the endogenous locus is a synthetic cytokine receptor complex. In some embodiments, the endogenous promoter of a particular locus is used.

[0430] In some embodiments, an exogenous promoter is operably connected to a gene encoding a synthetic cytokine receptor complex to promote expression. In some embodiments, the promoter is the EF1A promoter (also known as the EEF1A promoter). In some embodiments, the promoter is the MND promoter. In some embodiments, additional promoters may be included such that two or more promoters promote the expression of an exogenous gene of interest. In some embodiments, the two or more promoters may be the same or different. In some embodiments, the promoter is a dual promoter under the functional control of two promoters in which the synthetic cytokine receptor is. In some embodiments, the dual promoter is a dual EF1α promoter.

[0431] For example, in some embodiments, a cell comprises a disrupted B2M gene and a nucleotide sequence encoding a synthetic cytokine receptor within the disrupted B2M gene.

[0432] In some embodiments, the cells described herein (e.g., iPSCs, CILs) comprise (i) a disrupted B2M locus and (ii) a nucleotide sequence encoding a synthetic cytokine receptor complex (e.g., RACR) under the control of the endogenous B2M promoter and the EEF1A promoter.

[0433] In some embodiments, the cells described herein (e.g., iPSCs, CILs) comprise (i) a disrupted B2M locus and (ii) a nucleotide sequence encoding a synthetic cytokine receptor complex (e.g., RACR) inserted into the endogenous B2M gene and under the control of the endogenous B2M promoter and the EEF1A promoter.

[0434] In some embodiments, cells comprising (i) a disrupted B2M locus and (ii) a nucleotide sequence encoding a synthetic cytokine receptor complex (e.g., RACR) are produced by any of the methods described below.

[0435] A. Systems for genome editing In some embodiments, the system for editing the cells described herein includes a site-specific nuclease, such as the CRISPR / Cas system, and optionally a gRNA. In some embodiments, the system includes an engineered nuclease. In some embodiments, the system includes a site-specific nuclease. In some embodiments, the site-specific nuclease includes a CRISPR / Cas nuclease system. In some embodiments, the Cas nuclease is Cas9. In some embodiments, the nuclease is Mad7. In some embodiments, the guide RNA including the CRISPR / Cas system is a single guide RNA (sgRNA).

[0436] The sequences herein show exemplary gRNA targeting sequences. In some embodiments, the gRNA targeting sequence may contain one or more thymines within a complementary subsequence substituted by uracil. It will be understood by those skilled in the art that both uracil and thymine can be represented by "t" instead of "u" for uracil and "t" for thymine. In connection with ribonucleic acid, unless otherwise indicated, it will be understood that "t" is used to represent uracil.

[0437] 1. Nuclease a. CRISPR / Cas nuclease system The naturally occurring CRISPR / Cas system is a genetic defense system that provides a form of acquired immunity in prokaryotes. CRISPR is an acronym for clustered regularly interspaced short palindromic repeats ( C lustered R egularly I nterspaced S hort P alindromic RCRISPR is an abbreviation of "Clustered Regularly Interspaced Short Palindromic Repeats", and is a family of DNA sequences found in the genomes of bacteria and archaea that contain fragments of DNA (spacer DNA) similar to foreign DNA previously exposed to the cell by a virus that has infected or attacked the prokaryote. These fragments of DNA are used by prokaryotes, for example, to detect and destroy similar foreign DNA upon reintroduction from a similar virus during subsequent attacks. Transcription of the CRISPR locus results in the formation of an RNA molecule that contains spacer sequences that associate with and target Cas (CRISPR-associated) proteins that can recognize and cleave foreign exogenous DNA. A number of types and classes of CRISPR / Cas systems have been described (see, for example, Koonin et al., (2017) Curr Opin Microbiol 37:67-78).

[0438] Engineered versions of the CRISPR / Cas system have been developed in many forms to mutate or edit genomic DNA of cells from other species. A common approach using the CRISPR / Cas system involves heterologously expressing or introducing into the cell a site-specific nuclease (e.g., a Cas nuclease) in combination with a guide RNA (gRNA), resulting in a DNA cleavage event (e.g., formation of a single-strand break or a double-strand break (SSB or DSB)) in the backbone of the cell's genomic DNA at an accurately targetable position. The manner in which the DNA cleavage event is repaired by the cell provides an opportunity to edit the genome by addition, removal, or modification (substitution) of DNA nucleotides or sequences (e.g., genes).

[0439] In some embodiments, a system for editing a cell described herein includes a nuclease capable of inducing DNA cleavage within an endogenous target gene in the cell. In some embodiments, the DNA cleavage includes a double-strand break (DSB) induced by a nuclease capable of inducing DSB by cleaving both strands of double-stranded DNA at the cleavage site. In some embodiments, the DNA cleavage includes a single-strand break (SSB) at a cleavage site on the sense or antisense strand of the endogenous target gene. In some embodiments, the DNA cleavage includes an SSB at a cleavage site on the sense strand and an SSB at a cleavage site on the antisense strand, thereby resulting in a DSB. In some embodiments, the DSB is induced by a pair of recombinant nucleases, such as nickases, capable of inducing single-strand breaks (SSBs) in opposing DNA strands at different cleavage sites, for example, a cleavage site upstream of a gene variant on one strand of the target gene and a cleavage site downstream of the gene variant on the other strand. In some embodiments, the first of the pair of nickases forms a complex with a first guide RNA, such as a first sgRNA, to target cleavage of one strand, for example, the sense strand, and the second of the pair of nickases forms a complex with a second guide RNA, such as a second sgRNA, to target cleavage of the other strand, for example, the antisense strand. In some embodiments, the DSB is induced via SSBs for each of the opposing strands, i.e., the sense and antisense strands, of the endogenous target gene in the cell.

[0440] Generally, a gene is located in double-stranded DNA that includes a sense strand and an antisense strand that are complementary to each other. The sense strand is also called the coding strand because its sequence is the DNA version of the transcribed RNA sequence. The antisense strand is also called the template strand because its sequence is complementary to the transcribed RNA sequence.

[0441] i. Guide RNA (gRNA) The engineered CRISPR / Cas system comprises at least two components, namely, 1) a guide RNA (gRNA) molecule and 2) a Cas nuclease that interacts to form a gRNA / Cas nuclease complex. The gRNA comprises at least a user-defined targeting domain called a "spacer" that includes a nucleotide sequence and a CRISPR repeat sequence. In the engineered CRISPR / Cas system, the gRNA / Cas nuclease complex targets a specific target sequence of interest within a target nucleic acid (e.g., a genomic DNA molecule) by generating a gRNA that includes a spacer having a nucleotide sequence that can bind to the specific target sequence in a complementary manner (see Jinek et al., Science, 337, 816-821 (2012) and Deltcheva et al., Nature, 471, 602-607 (2011)). Thus, the spacer provides the targeting function of the gRNA / Cas nuclease complex.

[0442] In the naturally occurring type II CRISPR / Cas system, the "gRNA" is composed of two RNA strands, namely, 1) a CRISPR RNA (crRNA) that includes a spacer and a CRISPR repeat sequence and 2) a trans-activating CRISPR RNA (tracrRNA). In the type II CRISPR / Cas system, a portion of the crRNA that includes the CRISPR repeat sequence hybridizes with a portion of the tracrRNA to form a crRNA:tracrRNA duplex that interacts with a Cas nuclease (e.g., Cas9). As used herein, the term "split gRNA" or "modular gRNA" refers to a gRNA molecule that includes two RNA strands, wherein the first RNA strand incorporates the crRNA function and / or structure and the second RNA strand incorporates the tracrRNA function and / or structure, and the first and second RNA strands hybridize partially.

[0443] Thus, in some embodiments, the gRNA comprises two RNA molecules. In some embodiments, the gRNA comprises a CRISPR RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA). In some embodiments, the gRNA is a split gRNA. In some embodiments, the gRNA is a modular gRNA. In some embodiments, the split gRNA comprises, from 5' to 3', a first strand comprising a spacer and a first complementary region, a second strand comprising a second complementary region from 5' to 3', and optionally a tailed domain.

[0444] In some embodiments, the crRNA comprises a spacer comprising a nucleotide sequence that is complementary to and hybridizes with a sequence complementary to a target sequence on a target nucleic acid (e.g., a genomic DNA molecule). In some embodiments, the crRNA comprises a region that is complementary to and hybridizes with a portion of the tracrRNA.

[0445] In some embodiments, the target nucleic acid (e.g., an endogenous gene) is B2M. In some embodiments, the crRNA comprises the nucleotide sequence set forth in SEQ ID NO:18, or a nucleotide sequence having at least 80%, at least 85%, at least 90% or at least 95% sequence identity to the nucleotide sequence set forth in SEQ ID NO:18. In some embodiments, the crRNA comprises the nucleotide sequence set forth in SEQ ID NO:18.

[0446] In some embodiments, the target nucleic acid (e.g., an endogenous gene) is FKBP12. In some embodiments, the crRNA comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 19, 20, and 21, or a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the nucleotide sequence set forth in any one of SEQ ID NOs: 19, 20, and 21. In some embodiments, the crRNA comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 19, 20, and 21. In some embodiments, the crRNA comprises the nucleotide sequence set forth in SEQ ID NO: 19, or a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 19. In some embodiments, the crRNA comprises the nucleotide sequence set forth in SEQ ID NO: 19. In some embodiments, the crRNA comprises the nucleotide sequence set forth in SEQ ID NO: 20, or a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 20. In some embodiments, the crRNA comprises the nucleotide sequence set forth in SEQ ID NO: 20. In some embodiments, the crRNA comprises the nucleotide sequence set forth in SEQ ID NO: 21, or a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 21. In some embodiments, the crRNA comprises the nucleotide sequence set forth in SEQ ID NO: 21.

[0447] In some embodiments, the tracrRNA may comprise all or a portion of a wild-type tracrRNA sequence derived from a naturally occurring CRISPR / Cas system. In some embodiments, the tracrRNA may comprise a truncated or modified variant of the wild-type tracrRNA. The length of the tracrRNA may depend on the CRISPR / Cas system being used. In some embodiments, the tracrRNA may comprise a nucleotide length of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or more than 100 nucleotides. In certain embodiments, the tracrRNA is at least 26 nucleotides in length. In additional embodiments, the tracrRNA is at least 40 nucleotides in length. In some embodiments, the tracrRNA may comprise a particular secondary structure, such as, for example, one or more hairpin or stem-loop structures, or one or more bulge structures.

[0448] Single guide RNA (sgRNA) The engineered CRISPR / Cas nuclease system often combines crRNA and tracrRNA into a single RNA molecule referred to herein as a "single guide RNA" (sgRNA) by adding a linker between these components. Without being bound by theory, like the double-stranded crRNA and tracrRNA, the sgRNA forms a complex with a Cas nuclease (e.g., Cas9), guides the Cas nuclease to a target sequence, and activates the Cas nuclease to cleave a target nucleic acid (e.g., genomic DNA). Thus, in some embodiments, the gRNA can comprise a functionally linked crRNA and tracrRNA. In some embodiments, the sgRNA can comprise a crRNA covalently linked to a tracrRNA. In some embodiments, the crRNA and tracrRNA are covalently linked via a linker. In some embodiments, the sgRNA can comprise a stem-loop structure via base pairing between the crRNA and tracrRNA. In some embodiments, the sgRNA comprises, from 5' to 3', a spacer, a first complementary region, a linker domain, a second complementary region, and optionally a tail domain.

[0449] The sgRNA may or may not be modified. For example, a modified sgRNA can comprise one or more 2'-O-methyl phosphorothioate nucleotides.

[0450] By way of example, guide RNAs used in the CRISPR / Cas system, or other even smaller RNAs, are exemplified herein and can be readily synthesized by chemical means as described in the art. Although chemical synthesis procedures are constantly expanding, the purification of such RNAs by procedures such as high performance liquid chromatography (HPLC, which avoids the use of gels such as PAGE) tends to become more difficult as the length of the polynucleotide significantly increases beyond about 100 nucleotides. One approach used to generate longer RNAs is to generate two or more molecules that are ligated together. Much longer RNAs, such as those encoding Cas9 endonuclease, are even more readily generated enzymatically. During and / or after chemical synthesis and / or enzymatic generation of the RNA, various types of RNA modifications can be introduced, such as those described in the art, which improve stability, reduce the likelihood or extent of the innate immune response, and / or enhance other attributes.

[0451] Spacer In some embodiments, the gRNA comprises a spacer sequence. The spacer sequence is a sequence that defines a target site on a target nucleic acid (e.g., DNA). The target nucleic acid is a double-stranded molecule, one strand of which contains a target sequence adjacent to a PAM sequence and is referred to as the "PAM strand", and the second strand is referred to as the "non-PAM strand" and is complementary to the PAM strand and the target sequence. Both the gRNA spacer and the target sequence are complementary to the non-PAM strand of the target nucleic acid. In some embodiments, the spacer sequence corresponding to the target sequence adjacent to the PAM sequence is complementary to the non-PAM strand of the target nucleic acid. Thus, in some embodiments, the spacer sequence corresponding to the target sequence adjacent to the PAM sequence is identical to the PAM strand. The gRNA spacer sequence hybridizes to a complementary strand (e.g., the non-PAM strand of the target nucleic acid / target site). In some embodiments, the spacer is sufficiently complementary to the complementary strand of the target sequence (e.g., the non-PAM strand) to target the Cas nuclease to the target nucleic acid. In some embodiments, the spacer is at least 80%, at least 85%, at least 90% or at least 95% complementary to the non-PAM strand of the target nucleic acid. In some embodiments, the spacer is 100% complementary to the non-PAM strand of the target nucleic acid. In some embodiments, the spacer comprises 1, 2, 3, 4, 5, 6 or more nucleotides that are not complementary to the non-PAM strand of the target nucleic acid. In some embodiments, the spacer comprises one nucleotide that is not complementary to the non-PAM strand of the target nucleic acid. In some embodiments, the spacer comprises two nucleotides that are not complementary to the non-PAM strand of the target nucleic acid.

[0452] In some embodiments, the most 5'-terminal nucleotide of the gRNA comprises the most 5'-terminal nucleotide of the spacer. In some embodiments, the spacer is located at the 5'-end of the crRNA. In some embodiments, the spacer is located at the 5'-end of the sgRNA. In some embodiments, the spacer is about 15 to 50, about 20 to 45, about 25 to 40, or about 30 to 35 nucleotides in length. In some embodiments, the spacer is about 19 to 22 nucleotides in length. In some embodiments, the spacer is about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30 nucleotides in length. In some embodiments, the spacer is 19 nucleotides in length. In some embodiments, the spacer is 20 nucleotides in length, and in some embodiments, the spacer is 21 nucleotides in length.

[0453] In some embodiments, the nucleotide sequence of the spacer is designed or selected using a computer program. The computer program can use variables such as predicted melting temperature, secondary structure formation, predicted annealing temperature, sequence identity, genomic context, chromatin accessibility, %GC, genomic occurrence frequency (e.g., sequences that are identical or similar but differ at one or more spots as a result of mismatches, insertions, or deletions), methylation status, and / or the presence of SNPs.

[0454] In some embodiments, the spacer comprises at least one or more modified nucleotides such as those described herein. The present disclosure provides gRNA molecules comprising a spacer that may contain the nucleobase uracil (U), and any DNA encoding a gRNA comprising a spacer that contains the nucleobase uracil (U) contains the nucleobase thymine (T) at the corresponding position.

[0455] ii. Method for producing gRNA Methods for making gRNAs are known to those of skill in the art and include, without limitation, in vitro transcription (IVT), synthesis and / or chemical synthesis methods, or combinations thereof. Enzymatic (IVT) synthesis methods, solid-phase synthesis methods, liquid-phase synthesis methods, composite synthesis methods, small region synthesis, and ligation methods are utilized. In one aspect, the gRNA is made using an IVT enzymatic synthesis method. Methods for making polynucleotides by IVT are known in the art and are described in International Application PCT / US2013 / 30062. Accordingly, the present disclosure also includes polynucleotides, such as DNA, constructs, and vectors, used to transcribe the gRNAs described herein in vitro.

[0456] In some aspects, non-naturally modified nucleobases are introduced into the polynucleotide, such as the gRNA, during or after synthesis. In certain aspects, the modification is on the internucleoside linkage, purine or pyrimidine base, or sugar. In some aspects, the modification is introduced at the end of the polynucleotide using chemical synthesis or a polymerase enzyme. Examples of modified nucleic acids and their synthesis are disclosed in PCT Application No. PCT / US2012 / 058519. The synthesis of modified polynucleotides is also described in Verma and Eckstein, Annual Review of Biochemistry, vol. 76, 99-134 (1998).

[0457] In some aspects, enzymatic or chemical ligation methods are used to conjugate the polynucleotide or regions thereof with various functional moieties such as targeting or delivery agents, fluorescent labels, liquids, nanoparticles, etc. Conjugates of polynucleotides and modified polynucleotides are reviewed in Goodchild, Bioconjugate Chemistry, vol. 1(3), 165-187 (1990).

[0458] In some aspects, the disclosure provides a nucleic acid encoding a gRNA described herein, e.g., a vector. In some aspects, the nucleic acid is a DNA molecule. In other aspects, the nucleic acid is an RNA molecule. In some aspects, the nucleic acid comprises a nucleotide sequence encoding a crRNA. In some aspects, the nucleotide sequence encoding a crRNA comprises a spacer adjacent to all or part of a repeat sequence derived from a naturally occurring CRISPR / Cas system. In some aspects, the nucleic acid comprises a nucleotide sequence encoding a tracrRNA. In some aspects, the crRNA and tracrRNA are encoded by two separate nucleic acids. In other aspects, the crRNA and tracrRNA are encoded by a single nucleic acid. In some aspects, the crRNA and tracrRNA are encoded by opposite strands of a single nucleic acid. In other aspects, the crRNA and tracrRNA are encoded by the same strand of a single nucleic acid.

[0459] In some aspects, the gRNA provided by the disclosure is chemically synthesized by any means described in the art (see, e.g., WO 2005 / 01248). Although chemical synthesis procedures are constantly expanding, the purification of such RNAs by procedures such as high performance liquid chromatography (HPLC, which avoids the use of gels such as PAGE) tends to become more difficult as the length of the polynucleotide significantly increases beyond about 100 nucleotides. One approach used to generate longer RNAs is to generate two or more molecules that are ligated together.

[0460] In some embodiments, multiple guide RNAs can be used with the CRISPR / Cas nuclease system. Each guide RNA can contain a different targeting sequence such that the CRISPR / Cas system cleaves multiple target nucleic acids. In some embodiments, one or more guide RNAs can have the same or different properties, such as activity or stability, within the Cas9 RNP complex. When multiple guide RNAs are used, each guide RNA can be encoded on the same or different vectors. The promoters used to facilitate the expression of multiple guide RNAs can be the same or different.

[0461] The guide RNA can target any sequence of interest via the targeting sequence of the crRNA (e.g., the spacer sequence). In some embodiments, the degree of complementarity between the targeting sequence of the guide RNA and the target sequence on the target nucleic acid molecule is about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99% or about 100%. In some embodiments, the targeting sequence of the guide RNA and the target sequence on the target nucleic acid molecule are 100% complementary. In other embodiments, the targeting sequence of the guide RNA and the target sequence on the target nucleic acid molecule can contain at least one mismatch. For example, the targeting sequence of the guide RNA and the target sequence on the target nucleic acid molecule can contain 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mismatches. In some embodiments, the targeting sequence of the guide RNA and the target sequence on the target nucleic acid molecule can contain 1 to 6 mismatches. In some embodiments, the targeting sequence of the guide RNA and the target sequence on the target nucleic acid molecule can contain 5 or 6 mismatches.

[0462] The length of the targeting array can depend on the CRISPR-Cas system and the components used. For example, different Cas9 proteins from different bacterial species have various optimal targeting array lengths. Thus, the targeting array can include nucleotide lengths of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more than 50. In some embodiments, the targeting array can include lengths of 18-24 nucleotides. In some embodiments, the targeting array can include lengths of 19-21 nucleotides. In some embodiments, the targeting array can include a length of 20 nucleotides.

[0463] In some embodiments of the present disclosure, the CRISPR / Cas nuclease system includes at least one guide RNA. In some embodiments, the guide RNA and the Cas protein can form a ribonucleoprotein (RNP), such as a CRISPR / Cas complex. The guide RNA can direct the Cas protein to a target sequence on a target nucleic acid molecule (e.g., a genomic DNA molecule), and the Cas protein cleaves the target nucleic acid. In some embodiments, the CRISPR / Cas complex is a Cpf1 / guide RNA complex. In some embodiments, the CRISPR complex is a type II CRISPR / Cas9 complex. In some embodiments, the Cas protein is a Cas9 protein. In some embodiments, the CRISPR / Cas9 complex is a Cas9 / guide RNA complex. In some embodiments, the CRISPR / Cas complex is an engineered class 2 type V CRISPR system. In some embodiments, the endonuclease is Mad7.

[0464] iii. Cas nuclease In some aspects, the present disclosure provides compositions and systems that include site-specific nucleases (e.g., engineered CRISPR / Cas systems), where the site-specific nuclease is a Cas nuclease. The Cas nuclease can include at least one domain that interacts with a guide RNA (gRNA). Further, the Cas nuclease is guided to a target sequence by the guide RNA. The guide RNA interacts with the Cas nuclease and the target sequence such that the Cas nuclease can cleave the target sequence after being guided to the target sequence. In some aspects, the guide RNA provides specificity for cleavage of the target sequence, and the Cas nuclease is universal and pairs with various guide RNAs to cleave various target sequences.

[0465] In some aspects, the CRISPR / Cas system includes components derived from a type I, type II, or type III system. In an updated classification scheme for CRISPR / Cas loci, class 1 and class 2 CRISPR / Cas systems having types I-V or VI are defined (Makarova et al., (2015) Nat Rev Microbiol, 13(11):722-36; Shmakov et al., (2015) Mol Cell, 60:385-397). Class 2 CRISPR / Cas systems have a single protein effector. Type II, type V, and type VI Cas proteins are single-protein RNA-guided endonucleases referred to herein as "class 2 Cas nucleases." Class 2 Cas nucleases include, for example, Cas9 protein, Cpf1 protein, C2c1 protein, C2c2 protein, and C2c3 protein. The Cpf1 nuclease (Zetsche et al., (2015) Cell 163:1-13) is homologous to Cas9 and contains a RuvC-like nuclease domain.

[0466] In some embodiments, the Cas nuclease is derived from a type II CRISPR / Cas system (e.g., the Cas9 protein from the CRISPR / Cas9 system). In some embodiments, the Cas nuclease is derived from a class 2 CRISPR / Cas system (a single-protein Cas nuclease such as the Cas9 protein or the Cpf1 protein). Proteins of the Cas9 and Cpf1 families are enzymes having DNA endonuclease activity and can be directed to cleave a desired nucleic acid target by designing an appropriate guide RNA, as further described herein.

[0467] Type II CRISPR / Cas system components are derived from type II-A, type II-B, or type II-C systems. Cas9 and its orthologs are included. Non-limiting exemplary species from which Cas9 nuclease or other components are derived include Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus species, Staphylococcus aureus, Listeria innocua, Lactobacillus gasseri, Francisella novicida, Wolinella succinogenes, Sutterella wadsworthensis, Gammaproteobacteria, Neisseria meningitidis, Campylobacter jejuni, Pasteurella multocida, Fibrobacter succinogene, Rhodospirillum rubrum, Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides (Bacilluspseudomycoides), Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Lactobacillus buchneri, Treponema denticola, Microscilla marina, Burkholderiales bacteria, Polaromonas naphthalenivorans, Polaromonas species, Crocosphaera watsonii, Cyanothece species, Microcystis aeruginosa, Synechococcus species, Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosumThermus thermophilus), Marinobacter species, Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc species, Arthrospira maxima, Arthrospira platensis, Arthrospira species, Lyngbya species, Microcoleus chthonoplastes, Oscillatoria species, Petrotoga mobilis, Thermosipho africanus, Streptococcus pasteurianus, Neisseria cinerea, Campylobacter lari, Parvibaculum lavamentivorans, Corynebacterium diphtheria or Acaryochloris marina. In some embodiments, the Cas9 protein is derived from Streptococcus pyogenes (SpCas9). In some embodiments, the Cas9 protein is derived from Streptococcus thermophilus (StCas9). In some embodiments, the Cas9 protein is derived from Neisseria meningitidisderived from meningitides (NmCas9). In some embodiments, the Cas9 protein is derived from Staphylococcus aureus (SaCas9). In some embodiments, the Cas9 protein is derived from Campylobacter jejuni (CjCas9).

[0468] In some embodiments, the Cas nuclease can include multiple nuclease domains. For example, the Cas9 nuclease can include at least one RuvC-like nuclease domain (e.g., Cpf1) and at least one HNH-like nuclease domain (e.g., Cas9). In some embodiments, the Cas9 nuclease introduces a DSB into the target sequence. In some embodiments, the Cas9 nuclease is modified to contain only one functional nuclease domain. For example, the Cas9 nuclease is modified such that one of the nuclease domains is mutated or completely or partially deleted to reduce its nucleic acid cleavage activity. In some embodiments, the Cas9 nuclease is modified to not contain a functional RuvC-like nuclease domain. In other embodiments, the Cas9 nuclease is modified to not contain a functional HNH-like nuclease domain. In some embodiments where only one of the nuclease domains is functional, the Cas9 nuclease is a nickase that can introduce a single-strand break ("nick") into the target sequence. In some embodiments, the conserved amino acids within the Cas9 nuclease domain are substituted to reduce or alter nuclease activity. In some embodiments, the Cas nuclease nickase includes amino acid substitutions in the RuvC-like nuclease domain. Exemplary amino acid substitutions within the RuvC-like nuclease domain include D10A (based on the S. pyogenes Cas9 nuclease). In some embodiments, the nickase includes amino acid substitutions in the HNH-like nuclease domain. Exemplary amino acid substitutions within the HNH-like nuclease domain include E762A, H840A, N863A, H983A, and D986A (based on the S. pyogenes Cas9 nuclease). In some embodiments, the nuclease system described herein includes a nickase and a pair of guide RNAs that are complementary to the sense and antisense strands of the target sequence, respectively. The guide RNAs direct the nickase to target and introduce a DSB by generating nicks on opposite strands of the target sequence (i.e., double nicking).A chimeric Cas9 nuclease is used in which one domain or region of the protein is replaced by a portion of a different protein. For example, the Cas9 nuclease domain is replaced by a domain from a different nuclease such as Fok1. The Cas9 nuclease is a modified nuclease.

[0469] In some embodiments, the Cas nuclease is derived from a type I CRISPR / Cas system. In some embodiments, the Cas nuclease is a component of the type I CRISPR / Cas system's Cascade complex. For example, the Cas nuclease is Cas3 nuclease. In some embodiments, the Cas nuclease is derived from a type III CRISPR / Cas system. In some embodiments, the Cas nuclease is derived from a type IV CRISPR / Cas system. In some embodiments, the Cas nuclease is derived from a type V CRISPR / Cas system. In some embodiments, the Cas nuclease is derived from a type VI CRISPR / Cas system.

[0470] In some embodiments, the Cas nuclease is a Mad endonuclease. The CRISPR / Mad system is closely related to the type V (Cpf1-like) of the class 2 family of Cas enzymes. In some embodiments, the CRISPR-Mad system uses the Eubacterium rectale Mad7 endonuclease or a variant thereof. The Mad7-crRNA complex cleaves target DNA by identification of the PAM 5'-YTTN.

[0471] b. Engineered nuclease In some embodiments, the cells described herein are genetically engineered using a site-specific nuclease, and the site-specific nuclease is an engineered nuclease. Exemplary engineered nucleases are meganucleases (e.g., homing endonucleases), ZFNs, TALENs, and megaTALs.

[0472] Naturally occurring meganucleases can recognize and cleave double-stranded DNA sequences of about 12 to 40 base pairs and are generally classified into five families. In some embodiments, the meganuclease is selected from the LAGLIDADG family, the GIY-YIG family, the HNH family, the His-Cys box family, and the PD-(D / E)XK family. In some embodiments, the DNA binding domain of the meganuclease is engineered to recognize and bind to sequences other than its cognate target sequence. In some embodiments, the DNA binding domain of the meganuclease is fused to a heterologous nuclease domain. In some embodiments, a meganuclease such as a homing endonuclease is fused to a TAL module to create a hybrid protein such as a "megaTAL" protein. The megaTAL protein has improved DNA targeting specificity by recognizing the target sequence of the DNA binding domain of the meganuclease and the target sequence of the TAL module.

[0473] ZFN is a fusion protein that contains a zinc finger DNA-binding domain (the "zinc finger" or "ZF") and a nuclease domain. Each naturally occurring ZF can bind to three consecutive base pairs (a DNA triplet), and ZF repeats are combined to recognize a DNA target sequence and provide sufficient affinity. Thus, engineered ZF repeats are combined to recognize longer DNA sequences, such as 9 bp, 12 bp, 15 bp, or 18 bp. In some embodiments, the ZFN contains a ZF fused to a nuclease domain derived from a restriction endonuclease. For example, the restriction endonuclease is FokI. In some embodiments, the nuclease domain includes a dimerization domain, such as when the nuclease dimerizes to become active, and a pair of ZFNs that include ZF repeats, and the nuclease domain is designed to target a target sequence that includes two half target sequences recognized by each ZF repeat on opposite strands of a DNA molecule, with an intervening sequence (sometimes called a spacer in the literature) between them. For example, the intervening sequence is 5-7 bp in length. When both ZFNs of the pair bind, the nuclease domain can dimerize and introduce a DSB within the intervening sequence. In some embodiments, the dimerization domain of the nuclease domain includes a knob-into-hole motif to promote dimerization. For example, the ZFN includes a knob-into-hole motif in the dimerization domain of FokI.

[0474] The DNA binding domain of TALENs typically contains variable 34 or 35 amino acid...

Claims

1. Engineered stem cells comprising a synthetic cytokine receptor for a non-physiological ligand, wherein the cytokine receptor comprises a synthetic gamma chain polypeptide comprising a first dimerization domain, a first transmembrane domain, and an interleukin-2 receptor subunit gamma (IL-2RG) intracellular domain, and a synthetic beta chain polypeptide comprising a second dimerization domain, a second transmembrane domain, and an intracellular domain selected from the interleukin-2 receptor subunit beta (IL-2RB) intracellular domain, the interleukin-7 receptor subunit beta (IL-7RB) intracellular domain, and / or the interleukin-21 receptor subunit beta (IL-21RB) intracellular domain the engineered stem cells comprising the same.

2. The engineered stem cells according to claim 1, wherein the first dimerization domain and the second dimerization domain are extracellular domains.

3. The synthetic gamma chain polypeptide comprises, in order from the N-terminus to the C-terminus, the first dimerization domain, the first transmembrane domain, and the interleukin-2 receptor subunit gamma (IL-2RG) intracellular domain, and the synthetic beta chain polypeptide comprises, in order from the N-terminus to the C-terminus, the second dimerization domain, the second transmembrane domain, and the intracellular domain, the engineered stem cells according to claim 1 or claim 2.

4. The engineered stem cells according to any one of claims 1 to 3, wherein the IL-2RG intracellular domain comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:1 or the polypeptide sequence set forth in SEQ ID NO:

1.

5. The engineered stem cells according to any one of claims 1 to 4, wherein the first transmembrane domain comprises the IL-2RG transmembrane domain.

6. The engineered stem cells according to claim 5, wherein the IL-2RG transmembrane domain comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:8 or 31, or the polypeptide sequence set forth in SEQ ID NO:8 or 31.

7. The engineered stem cells according to any one of claims 1 to 6, wherein the beta chain intracellular domain comprises the IL-2RB intracellular domain.

8. The engineered stem cell according to claim 7, wherein the intracellular domain of IL-2RB comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:2, or a polypeptide sequence set forth in SEQ ID NO:

2.

9. The engineered stem cell according to any one of claims 1 to 6, wherein the intracellular domain of the beta chain comprises the intracellular domain of IL-7RB.

10. The engineered stem cell according to claim 9, wherein the intracellular domain of IL-7RB comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:3, or a polypeptide sequence set forth in SEQ ID NO:

3.

11. The engineered stem cell according to any one of claims 1 to 6, wherein the intracellular domain of the beta chain comprises the intracellular domain of IL-21RB.

12. The engineered stem cell according to claim 11, wherein the intracellular domain of IL-21RB comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:4, or a polypeptide sequence set forth in SEQ ID NO:

4.

13. The engineered stem cell according to any one of claims 1 to 12, wherein the second transmembrane domain comprises a transmembrane domain derived from the same beta chain intracellular domain.

14. The engineered stem cell according to any one of claims 1 to 8 and 13, wherein the second transmembrane domain is a transmembrane domain of IL-2RB that comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:35 or 36, or a polypeptide sequence set forth in SEQ ID NO:35 or 36.

15. The synthetic gamma chain polypeptide contains an IL-2RG TM domain comprising the sequence set forth in SEQ ID NO:8 or 31 and an IL-2RG intracellular domain comprising the sequence set forth in SEQ ID NO:1, and the synthetic beta chain polypeptide contains an IL-2RB TM domain comprising the sequence set forth in SEQ ID NO:35 or 36 and an IL-2RB intracellular domain comprising the sequence set forth in SEQ ID NO:2, The engineered stem cell according to any one of claims 1 to 8, 13 and 14.

16. The first dimerization domain and the second dimerization domain are heterodimerization domains selected from FK506-binding protein (FKBP) of size 12 kD and FKBP12-rapamycin binding (FRB) domain, and / or the non-physiological ligand is rapamycin or a rapalog, The engineered stem cells according to any one of claims 1 to 15.

17. The engineered stem cells according to claim 16, wherein the FRB domain comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:6 or SEQ ID NO:

7.

18. The engineered stem cells according to claim 16, wherein the FRB domain comprises the polypeptide sequence set forth in SEQ ID NO:6 or SEQ ID NO:

7.

19. The first dimerization domain and the second dimerization domain are heterodimerization domains selected from FK506-binding protein (FKBP) of size 12 kD and calcineurin domain, and / or the non-physiological ligand is FK506 or an analog thereof, The engineered stem cells according to any one of claims 1 to 15.

20. The engineered stem cells according to any one of claims 1 to 16, wherein the FKBP domain comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:5 or SEQ ID NO:

30.

21. The engineered stem cells according to any one of claims 1 to 16, wherein the FKBP domain comprises the polypeptide sequence set forth in SEQ ID NO:5 or SEQ ID NO:

30.

22. The engineered stem cell according to any one of claims 1 to 8 and 13 to 21, wherein the synthetic gamma chain polypeptide has the amino acid sequence set forth in SEQ ID NO:28, or an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:28, and the synthetic beta chain polypeptide has the amino acid sequence set forth in SEQ ID NO:33, or an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

33.

23. The engineered stem cell according to any one of claims 1 to 8 and 13 to 22, wherein the synthetic gamma chain polypeptide has the amino acid sequence set forth in SEQ ID O:28, and the synthetic beta chain polypeptide has the amino acid sequence set forth in SEQ ID NO:

33.

24. The first dimerization domain and the second dimerization domain are i) FK506-binding protein (FKBP) of size 12 kD; ii) Cyclophilin A (CypA); or iii) Gyrase B (CyrB) selected homodimerization domains, and the non-physiological ligands are, respectively, i) FK1012, AP1510, AP1903 or AP20187 or analogs thereof; ii) Cyclosporin-A (CsA) or analogs thereof; or iii) Coumermycin or analogs thereof is, The engineered stem cell according to any one of claims 1 to 15.

25. The engineered stem cell according to any one of claims 1 to 24, wherein the stem cell is a pluripotent stem cell.

26. The engineered stem cell according to any one of claims 1 to 25, wherein the stem cell is an induced pluripotent stem cell (iPSC).

27. The engineered stem cell according to any one of claims 1 to 26, wherein the stem cell is resistant to rapamycin-mediated mTOR inhibition.

28. The engineered stem cell according to any one of claims 1 to 27, wherein the stem cell expresses a cytosolic polypeptide that binds to the non-physiological ligand.

29. The engineered stem cell according to any one of claims 1 to 28, wherein the non-physiological ligand is rapamycin or a rapalog, and the stem cell expresses a cytosolic FRB domain or a variant thereof.

30. The engineered stem cell according to claim 29, wherein the cytosolic FRB domain comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:6 or SEQ ID NO:

7.

31. The engineered stem cell according to claim 29, wherein the cytosolic FRB domain comprises a polypeptide sequence that is at least 98% identical to SEQ ID NO:6 or SEQ ID NO:

7.

32. The engineered stem cell according to any one of claims 1 to 31, wherein the stem cell comprises a disrupted FKBP12 gene that reduces the expression of FKBP12.

33. The engineered stem cell according to any one of claims 1 to 32, wherein the stem cell comprises a knockout of the FKBP12 gene.

34. The engineered stem cell according to any one of claims 1 to 33, wherein the stem cell comprises a nucleotide sequence encoding the synthetic cytokine receptor inserted into the genome of the stem cell.

35. The engineered stem cell according to claim 34, wherein the nucleotide sequence encoding the synthetic cytokine receptor is inserted into a non-target locus within the genome of the stem cell.

36. The engineered stem cell according to claim 34, wherein the nucleotide sequence encoding the synthetic cytokine receptor is inserted into an endogenous gene of the stem cell.

37. The engineered stem cell according to claim 36, wherein the insertion reduces the expression of the endogenous gene within the locus.

38. The engineered stem cell according to claim 36 or claim 37, wherein the insertion knocks out the endogenous gene within the locus.

39. The engineered stem cell according to any one of claims 36 to 38, wherein the insertion is by homologous recombination repair.

40. The engineered stem cell according to any one of claims 36 to 39, wherein the endogenous gene is a housekeeping gene, a blood lineage-specific locus, or an immune-related gene.

41. The endogenous gene is a housekeeping gene, and the housekeeping gene is selected from eukaryotic translation elongation factor 1 alpha (EEF1A), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ubiquitin C (UBC), and actin beta (ACTB), the engineered stem cell according to claim 40.

42. The endogenous gene is a blood system-specific locus, and the blood system-specific locus is selected from protein tyrosine phosphatase receptor type C (PTPRC), IL2RG, and IL2RB, the engineered stem cell according to claim 40.

43. The immune-related gene is selected from the beta-2-microglobulin (B2M) gene, T cell receptor alpha constant (TRAC) gene, and signal regulatory protein alpha (SIRPA) gene, the engineered stem cell according to claim 40.

44. The endogenous gene is B2M, the engineered stem cell according to any one of claims 36-40 and 43.

45. The stem cell includes a B2M knockout, the engineered stem cell according to any one of claims 1-43.

46. The cell has a disruption of the gene encoding FKBP12, the engineered stem cell according to any one of claims 1-44.

47. The disruption is an FKBP12 knockout that inactivates the gene encoding FKBP12, the engineered stem cell according to claim 46.

48. The stem cell includes a B2M knockout and an FKBP12 knockout, the engineered stem cell according to any one of claims 1-44.

49. The engineered stem cell according to any one of claims 1-48, comprising a chimeric antigen receptor (CAR).

50. The CAR is an anti-FITC CAR, the engineered stem cell according to claim 49.

51. The binding of the non-physiological ligand to the synthetic cytokine receptor activates the synthetic cytokine receptor in the stem cell to induce the differentiation of the engineered stem cell in the cell population, the engineered stem cell according to any one of claims 1-50.

52. A cell population comprising the engineered stem cell according to any one of claims 1-51.

53. contacting a population of stem cells with a recombinant vector comprising (i) a guide RNA (gRNA) targeting a target site within an endogenous gene, (ii) an RNA-guided endonuclease, and (iii) a nucleotide sequence encoding a synthetic cytokine receptor for a non-physiological ligand, thereby inserting the nucleotide sequence into the endogenous gene, A method of genetically engineering stem cells to express a synthetic cytokine receptor, comprising: wherein the cytokine receptor is a synthetic gamma chain polypeptide comprising a first dimerization domain, a first transmembrane domain, and an intracellular domain of interleukin-2 receptor subunit gamma (IL-2RG), and a synthetic beta chain polypeptide comprising a second dimerization domain, a second transmembrane domain, and an intracellular domain selected from the intracellular domain of interleukin-2 receptor subunit beta (IL-2RB), the intracellular domain of interleukin-7 receptor subunit beta (IL-7RB), and / or the intracellular domain of interleukin-21 receptor subunit beta (IL-21RB), the method as described above.

54. The method according to claim 53, wherein the nucleotide sequence is inserted via homologous recombination repair (HDR).

55. The method according to claim 54, wherein the vector comprises, from 5' to 3', (a) a nucleotide sequence homologous to a region located upstream of the target site, (b) a nucleotide sequence encoding a synthetic cytokine receptor for a non-physiological ligand, and (c) a nucleotide sequence homologous to a region located downstream, in a nucleic acid.

56. The method according to claim 53, wherein the nucleotide sequence is inserted via non-homologous end joining (NHEJ).

57. The method according to any one of claims 53 to 56, wherein the RNA-guided endonuclease is selected from Cas endonuclease, Mad endonuclease, and Cpf1 endonuclease.

58. The method according to any one of claims 53 to 57, wherein the RNA-guided endonuclease is Cas9.

59. The method according to any one of claims 53 to 57, wherein the RNA-guided endonuclease is Mad7.

60. The method according to any one of claims 53 to 59, wherein the endogenous gene is selected from B2M, TRAC, and SIRPA.

61. The method according to any one of claims 53 to 60, wherein the endogenous gene is B2M.

62. The method according to any one of claims 53 to 61, wherein the gRNA comprises the sequence set forth in SEQ ID NO:

18.

63. The nucleotide sequence homologous to the region located upstream of the target site comprises a nucleic acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 22, and the nucleotide sequence homologous to the region located downstream comprises a nucleic acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:

23. The method according to any one of claims 55 to 62.

64. The nucleotide sequence encoding the synthetic cytokine receptor comprises a first nucleic acid sequence encoding a gamma chain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 37, and a second nucleic acid sequence encoding a beta chain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:

38. The method according to any one of claims 53 to 63.

65. The method according to claim 64, wherein the first nucleic acid sequence and the second nucleic acid sequence are separated by a cleavable linker or an IRES.

66. The method according to claim 65, wherein the cleavable linker is optionally a protein quantification reporter linker (PQR) described in SEQ ID NO:

42. **Claim 67** The method according to any one of claims 53 to 66, wherein the nucleotide sequence encoding the synthetic cytokine receptor for the non-physiological ligand is under the functional control of a heterologous promoter. **Claim 68** The method according to claim 67, wherein the heterologous promoter is an EF1α promoter or an MND promoter. **Claim 69** The method according to any one of claims 53 to 68, wherein the nucleotide sequence encoding the synthetic cytokine receptor comprises a polyadenylation sequence. **Claim 70** The method according to any one of claims 53 to 69, wherein the recombinant vector comprises the sequence described in SEQ ID NO: 40, or a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence described in SEQ ID NO:

40. **Claim 71** The method according to any one of claims 53 to 70, further comprising the step of manipulating a population of stem cells to be resistant to rapamycin-mediated mTOR inhibition. **Claim 72** The method according to claim 71, wherein the step of manipulating the population of stem cells to be resistant to rapamycin comprises knocking out the FKBP12 gene. **Claim 73** The method according to claim 72, further comprising the step of contacting the population of stem cells with an RNA-guided endonuclease and a guide RNA (gRNA) targeting a target site within the FKBP12 gene. **Claim 74** The method according to claim 73, wherein the RNA-guided endonuclease is selected from Cas endonuclease, Mad endonuclease and Cpf1 endonuclease. **Claim 75** The method according to claim 73 or claim 74, wherein the RNA-guided endonuclease is Cas9. **Claim 76** The method according to claim 73 or claim 74, wherein the RNA-guided endonuclease is Mad7. **Claim 77** The method according to any one of claims 73 to 76, wherein the step of further contacting is performed simultaneously with the contacting step in (i), optionally in combination with the same RNA-guided endonuclease, with a guide RNA (gRNA) targeting a target site within an endogenous gene.

78. The method according to any one of claims 73 to 77, wherein the gRNA comprises one or more gRNAs selected from gRNAs comprising the sequences set forth in SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO:

21.

79. The method according to claim 78, wherein the one or more gRNAs is a pool of gRNAs comprising two or three gRNAs.

80. The method according to any one of claims 53 to 79, further comprising the step of introducing a chimeric antigen receptor (CAR) into the population of stem cells.

81. The method according to claim 80, wherein the CAR is an anti-FITC CAR.

82. The method according to any one of claims 53 to 81, wherein the stem cells are pluripotent stem cells.

83. The method according to any one of claims 53 to 82, wherein the stem cells are iPSCs.

84. A cell population produced by the method according to any one of claims 53 to 83.

85. A pharmaceutical composition comprising the cell population according to claim 52 or claim 84.

86. (a) culturing a cell population comprising the engineered iPSCs according to any one of claims 1 to 51 or claim 84 under conditions that allow for the formation of aggregates; (b) culturing the cells produced in (a) under conditions that induce mesoderm formation in a plurality of the cells, wherein the start of the culturing step in (b) is day 0; and (c) culturing the cells produced in (b) under conditions that differentiate the cells into a population of hematopoietic progenitors (HP) comprising wherein at least a portion of one or more of steps (a) to (c) is performed in the presence of a non-physiological ligand of a synthetic cytokine receptor. A method for generating hematopoietic progenitor (HP) cells.

87. A method for generating cytotoxic innate lymphoid system (iCIL) cells, comprising culturing a cell population comprising engineered iPSCs according to any one of claims 1 to 51 under conditions that allow differentiation of the iPSCs into cytotoxic innate lymphoid system (iCIL), wherein a non-physiological ligand of a synthetic cytokine receptor is added during at least a part of the culturing step.

88. The culturing step comprises: (a) culturing the cell population comprising engineered iPSCs under conditions that allow formation of aggregates; (b) culturing the cells produced in (a) under conditions that induce mesoderm formation in a plurality of the cells, wherein the start of the culturing in (b) is on day 0; (c) culturing the cells produced in (b) under conditions that allow differentiation of the cells into a population of hematopoietic precursors (HP); and (d) culturing the cells produced in (c) under conditions that allow generation of iCIL cells and at least a part of one or more of steps (a) to (d) is performed in the presence of the non-physiological ligand of the synthetic cytokine receptor, The method according to claim 87.

89. (a) culturing a cell population comprising engineered iPSCs according to any one of claims 1 to 51 under conditions that allow formation of aggregates; (b) culturing the cells produced in (a) under conditions that induce mesoderm formation in a plurality of the cells, wherein the start of the culturing in (b) is on day 0; (c) culturing the cells produced in (b) under conditions that allow differentiation of the cells into a population of hematopoietic precursors (HP); and (d) culturing the cells produced in (c) under conditions that allow generation of iCIL cells and at least a part of one or more of steps (a) to (d) is performed in the presence of a non-physiological ligand of a synthetic cytokine receptor, A method for generating cytotoxic innate lymphoid system (iCIL) cells.

90. The method according to any one of claims 86 to 89, wherein the culturing is performed in a container treated to promote cell adhesion and proliferation.

91. The method according to claim 90, wherein the container is Matrigel.

92. The method according to any one of claims 86 to 89, wherein the culturing is performed in a non-adherent culture container.

93. The method according to claim 92, wherein the non-adherent culture container is an Aggrewell (trademark) plate.

94. The method according to any one of claims 86 and 88 to 93, wherein the aggregate in (a) is an embryoid body (EB).

95. The method according to any one of claims 86 to 89, wherein the culture is performed in suspension.

96. The method according to any one of claims 86 to 89 and 94, wherein the culture is performed in a culture vessel that has not been treated to promote cell adhesion and proliferation.

97. Step (a) comprises (i) performing a first incubation that includes culturing a population of engineered stem cells under conditions that form a first aggregate; (ii) contacting the aggregate with a dissociating agent to form a population of dissociated cells; and (iii) performing a second incubation that includes culturing the population of dissociated cells under conditions that form a second aggregate The method according to claim 94 or claim 95.

98. The method according to any one of claims 86 to 97, wherein the culture in (b) is in a medium containing one or more of BMP4, FGF2, VEGF, and a Rock inhibitor, and optionally, the Rock inhibitor is Y27632.

99. The method according to any one of claims 86 to 98, wherein the culture in (b) is in a medium containing BMP4, FGF2, VEGF, and Y27632.

100. The method according to any one of claims 86 to 98, wherein the culture in (b) is in a medium containing BMP4, FGF2, and VEGF.

101. The method according to any one of claims 86 to 100, wherein the culture in (b) is in a medium containing the non-physiological ligand.

102. The method according to any one of claims 86 to 96 and 101, wherein the culture in (b) is in a medium containing the non-physiological ligand and does not contain any additional growth factors.

103. The method according to any one of claims 86 to 102, wherein the culture in (b) is for 2 to 4 days, optionally 3 days or about 3 days.

104. The method according to any one of claims 86 to 103, wherein the culture in (c) is in a medium containing one or more of BMP4, FGF2, VEGF, TPO, SCF, and LDL.

105. The method according to any one of claims 86 to 103, wherein the culture in (c) is in a medium containing one or more of BMP4, FGF2, VEGF, and LDL.

106. The method according to any one of claims 86 to 103, wherein the culture in (c) is in a medium containing BMP4 and FGF2.

107. The method according to claim 106, wherein the culture with BMP4 and FGF2 in (c) is over the 3rd to 15th days.

108. The method according to any one of claims 86 to 106, wherein the culture in (c) contains a PI3K inhibitor.

109. The method according to claim 108, wherein the PI3K inhibitor is LY2940002.

110. The method according to claims 108 and 109, wherein the PI3K inhibitor is added during part of the culture in (c).

111. The method according to any one of claims 108 to 110, wherein the PI3K inhibitor is added from about the 6th day to the 15th day.

112. The method according to any one of claims 86 to 109, wherein the culture in (c) is in a medium not containing SCF and TPO.

113. The method according to any one of claims 86 to 112, wherein the culture in (c) is in a medium containing the non-physiological ligand.

114. The method according to any one of claims 86 to 102 and 112, wherein the culture in (c) is in a medium containing the non-physiological ligand and not containing any additional growth factor, cytokine, or both.

115. The method according to any one of claims 86 to 112, wherein the culture in (c) is for 3 days to 15 days.

116. The method according to any one of claims 86 to 115, wherein during at least part of the culture in (c), the medium contains an aryl hydrocarbon receptor (AHR) antagonist, such as a pyrido-[4,5-b]-indole derivative, such as or both.

117. The method according to claim 116, wherein the AHR antagonist is StemRegenin 1 (SR1).

118. The method according to claim 116 or claim 117, wherein the pyrido-[4,5-b]-indole derivative is UM729.

119. The method according to any one of claims 116 to 118, wherein SR1 and UM729 are added to the culture in (c) starting on the 6th to 9th days.

120. The method according to claim 116 or claim 119, wherein SR1 and UM729 are added to the culture in (c) starting on about the 6th day.

121. The method according to any one of claims 88 to 119, wherein the culture in (d) is in a medium containing one or more of stem cell factor (SCF), FLT3L, IL-7, IL-12, IL-15, SR-1, and UM729.

122. The method according to any one of claims 88 to 121, wherein the culture in (d) is in a medium containing the non-physiological ligand.

123. The method according to any one of claims 88 to 120 and 122, wherein the culture in (d) is in a medium containing the non-physiological ligand and does not contain any additional growth factor, cytokine, or both.

124. The method according to any one of claims 88 to 123, wherein the culture in (d) is over a period from the 15th day to the 40th day.

125. The method according to any one of claims 88 to 123, wherein the culture in (d) is over the 15th day and the 30th day.

126. A method for generating cytotoxic innate lymphoid (iCIL) cells, comprising: (a) culturing a cell population comprising engineered iPSCs according to any one of claims 1 to 48 under conditions for forming aggregates; (b) culturing the cells produced in (a) in a medium containing one or more selected from the group consisting of BMP4, VEGF, FGF2, and ROCKi to induce mesoderm formation in a plurality of the cells, wherein the start of the culturing step in (b) is on day 0; (c) culturing the cells produced in (b) in a medium containing BMP4, FGF2, and LY2940002 to differentiate the cells into a population of hematopoietic precursors (HP), wherein the start of the culturing step in (c) is on day 3; and (d) culturing the cells produced in (d) in a medium containing SCF and IL-15 to generate iCIL cells, wherein the start of the culturing step in (b) is on day 15, comprising, wherein at least a part of one or more of steps (a) to (d) is performed in the presence of a non-physiological ligand of a synthetic cytokine receptor. The above method.

127. The method according to claim 126, wherein SR1 and UM729 are added to the culture in (c) starting on the days from the 6th day to the 9th day.

128. The method according to claim 126 or claim 127, wherein SR1 and UM729 are added to the culture in (c) starting on about day 6.

129. A method for generating cytotoxic innate lymphoid cell (iCIL) cells, comprising: contacting a cell population comprising the engineered stem cells according to any one of claims 1 to 48 with the non-physiological ligand for a first period sufficient to generate CLP; and contacting the CLP with a differentiation medium for a second period sufficient to generate iCIL. The method as described above.

130. The method according to claim 129, wherein the differentiation medium comprises stem cell factor (SCF), FLT3L, IL-7, IL-12, IL-15, SR-1, and UM729.

131. The method according to claim 130, wherein the differentiation medium comprises the non-physiological ligand.

132. The method according to any one of claims 129 to 131, wherein the first period is 1 to 15 days and the second period is 1 to 15 days.

133. The method according to any one of claims 129 to 132, further comprising contacting the iCIL with a pre-activation medium comprising IL-7, IL-12, IL-15, IL-18 and IL-21 for a third period sufficient to generate mature iCIL.

134. The method according to claim 133, wherein the pre-activation medium comprises the non-physiological ligand.

135. The method according to claim 133 or 134, wherein the third period is 1 to 10 days.

136. The method according to claim 133 or claim 134, wherein mature iCIL expresses NKp46, NKG2D, LFA1, DNAM1, CD16 and CD56.

137. The method according to any one of claims 86 to 136, wherein the non-physiological ligand is rapamycin or a rapamycin analog.

138. The method according to claim 137, wherein the rapamycin analog is a rapalog.

139. The method according to any one of claims 86 to 138, wherein the non-physiological ligand is added to the medium at a concentration of 5 nM to 200 nM, 5 nM to 150 nM, 5 nM to 100 nM, 5 nM to 50 nM, 5 nM to 20 nM, 5 nM to 10 nM, 10 nM to 200 nM, 10 nM to 150 nM, 10 nM to 100 nM, 10 nM to 50 nM, 10 nM to 20 nM, 20 nM to 200 nM, 20 nM to 150 nM, 20 nM to 100 nM, 20 nM to 50 nM, 50 nM to 200 nM, 50 nM to 150 nM, 50 nM to 100 nM, 100 nM to 200 nM, 100 nM to 150 nM, and 150 nM to 200 nM.

140. The method according to any one of claims 86 to 139, wherein the non-physiological ligand is added to the medium at a concentration of 10 nM or about 10 nM.

141. The method according to any one of claims 86 to 139, wherein the non-physiological ligand is added to the medium at a concentration of 100 nM or about 100 nM.

142. The method according to any one of claims 86 to 138, wherein the non-physiological ligand is added to the medium at a concentration of 2.5 nM to 10 nM.

143. The method according to any one of claims 86 to 138 and 142, wherein the non-physiological ligand is added to the medium at a concentration of 3 nM to 7 nM.

144. Hematopoietic progenitor (HP) cells produced by the method according to any one of claims 86 and 90 to 119.

145. The HP cells according to claim 144, wherein the HP cells have lower expression of HLF, HOXA9 and / or CD133 compared to CD34+ cord blood cells.

146. The HP cells according to claim 145, wherein the expression of HLF, HOXA9 and / or CD133 in the HP cells is 1 / 8, 1 / 7, 1 / 6, 1 / 5, 1 / 4, 1 / 3, 1 / 2, or 1 / 1 compared to CD34+ cord blood cells.

147. The HP cells according to claim 145 or claim 146, wherein the CD34+ cord blood cells contain hematopoietic stem cells (HSC).

148. Cytotoxic natural lymphocyte system (iCIL) cells produced by the method according to any one of claims 87 to 136.

149. 52. A hematopoietic progenitor (HP) cell differentiated from the pluripotent stem cell of any one of claims 1 to 51, wherein the HP comprises a synthetic cytokine receptor.

150. A cytotoxic innate lymphoid cell (iCIL) differentiated from the pluripotent stem cell of any one of claims 1 to 51, wherein the iCIL comprises a synthetic cytokine receptor.

151. 96. A population of hematopoietic progenitor (HP) cells produced by the method of any one of claims 86 and 74-95.

152. 152. The population of claim 151, wherein said population of HP cells comprises lower expression of HLF, HOXA9 and / or CD133 compared to a population of CD34+ cord blood cells.

153. 154. The population of claim 153, wherein said expression of HLF, HOXA9 and / or CD133 in HP cells is eight-fold, seven-fold, six-fold, five-fold, four-fold, three-fold, half-fold, or one-fold lower than a population of CD34+ cord blood cells.

154. The population of claim 152 or claim 153, wherein said population of CD34+ umbilical cord blood cells comprises hematopoietic stem cells (HSCs).

155. 142. A population of cytotoxic innate lymphoid (iCIL) cells produced by the method of any one of claims 87-141.

156. 151. A population of cells comprising the iCIL of claim 148 or claim 150.

157. The population of iCILs of claim 148 or claim 150 or of iCILs of claim 155 or claim 156, wherein said iCILs comprise a B2M knockout.

158. The population of iCILs of claim 148 or claim 150 or of iCILs of claim 155 or claim 156, wherein said iCILs comprise a B2M knockout and an FKBP12 knockout.

159. 159. A pharmaceutical composition comprising the iCIL or population of iCILs of any one of claims 148-158.

160. A method for expanding cytotoxic innate lymphoid cells (iCILs), comprising contacting an iCIL or population of iCILs described in any one of claims 148-158 or a pharmaceutical composition of claim 159 with said non-physiological ligand of said synthetic cytokine receptor.

161. A method of killing cancer cells or inhibiting the growth of cancer cells, the method comprising contacting the cancer cells with the non-physiological ligand of the synthetic cytokine receptor together with the iCIL or population of iCILs according to any one of claims 148 to 158 or the pharmaceutical composition according to claim 159.

162. The method according to claim 160 or claim 161, which is carried out in vitro or ex vivo.

163. The method according to any one of claims 160 to 162, wherein the non-physiological ligand is rapamycin or a rapamycin analog.

164. The method according to claim 163, wherein the rapamycin analog is a rapalog.

165. The method according to any one of claims 160 to 164, wherein the non-physiological ligand is contacted at a concentration of 5 nM to 200 nM, 5 nM to 150 nM, 5 nM to 100 nM, 5 nM to 50 nM, 5 nM to 20 nM, 5 nM to 10 nM, 10 nM to 200 nM, 10 nM to 150 nM, 10 nM to 100 nM, 10 nM to 50 nM, 10 nM to 20 nM, 20 nM to 200 nM, 20 nM to 150 nM, 20 nM to 100 nM, 20 nM to 50 nM, 50 nM to 200 nM, 50 nM to 150 nM, 50 nM to 100 nM, 100 nM to 200 nM, 100 nM to 150 nM, and 150 nM to 200 nM.

166. The method according to any one of claims 160 to 165, wherein the non-physiological ligand is contacted at a concentration of 10 nM or about 10 nM.

167. The method according to any one of claims 160 to 165, wherein the non-physiological ligand is contacted at a concentration of 100 nM or about 100 nM.

168. The method according to any one of claims 160 to 164, wherein the non-physiological ligand is added to the medium at a concentration of 2.5 nM to 10 nM.

169. The method according to any one of claims 160 to 164 and 168, wherein the non-physiological ligand is added to the medium at a concentration of 3 nM to 7 nM.

170. The method according to any one of claims 160 to 164 and 168 to 169, wherein the non-physiological ligand is added to the medium at a concentration of 3.1 nM or about 3.1 nM.

171. The method according to any one of claims 160, 161, and 163 to 166, wherein the method is performed in vivo on a subject and the non-physiological ligand is administered to the subject.

172. A method for treating cancer in a subject, comprising administering to the subject an effective amount of the cell population according to any one of claims 1 to 51 and 84 or the pharmaceutical composition according to claim 85.

173. A method for treating cancer in a subject, comprising administering to the subject an effective amount of the iCIL or population of iCILs according to any one of claims 148 to 158 or the pharmaceutical composition according to claim 159.

174. The method according to any one of claims 171 to 173, wherein the subject has not been subjected to lymphodepletion therapy prior to administration of the iCIL, population of iCILs, or the pharmaceutical composition.

175. The method according to any one of claims 160 to 174, wherein the iCIL expresses a CAR that targets cancer cells in the subject.

176. The method according to claim 175, wherein the CAR is an anti-FITC CAR, and a FITC-ligand has been administered to the subject to tag cancer cells in the subject, and the ligand specifically binds to a molecule expressed on the tumor.

177. The method according to claim 176, wherein the FITC-ligand is FITC-folate.

178. The method according to any one of claims 171 to 177, comprising administering the non-physiological ligand of the synthetic cytokine receptor to the subject.

179. The method according to any one of claims 171 to 178, wherein the non-physiological ligand is rapamycin or a rapamycin analog.

180. The method according to claim 179, wherein the rapamycin analog is a rapalog.

181. The method according to any one of claims 171 to 180, wherein the non-physiological ligand is administered at a dose of 1 mg to 100 mg, optionally 10 to 100 mg, optionally 10 mg or about 10 mg, 20 mg or about 20 mg, 25 mg or about 25 mg, 30 mg or about 30 mg, 40 mg or about 40 mg, 50 mg or about 50 mg, or any value between any of the foregoing.

182. The method according to any one of claims 171 to 181, wherein a plurality of doses of the non-physiological ligand are administered to the subject.

183. The method according to claim 182, wherein after administering the iCIL population or a composition thereof to the subject, the plurality of doses are administered intermittently or at regular intervals, optionally over a predetermined period.

184. The method according to any one of claims 171 to 183, wherein 2 to 8 doses of the non-physiological ligand are administered to the subject.

185. The method according to any one of claims 171 to 181, wherein a single dose of the non-physiological ligand is administered to the subject.

186. The iCIL population or composition thereof is administered at a dose of 1×10 8 cells or about 1×10 8 cells of iCIL cells to 100×10 9 cells or about 100×10 9 cells of iCIL cells, according to the method of any one of claims 172 to 185.

187. The iCIL population or composition thereof is administered at a dose of more than 5×10 9 cells or about more than 5×10 9 cells, and optionally, the dose is from 5×10 9 cells or about 5×10 9 cells to 100×10 9 cells or about 100×10 9 cells, the method according to any one of claims 172 to 186.

188. The method according to any one of claims 171 to 187, wherein a plurality of doses of the iCIL cells are administered to the subject.

189. The method according to claim 188, wherein the plurality of doses of the iCIL cells are administered intermittently or at regular intervals, optionally over a predetermined period.

190. The method according to any one of claims 171 to 189, wherein 2 to 8 doses of the iCIL cells are administered to the subject.

191. The method according to any one of claims 171 to 187, wherein a single dose of the iCIL cells is administered to the subject.

192. A kit comprising the engineered stem cells according to any one of claims 1 to 51 and 84, and instructions for differentiating a cell population into cytotoxic natural lymphocyte-like cells.

193. A kit comprising 148 to 158 iCIL or a population of iCIL or the pharmaceutical composition according to claim 159, and instructions for administering to a subject in need thereof.

194. The kit according to claim 192 or claim 193, further comprising a container containing the non-physiological ligand and instructions for administering the non-physiological ligand to the subject after administration of the cell population.

195. The kit according to any one of claims 192 to 194, wherein the subject has cancer.

196. A population of induced cytotoxic natural lymphocyte-like (iCIL) cells, wherein the iCIL are mature iCIL expressing CD56 and LFA1, and at least 25% of the iCIL express cytotoxic receptors, 75% or less of the iCIL express dysfunctional receptors, and / or at least 25% of the iCIL are proliferative, said population.

197. The population according to claim 196, wherein at least 25% of said iCIL expresses a cytotoxic receptor. **Claim 198** The population according to claim 196 or claim 197, wherein said cytotoxic receptor is one or more of NKp30, NKp46 and NKG2D. **Claim 199** The population according to any one of claims 196 to 198, wherein at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55% or at least 60% of said iCIL expresses NKp30+. **Claim 200** The population according to any one of claims 196 to 199, wherein at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80% of said iCIL expresses NKp46. **Claim 201** The population according to any one of claims 196 to 200, wherein at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% of said iCIL expresses NKG2D. **Claim 202** The population according to any one of claims 196 to 201, wherein 75% or less of said iCIL expresses a dysfunctional receptor. **Claim 203** The population according to any one of claims 196 to 202, wherein said dysfunctional receptor is one or more of KLRG1, CD73 and CD38. **Claim 204** The population according to any one of claims 196 to 203, wherein 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less or 5% or less of said iCIL expresses KLRG1. **Claim 205** The population according to any one of claims 196 to 204, wherein 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less or 1% or less of said iCIL expresses CD73. **Claim 206** The population according to any one of claims 196 to 205, wherein 75% or less, 65% or less, 55% or less, 45% or less, 35% or less, 25% or less, 15% or less or 5% or less of said iCIL expresses CD38. **Claim 207** The population according to any one of claims 196 to 206, wherein at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55% or at least 60% of the iCILs are proliferative.

208. The population according to any one of claims 196 to 207, wherein the proliferative iCILs are CD56bright CD57-.

209. The iCILs further comprise a synthetic cytokine receptor for a non-physiological ligand, and the cytokine receptor comprises a synthetic gamma chain polypeptide comprising a first dimerization domain, a first transmembrane domain, and an interleukin-2 receptor subunit gamma (IL-2RG) intracellular domain, and a synthetic beta chain polypeptide comprising a second dimerization domain, a second transmembrane domain, and an intracellular domain selected from an interleukin-2 receptor subunit beta (IL-2RB) intracellular domain, an interleukin-7 receptor subunit beta (IL-7RB) intracellular domain, and / or an interleukin-21 receptor subunit beta (IL-21RB) intracellular domain The population according to any one of claims 196 to 208.

210. The population according to claim 209, wherein the first dimerization domain and the second dimerization domain are extracellular domains.

211. The synthetic gamma chain polypeptide comprises, in order from the N-terminus to the C-terminus, the first dimerization domain, the first transmembrane domain, and the interleukin-2 receptor subunit gamma (IL-2RG) intracellular domain, and the synthetic beta chain polypeptide comprises, in order from the N-terminus to the C-terminus, the second dimerization domain, the second transmembrane domain, and the intracellular domain, The population according to claim 209 or claim 210.

212. The population according to any one of claims 209 to 211, wherein the IL-2RG intracellular domain comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:1, or the polypeptide sequence set forth in SEQ ID NO:

1.

213. The population according to any one of claims 209 to 212, wherein the first transmembrane domain comprises the IL-2RG transmembrane domain.

214. The population according to claim 213, wherein the IL-2RG transmembrane domain comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO: 8 or 31, or comprises the polypeptide sequence set forth in SEQ ID NO: 8 or 31.

215. The population according to any one of claims 209 to 214, wherein the beta chain intracellular domain comprises the IL-2RB intracellular domain.

216. The population according to claim 215, wherein the IL-2RB intracellular domain comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO: 2, or comprises the polypeptide sequence set forth in SEQ ID NO:

2.

217. The population according to any one of claims 209 to 214, wherein the beta chain intracellular domain comprises the IL-7RB intracellular domain.

218. The population according to claim 217, wherein the IL-7RB intracellular domain comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO: 3, or comprises the polypeptide sequence set forth in SEQ ID NO:

3.

219. The population according to any one of claims 209 to 214, wherein the beta chain intracellular domain comprises the IL-21RB intracellular domain.

220. The population according to claim 219, wherein the IL-21RB intracellular domain comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO: 4, or comprises the polypeptide sequence set forth in SEQ ID NO:

4.

221. The population according to any one of claims 209 to 220, wherein the second transmembrane domain comprises a transmembrane domain derived from the same beta chain intracellular domain.

222. The population according to any one of claims 209 to 216 and 221, wherein the second transmembrane domain is a transmembrane domain of IL-2RB that comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO: 35 or 36, or comprises the polypeptide sequence set forth in SEQ ID NO: 35 or 36.

223. The synthetic gamma chain polypeptide contains an IL-2RG TM domain comprising the sequence set forth in SEQ ID NO: 8 or 31 and an IL-2RG intracellular domain comprising the sequence set forth in SEQ ID NO: 1, and The synthetic beta-chain polypeptide contains an IL-2RB TM domain comprising the sequence set forth in SEQ ID NO: 35 or 36 and an IL-2RB intracellular domain comprising the sequence set forth in SEQ ID NO:

2. The population according to any one of claims 209 to 216, 221 and 222. **Claim 224** The first dimerization domain and the second dimerization domain are heterodimerization domains selected from a 12 kD FK506-binding protein (FKBP) and an FKBP12-rapamycin binding (FRB) domain, and / or The non-physiological ligand is rapamycin or a rapalog. The population according to any one of claims 209 to 223. **Claim 225** The population according to claim 224, wherein the FRB domain comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO: 6 or SEQ ID NO:

7. **Claim 226** The population according to claim 224, wherein the FRB domain comprises the polypeptide sequence set forth in SEQ ID NO: 6 or SEQ ID NO:

7. **Claim 227** The first dimerization domain and the second dimerization domain are heterodimerization domains selected from a 12 kD FK506-binding protein (FKBP) and a calcineurin domain, and / or The non-physiological ligand is FK506 or an analog thereof. The population according to any one of claims 209 to 223. **Claim 228** The population according to any one of claims 209 to 224, wherein the FKBP domain comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO: 5 or SEQ ID NO:

30. **Claim 229** The population according to any one of claims 209 to 224, wherein the FKBP domain comprises the polypeptide sequence set forth in SEQ ID NO: 5 or SEQ ID NO:

30. **Claim 230** The population according to any one of claims 209 to 216 and 221 to 229, wherein the synthetic gamma chain polypeptide has the amino acid sequence set forth in SEQ ID NO: 28, or an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 28, and the synthetic beta chain polypeptide has the amino acid sequence set forth in SEQ ID NO: 33, or an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

33.

231. The population according to any one of claims 209 to 216 and 221 to 230, wherein the synthetic gamma chain polypeptide has the amino acid sequence set forth in SEQ ID NO: 28, and the synthetic beta chain polypeptide has the amino acid sequence set forth in SEQ ID NO:

33.

232. The first dimerization domain and the second dimerization domain are i) FK506-binding protein (FKBP) of size 12 kD; ii) cyclophilin A (CypA); or iii) gyrase B (CyrB) selected homodimerization domains, and the non-physiological ligands are, respectively, i) FK1012, AP1510, AP1903 or AP20187 or an analog thereof; ii) cyclosporin-A (CsA) or an analog thereof; or iii) coumermycin or an analog thereof is, the population according to any one of claims 209 to 223.

233. about 1×10 6 to about 1×10 12 iCILs, about 1×10 6 to about 1×10 10 iCILs, about 1×10 6 to about 1×10 8 iCILs, about 1×10 8 to about 1×10 12 iCILs, about 1×10 8 to about 1×10 10 iCILs, or about 1×10 10 to about 1×10 12 iCILs, a population according to any one of claims 196 to 232.

234. The volume of the population is from about 1 mL to about 100 mL, from about 1 mL to about 80 mL, from about 1 mL to about 60 mL, from about 1 mL to about 40 mL, from about 1 mL to about 20 mL, from about 1 mL to about 10 mL, from about 10 mL to about 100 mL, from about 10 mL to about 80 mL, from about 10 mL to about 60 mL, from about 10 mL to about 40 mL, from about 10 mL to about 20 mL, from about 20 mL to about 100 mL, from about 20 mL to about 80 mL, from about 20 mL to about 60 mL, from about 20 mL to about 40 mL, from about 40 mL to about 100 mL, from about 40 mL to about 80 mL, from about 40 mL to about 60 mL, from about 60 mL to about 100 mL, from about 60 mL to about 80 mL, or from about 80 mL to about 100 mL, the population according to any one of claims 196 to 233.

235. A pharmaceutical composition comprising the population of iCIL according to any one of claims 196 to 234.

236. The pharmaceutical composition according to claim 235, further comprising a cryoprotectant.

237. A cryopreservation composition comprising the population of iCIL according to any one of claims 196 to 234.

238. About 1×10 6 to about 1×10 12 iCILs, about 1×10 6 to about 1×10 10 iCILs, about 1×10 6 to about 1×10 8 iCILs, about 1×10 8 to about 1×10 12 iCILs, about 1×10 8 to about 1×10 10 iCILs, or about 1×10 10 to about 1×10 12 iCILs, the composition according to any one of claims 235 to 237.

239. The volume of the composition is from about 1 mL to about 100 mL, from about 1 mL to about 80 mL, from about 1 mL to about 60 mL, from about 1 mL to about 40 mL, from about 1 mL to about 20 mL, from about 1 mL to about 10 mL, from about 10 mL to about 100 mL, from about 10 mL to about 80 mL, from about 10 mL to about 60 mL, from about 10 mL to about 40 mL, from about 10 mL to about 20 mL, from about 20 mL to about 100 mL, from about 20 mL to about 80 mL, from about 20 mL to about 60 mL, from about 20 mL to about 40 mL, from about 40 mL to about 100 mL, from about 40 mL to about 80 mL, from about 40 mL to about 60 mL, from about 60 mL to about 100 mL, from about 60 mL to about 80 mL, or from about 80 mL to about 100 mL, the composition according to any one of claims 235 to 238.

240. A method of killing target cells or inhibiting the growth of target cells, comprising the step of contacting the target cells with the population of iCIL according to any one of claims 196 to 234, or the composition according to any one of claims 235 to 239.

241. The method according to claim 240, wherein the target cells are cancer cells.

242. The method according to claim 240 or claim 241, wherein the iCIL further comprises the synthetic cytokine receptor for the non-physiological ligand, and the method comprises the step of contacting the target cells with the non-physiological ligand of the synthetic cytokine receptor.

243. The method according to any one of claims 240 to 242, which is performed in vitro or ex vivo.

244. The method according to claim 242 or claim 243, wherein the non-physiological ligand is rapamycin or a rapamycin analog.

245. The method according to claim 244, wherein the rapamycin analog is a rapalog.

246. The method according to any one of claims 242 to 245, wherein the non-physiological ligand is contacted at a concentration of 5 nM to 200 nM, 5 nM to 150 nM, 5 nM to 100 nM, 5 nM to 50 nM, 5 nM to 20 nM, 5 nM to 10 nM, 10 nM to 200 nM, 10 nM to 150 nM, 10 nM to 100 nM, 10 nM to 50 nM, 10 nM to 20 nM, 20 nM to 200 nM, 20 nM to 150 nM, 20 nM to 100 nM, 20 nM to 50 nM, 50 nM to 200 nM, 50 nM to 150 nM, 50 nM to 100 nM, 100 nM to 200 nM, 100 nM to 150 nM, or 150 nM to 200 nM.

247. The method according to any one of claims 242 to 246, wherein the non-physiological ligand is contacted at a concentration of 10 nM or about 10 nM.

248. The method according to any one of claims 242 to 246, wherein the non-physiological ligand is contacted at a concentration of 100 nM or about 100 nM.

249. The method according to any one of claims 242 to 245, wherein the non-physiological ligand is added to the medium at a concentration of 2.5 nM to 10 nM.

250. The method according to any one of claims 242 to 245 and 249, wherein the non-physiological ligand is added to the medium at a concentration of 3 nM to 7 nM.

251. The method according to any one of claims 240 to 242 and 244 to 248, wherein the method is performed in vivo on a subject, and the population or composition of iCIL is administered to the subject.

252. The method according to claim 251, wherein the iCIL further comprises the synthetic cytokine receptor for the non-physiological ligand, and the method comprises the step of administering the non-physiological ligand to the subject.

253. A method of inducing natural killer (NK) cell-mediated cell killing in a subject, comprising administering to the subject an effective amount of a population of iCILs according to any one of claims 196 to 234 or a composition according to any one of claims 235 to 239.

254. A method of treating cancer in a subject, comprising administering to the subject an effective amount of a population of iCILs according to any one of claims 196 to 234 or a composition according to any one of claims 235 to 239.

255. The method according to any one of claims 251 to 254, wherein the subject has not been subjected to lymphodepletion therapy prior to the step of administering the population of iCILs or its composition.

256. The method according to any one of claims 251 to 255, wherein the iCIL expresses a CAR that targets cancer cells in the subject.

257. The method according to claim 256, wherein the CAR is an anti-FITC CAR, and a FITC-ligand has been administered to the subject to tag cancer cells in the subject, and the ligand specifically binds to a molecule expressed on the tumor.

258. The method according to claim 257, wherein the FITC-ligand is FITC-folate.

259. The method according to any one of claims 253 to 258, wherein the iCIL further comprises the synthetic cytokine receptor for the non-physiological ligand, and the method comprises administering to the subject the non-physiological ligand of the synthetic cytokine receptor.

260. The method according to any one of claims 252 and 255 to 259, wherein the non-physiological ligand is rapamycin or a rapamycin analog.

261. The method according to claim 260, wherein the rapamycin analog is a rapalog.

262. The method according to any one of claims 252 and 255 to 261, wherein the non-physiological ligand is administered at a dose of 1 mg to 100 mg, optionally 10 to 100 mg, optionally 10 mg or about 10 mg, 20 mg or about 20 mg, 25 mg or about 25 mg, 30 mg or about 30 mg, 40 mg or about 40 mg, 50 mg or about 50 mg, or any value between any of the foregoing.

263. The method according to any one of claims 252 and 255 to 262, wherein a plurality of doses of the non - physiological ligand are administered to the subject.

264. The method according to claim 263, wherein after administering the population of iCIL or a composition thereof to the subject, the plurality of doses are administered intermittently or at regular intervals, optionally over a predetermined period.

265. The method according to any one of claims 252 and 255 to 264, wherein 2 to 8 doses of the non - physiological ligand are administered to the subject.

266. The method according to any one of claims 252 and 255 to 262, wherein a single dose of the non - physiological ligand is administered to the subject.

267. The population of iCIL or its composition is from 1×10 8 or about 1×10 8 iCIL cells, and is administered at a dose of 100×10 9 or about 100×10 9 iCIL cells, according to the method of any one of claims 251 to 266.

268. The population of iCIL or a composition thereof is administered at a dose of more than 5×10 9 cells or more than about 5×10 9 cells, and optionally, the dose is from 5×10 9 cells or about 5×10 9 cells to 100×10 9 cells or about 100×10 9 cells. The method according to any one of claims 251 to 267.

269. The method according to any one of claims 251 to 268, wherein a plurality of doses of the iCIL cells are administered to the subject.

270. The method according to claim 269, wherein the plurality of doses of iCIL cells are administered intermittently or at regular intervals, optionally over a predetermined period.

271. The method according to any one of claims 251 to 270, wherein 2 to 8 doses of the iCIL cells are administered to the subject.

272. The method according to any one of claims 251 to 268, wherein a single dose of the iCIL cells is administered to the subject.

273. A kit comprising a population of iCIL according to any one of claims 196 to 234, or a composition according to any one of claims 235 to 239, and instructions for administering the population of iCIL or a composition thereof to a subject in need thereof.

274. The kit according to claim 273, wherein the iCIL further comprises the synthetic cytokine receptor for the non - physiological ligand, and the kit further comprises a container containing the non - physiological ligand and instructions for administering the non - physiological ligand to the subject after administering the population of iCIL or a composition thereof.

275. The kit according to claim 273 or claim 274, wherein the subject has cancer.

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