Engineered stem cells and uses thereof

Abstract

Provided herein are compositions and methods for cell populations comprising engineered stem cells comprising synthetic cytokine receptors for non-physiological ligands. The non-physiological ligands activate synthetic cytokine receptors in engineered stem cells to induce differentiation of stem cells and expansion and / or activation of cytotoxic innate lymphocytes thus produced.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 351,144, filed on June 10, 2022, entitled “ENGINEERED STEM CELLS AND USES THEREOF,” U.S. Provisional Application No. 63 / 392,861, filed on July 27, 2022, entitled “ENGINEERED STEM CELLS AND USES THEREOF,” U.S. Provisional Application No. 63 / 411,065, filed on September 28, 2022, entitled “ENGINEERED STEM CELLS AND USES THEREOF,” U.S. Provisional Application No. 63 / 422,882, filed on November 4, 2022, entitled “ENGINEERED STEM CELLS AND USES THEREOF,” U.S. Provisional Application No. 63 / 447,337, filed on February 21, 2023, and U.S. Provisional Application No. 63 / 451,536, filed on March 10, 2023, entitled “ENGINEERED STEM CELLS AND USES THEREOF,” the contents of each of which are incorporated herein by reference in their entirety.

[0003] Incorporation by Reference into the Sequence Listing

[0004] This application is submitted in electronic format along with a sequence listing. The sequence listing is provided as a file named 260132000340SeqList.xml, created on June 9, 2023, and 83,181 bytes in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety. Technical Field

[0005] The present disclosure provides compositions and methods related to cell populations comprising engineered stem cells containing synthetic cytokine receptors for non-physiological ligands that activate the synthetic cytokine receptors in the engineered stem cells to induce differentiation of the stem cells and expansion and / or activation of the resulting cytotoxic innate lymphoid cells. Background Art

[0006] Cytotoxic innate lymphoid cells (CILs) are a type of immune cell that can be used in immunotherapy, including cancer immunotherapy. One type of CIL is a natural killer (NK) cell, which is typically identified as a cell that is positive for the cell surface protein CD56 (CD56+) and other markers and has cytotoxic activity.

[0007] CIL cells for 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)), which are cells derived from somatic cells (usually fibroblasts or peripheral blood mononuclear cells [PBMC]), and human embryonic stem cells (hESC), any of which can be induced to proliferate indefinitely and differentiate into other cell types under appropriate differentiation conditions. From iPSC, CIL cells can be obtained as follows: iPSCs are sequentially differentiated into hematopoietic progenitor cells (HPCs), also known as hematopoietic stem cells (HSCs); HPCs are differentiated into common lymphoid progenitor cells (CLPs); and CLPs are then differentiated into CIL cells - called iPSC-derived cytotoxic innate lymphoid cells (iPSC-CIL). In general, iPSC-CIL cells express CD56 and have cytotoxic activity like NK cells; however, iPSC-CIL cells may differ from NK cells in phenotype and other aspects.

[0008] Methods are known for differentiating iPSCs into CD34+ HPCs using embryoid bodies (EBs) or single-cell iPSC culture on feeder cells. The CD34+ HPCs can then be differentiated into CLPs.

[0009] There remains a need in the art for compositions and methods related to engineering stem cells, methods of making such cells, methods of differentiating such cells into CILs, and methods of using them for immunotherapy. Summary of the Invention

[0010] The present disclosure is based in part on the discovery that stem cells engineered to express synthetic cytokine receptors can improve or enhance differentiation into hematopoietic progenitor cells and CLPs in response to the receptor's cognate non-physiological ligand. These progenitor cells then differentiate into engineered CIL cells. As demonstrated herein, CRISPR is used to genetically engineer stem cells to express synthetic cytokine receptors and, in some embodiments, simultaneously disrupt genes (such as beta-2-microglobulin) to avoid immune rejection and / or provide resistance to rapamycin.

[0011] According to the methods described herein, large quantities of CIL cells with ideal functional properties can be generated from engineered stem cells. Non-limiting advantages of certain embodiments include the ability of the CIL cells described herein to differentiate without the use of exogenous factors (e.g., without the use of SCF, TPO, BMP4, FGF and / or VEGF) or supplementation of exogenous factors. In addition, CIL cells expressing synthetic cytokine receptors provide the ability to expand the CIL cells described herein without the use of exogenous factors (e.g., without the use of IL-2, IL-7, IL-15 and / or IL-21). The CIL cells and related compositions described herein can be used for ex vivo expansion immunotherapy.

[0012] Thus, in some aspects, the present disclosure provides an engineered stem cell comprising a synthetic cytokine receptor for a non-physiological ligand, wherein the cytokine receptor comprises: a synthetic γ chain polypeptide and a synthetic β chain polypeptide, the synthetic γ chain polypeptide comprising a first dimerization domain, a first transmembrane domain and an interleukin-2 receptor subunit γ (IL-2RG) intracellular domain; and the synthetic β chain polypeptide comprising a second dimerization domain, a second transmembrane domain and an intracellular domain, the intracellular domain being selected from the group consisting of the interleukin-2 receptor subunit β (IL-2RB) intracellular domain, the interleukin-7 receptor subunit β (IL-7RB) intracellular domain and / or the interleukin-21 receptor subunit β (IL-21RB) intracellular domain.

[0013] In some embodiments, the first dimerization domain and the second dimerization domain are extracellular domains. In some embodiments, the synthetic γ chain polypeptide comprises, in N- to C-terminal order, a first dimerization domain, a first transmembrane domain, and an interleukin-2 receptor subunit γ (IL-2RG) intracellular domain, and the synthetic β chain polypeptide comprises, in N- to C-terminal order, a second dimerization domain, a second transmembrane domain, and an intracellular domain.

[0014] In some embodiments, the IL-2RG intracellular domain comprises a polypeptide sequence at least 95% identical to, or as 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 at least 95% identical to, or as set forth in, SEQ ID NO: 8 or 31.

[0015] In some embodiments, the beta chain intracellular domain comprises an IL-2RB intracellular domain. In some embodiments, the IL-2RB intracellular domain comprises a polypeptide sequence at least 95% identical to SEQ ID NO: 2, or a polypeptide sequence as set forth in SEQ ID NO: 2.

[0016] 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 at least 95% identical to SEQ ID NO: 3, or a polypeptide sequence as set forth in SEQ ID NO: 3.

[0017] 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 at least 95% identical to SEQ ID NO: 4, or a polypeptide sequence as set forth in SEQ ID NO: 4.

[0018] In some embodiments, the second transmembrane domain comprises a transmembrane domain from the same beta chain intracellular domain. In some embodiments, the second transmembrane domain is the intracellular domain of IL-2RB, which comprises a polypeptide sequence at least 95% identical to SEQ ID NO: 35 or 36, or a polypeptide sequence as set forth in SEQ ID NO: 35 or 36.

[0019] In some embodiments, the synthetic γ chain polypeptide contains an IL-2RG TM (transmembrane) 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 β 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.

[0020] In some embodiments, the first dimerization domain and the second dimerization domain are heterologous dimerization domains selected from the 12kD size FK506 binding protein (FKBP) and FKBP12-rapamycin binding (FRB) domain. In some embodiments, the non-physiological ligand is rapamycin or a rapamycin analog (rapalog). In some embodiments, the FRB domain comprises a polypeptide sequence at least 95% identical to SEQ ID NO: 6 or SEQ ID NO: 7. In some embodiments, the FRB domain comprises a polypeptide sequence as shown in SEQ ID NO: 6 or SEQ ID NO: 7. In some embodiments, the FKBP domain comprises a polypeptide sequence at least 95% identical to SEQ ID NO: 5, SEQ ID NO: 49 or SEQ ID NO: 30.

[0021] In some embodiments, the FKBP domain comprises a polypeptide sequence as 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 heterologous dimerization domains selected from a 12 kD FK506 binding protein (FKBP) and a 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 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 a polypeptide sequence as set forth in SEQ ID NO: 5, SEQ ID NO: 49, or SEQ ID NO: 30.

[0022] In some arbitrary 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 NO: 28, and the synthetic beta chain polypeptide has the amino acid sequence set forth in SEQ ID NO: 33.

[0023] In some embodiments, the first dimerization domain and the second dimerization domain are homologous dimerization domains selected from the group consisting of: i) 12 kD FK506 binding protein (FKBP); ii) cyclophilin A (CypA); or iii) gyrase B (CyrB); and the non-physiological ligand is: i) FK1012, AP1510, AP1903 or AP20187, or an analog thereof; ii) cyclosporine-A (CsA) or an analog thereof; or iii) coumarins or an analog thereof, respectively.

[0024] In some arbitrary embodiments, the stem cell is a pluripotent stem cell. In some embodiments, the stem cell is an induced pluripotent stem cell (iPSC).

[0025] In some embodiments, the stem cells are resistant to rapamycin-mediated inhibition of mTOR.

[0026] In some embodiments, the stem cell expresses a cytoplasmic polypeptide that binds to a non-physiological ligand. In some embodiments, the non-physiological ligand is rapamycin or a rapamycin analog, and the stem cell expresses a cytoplasmic FRB domain or a variant thereof. In some embodiments, the cytoplasmic 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 cytoplasmic FRB domain comprises a polypeptide sequence that is at least 98% identical to SEQ ID NO: 6 or SEQ ID NO: 7.

[0027] In some embodiments, the stem cells comprise a disrupted FKBP12 gene, which reduces expression of FKBP12. In some embodiments, the stem cells comprise a knockout of the FKBP12 gene.

[0028] In some embodiments, the stem cell comprises a nucleotide sequence encoding a synthetic cytokine receptor inserted into the stem cell genome. In some embodiments, the nucleotide sequence encoding the synthetic cytokine receptor is inserted into a non-target locus in the stem cell genome. In some embodiments, the nucleotide sequence encoding the synthetic cytokine receptor is inserted into an endogenous gene of the stem cell. In some embodiments, the insertion reduces the expression of the endogenous gene in the locus. In some embodiments, the insertion knocks out the endogenous gene in the locus. In some embodiments, the insertion is carried out by homology-directed repair.

[0029] 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 1α (EEF1A), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ubiquitin C (UBC), and actin β (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 beta-2-microglobulin (B2M) gene, T cell receptor alpha constant (TRAC) gene, and signal regulatory protein alpha (SIRPA) gene. In some embodiments, the endogenous gene is B2M. In some embodiments, stem cells include B2M knockout. In some embodiments, the cell has a destruction of the gene encoding FKBP12. In some embodiments, the disruption is an FKBP12 knockout that inactivates the gene encoding FKBP12.

[0030] In some embodiments, the stem cells comprise a B2M knockout and a FKBP12 knockout.

[0031] In some embodiments, the stem cell comprises a chimeric antigen receptor (CAR). In some embodiments, the CAR is an anti-FITC CAR.

[0032] In some embodiments, binding of a non-physiological ligand to the synthetic cytokine receptor activates the synthetic cytokine receptor in the stem cells to induce differentiation of the engineered stem cells in the cell population.

[0033] Also provided herein are cell populations comprising any of the provided engineered stem cells.

[0034] Thus, in some aspects, the present disclosure provides a cell population comprising engineered stem cells comprising a synthetic cytokine receptor for a non-physiological ligand, wherein the cytokine receptor comprises: a synthetic γ chain polypeptide and a synthetic β chain polypeptide, the synthetic γ chain polypeptide comprising a first dimerization domain, a first transmembrane domain, and an interleukin-2 receptor subunit γ (IL-2RG) intracellular domain; and the synthetic β chain polypeptide comprising a second dimerization domain, a second transmembrane domain, and an intracellular domain, the intracellular domain being selected from the group consisting of an interleukin-2 receptor subunit β (IL-2RB) intracellular domain, an interleukin-7 receptor subunit β (IL-7RB) intracellular domain, and / or an interleukin-21 receptor subunit β (IL-21RB) intracellular domain; wherein the non-physiological ligand activates the synthetic cytokine receptor in the stem cell to induce differentiation of the stem cell.

[0035] In some embodiments, the beta chain intracellular domain comprises an IL-2RB intracellular domain. In some embodiments, the IL-2RB intracellular domain comprises a polypeptide sequence at least 95% identical to SEQ ID NO: 2, or a polypeptide sequence as set forth in SEQ ID NO: 2.

[0036] 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 at least 95% identical to SEQ ID NO: 3, or a polypeptide sequence as set forth in SEQ ID NO: 3.

[0037] 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 at least 95% identical to SEQ ID NO: 4, or a polypeptide sequence as set forth in SEQ ID NO: 4.

[0038] In some or any of the foregoing embodiments, the first dimerization domain and the second dimerization domain are extracellular domains; the synthetic γ chain polypeptide comprises, in N- to C-terminal order, a first dimerization domain, a first transmembrane domain, and an interleukin-2 receptor subunit γ (IL-2RG) intracellular domain, and the synthetic β chain polypeptide comprises, in N- to C-terminal order, a second dimerization domain, a second transmembrane domain, and an intracellular domain.

[0039] In some or any of the above embodiments, the first dimerization domain and the second dimerization domain are heterologous dimerization domains selected from the group consisting of a 12 kD FK506 binding protein (FKBP) and a FKBP12-rapamycin binding (FRB) domain; and / or wherein the non-physiological ligand is rapamycin or a rapamycin analog. In some embodiments, the FKBP domain comprises a polypeptide sequence at least 95% identical to SEQ ID NO: 5. In some embodiments, the FKBP domain comprises a polypeptide sequence as shown in SEQ ID NO: 5. In some embodiments, the FKBP domain comprises a polypeptide sequence at least 95% identical to SEQ ID NO: 49. In some embodiments, the FKBP domain comprises a polypeptide sequence as shown in SEQ ID NO: 49. In some embodiments, the FRB domain comprises a polypeptide sequence at least 95% identical to SEQ ID NO: 6 or SEQ ID NO: 7. In some embodiments, the FRB domain comprises a polypeptide sequence as shown in SEQ ID NO: 6 or SEQ ID NO: 7.

[0040] In some or any of the above embodiments, the first dimerization domain and the second dimerization domain are heterologous dimerization domains selected from a 12 kD FK506 binding protein (FKBP) and a calcineurin domain; and / or wherein the non-physiological ligand is FK506 or an analog thereof. In some embodiments, the FKBP domain comprises a polypeptide sequence at least 95% identical to SEQ ID NO: 5. In some embodiments, the FKBP domain comprises a polypeptide sequence as set forth in SEQ ID NO: 5. In some embodiments, the FKBP domain comprises a polypeptide sequence at least 95% identical to SEQ ID NO: 49. In some embodiments, the FKBP domain comprises a polypeptide sequence as set forth in SEQ ID NO: 49.

[0041] In some or any of the above embodiments, the first dimerization domain and the second dimerization domain are homologous dimerization domains selected from:

[0042] i) FK506 binding protein (FKBP) with a size of 12 kD;

[0043] ii) Cyclophilin A (CypA); or

[0044] iii) gyrase B (CyrB);

[0045] And the non-physiological ligands are:

[0046] i) FK1012, AP1510, AP1903 or AP20187 or their analogs;

[0047] ii) cyclosporine-A (CsA) or an analogue thereof; or

[0048] iii) Coumarin or its analogues.

[0049] In some or any of the above embodiments, the stem cell expresses a cytoplasmic polypeptide that binds to a non-physiological ligand. In some embodiments, the non-physiological ligand is rapamycin or a rapamycin analog, and the stem cell expresses a cytoplasmic FRB domain or a variant thereof. In some embodiments, the cytoplasmic 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 cytoplasmic FRB domain comprises a polypeptide sequence that is at least 98% identical to SEQ ID NO: 6 or SEQ ID NO: 7.

[0050] In some or any of the above embodiments, the stem cells are induced pluripotent stem cells (iPSCs).

[0051] In some or any of the above embodiments, the stem cell comprises a nucleotide sequence encoding a synthetic cytokine receptor. In some embodiments, the nucleotide sequence is inserted into the endogenous gene of the stem cell. 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 1α (EEF1A), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ubiquitin C (UBC) and actin β (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 the destroyed gene of the stem cell. In some embodiments, the destroyed gene is selected from destroyed beta-2-microglobulin (B2M) gene, destroyed T cell receptor alpha constant (TRAC) gene and destroyed signal regulatory protein alpha (SIRPA) gene.

[0052] In some or any of the above embodiments, the stem cells comprise a disrupted B2M gene. In some embodiments, the stem cells comprise reduced expression of B2M. In some embodiments, the stem cells are B2M knockout.

[0053] In some or any of the above embodiments, the stem cells are resistant to rapamycin. In some embodiments, the rapamycin-resistant stem cells comprise a disrupted FKBP12 gene. In some embodiments, the stem cells comprise reduced expression of FKBP12. In some embodiments, the stem cells are FKBP12 knocked out.

[0054] In some or any of the above embodiments, the non-physiological ligand activates a cytokine receptor synthesized in a stem cell to induce the stem cell to differentiate into a hematopoietic progenitor cell. In some embodiments, the non-physiological ligand activates a cytokine receptor synthesized in a stem cell to induce the stem cell to differentiate into a common lymphoid progenitor cell (CLPs) or a common myeloid progenitor cell (CMP).

[0055] In some or any of the above embodiments, the stem cell comprises a chimeric antigen receptor (CAR).

[0056] In some aspects, the present disclosure provides a method of generating cytotoxic innate lymphoid (iCIL) cells, comprising contacting a cell population of any one of the embodiments provided herein with a non-physiological ligand for a first period of time sufficient to generate CLPs, and contacting the CLPs with a differentiation medium for a second period of time sufficient to generate iCILs.

[0057] 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.

[0058] In some embodiments, the first time period is 1-15 days, and the second time period is 1-15 days. In some embodiments, the method comprises contacting the iCIL with a pre-activation medium comprising IL-7, IL-12, IL-15, IL-18, and IL-21 for a third time period sufficient to generate mature iCIL. In some embodiments, the pre-activation medium comprises a non-physiological ligand. In some embodiments, the third time period is 1-10 days.

[0059] In some or any of the above embodiments, the mature iCIL expresses NKp46, NKG2D, LFA1, DNAM1, CD16, and CD56.

[0060] In some aspects, the present disclosure provides a method of genetically engineering stem cells to express a synthetic cytokine receptor, comprising: contacting a population of stem cells with: (i) a guide RNA (gRNA) that targets a target site in an endogenous gene, (ii) an RNA-guided endonuclease, and (iii) a recombinant vector comprising a nucleotide sequence encoding a synthetic cytokine receptor for a non-physiological ligand,

[0061] Thereby, the nucleotide sequence is inserted into the endogenous gene;

[0062] Wherein said cytokine receptors include:

[0063] 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

[0064] A synthetic β 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 β (IL-2RB), the intracellular domain of interleukin-7 receptor subunit β (IL-7RB) and / or the intracellular domain of interleukin-21 receptor subunit β (IL-21RB).

[0065] In some or embodiments, the nucleotide sequence is inserted by homology directed repair (HDR). In some embodiments, the recombinant vector comprises 5' and 3' homology arms flanked by nucleotide sequences encoding synthetic cytokine receptors, wherein the 3' homology arms are homologous to the region upstream of the gRNA target site, and the 5' homology arms are homologous to the region downstream of the gRNA target site. In some or embodiments, the method includes contacting the cell with a vector comprising a nucleic acid, the nucleic acid comprising from 5' to 3' (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 in the target site of the endogenous gene, and the nucleic acid is exchanged with the homologous nucleotide sequence of the endogenous gene.

[0066] In some or embodiments, the nucleotide sequence is inserted by non-homologous end joining (NHEJ).

[0067] 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.

[0068] 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, wherein disrupting the target gene comprises contacting the population of stem cells with (i) a gRNA targeted to a target site in the target gene and (ii) an RNA-guided endonuclease. In some embodiments, the endogenous target gene is selected from B2M, TRAC, and SIRPA.

[0069] In some or any embodiments, the endogenous gene is B2M. In some embodiments, the gRNA comprises the sequence shown in SEQ ID NO: 18.

[0070] In some or any embodiments, 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; 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.

[0071] In some or any embodiments, the nucleotide sequence encoding the synthetic cytokine receptor comprises a first nucleic acid sequence encoding a gamma chain and a second nucleic acid sequence encoding a beta chain, wherein the first nucleic acid sequence has 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 the second nucleic acid sequence has 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 a first nucleic acid set forth in SEQ ID NO: 37 and a second nucleic acid set forth in SEQ ID NO: 38.

[0072] 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 quantitative reporter linker (PQR). In some embodiments, the PQR linker has the sequence shown in SEQ ID NO:42.

[0073] In some or any embodiments, the nucleotide sequence encoding the synthetic cytokine receptor for a non-physiological ligand is under the operable control of a heterologous promoter. In some embodiments, the heterologous promoter is the EF1α promoter or the MND promoter. In some embodiments, the promoter is a dual promoter, wherein the synthetic cytokine receptor is under the operable control of two promoters. In some embodiments, the dual promoter is a dual EF1α promoter.

[0074] In some or any embodiments, the nucleotide sequence encoding the synthetic cytokine receptor comprises a polyadenylation sequence.

[0075] 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.

[0076] In some or any of the above embodiments, the method comprises engineering a population of stem cells to make them resistant to rapamycin. In some embodiments, resistance to rapamycin is rapamycin-mediated mTOR inhibition. In some embodiments, the population of stem cells resistant to rapamycin comprises destroying the FKBP12 gene in the stem cells. In some embodiments, the population of stem cells resistant to rapamycin has reduced expression of FKBP12. In some embodiments, the population of stem cells resistant to rapamycin comprises knocking out the FKBP12 gene.

[0077] In some embodiments, stem cells are engineered with CRISPR-Cas and gRNA targeting the FKBP12 gene to destroy FKBP12 in the cell. In some or any embodiments, the method includes further contacting the stem cell population with a guide RNA (gRNA) targeting a target site in the FKBP12 gene. In some 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. In some embodiments, the further contact is performed simultaneously with (i) contact with a guide RNA (gRNA) targeting a target site in an endogenous gene, optionally in combination with the same RNA-guided endonuclease.

[0078] In some embodiments, the gRNA comprises one or more gRNAs selected from the group consisting of gRNAs comprising the sequence shown in SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21. In some embodiments, the one or more gRNAs are a gRNA pool comprising 2 or 3 gRNAs.

[0079] In some or any embodiments, the method further comprises introducing a chimeric antigen receptor (CAR) into the stem cell population. In some embodiments, the CAR is an anti-FITC CAR.

[0080] In some or any embodiments, the stem cells are pluripotent stem cells.

[0081] In some or any of the above embodiments, the stem cells are iPSCs.

[0082] In some or any embodiments, provided herein is a method for generating hematopoietic progenitor (HP) cells, the method comprising a) culturing a cell population comprising any engineered iPSC 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 culturing in b) is day 0; c) culturing the cells produced in b) under conditions that differentiate the cells into a hematopoietic progenitor (HP) population, wherein at least a portion of one or more of steps a)-c) is performed in the presence of a non-physiological ligand for a synthetic cytokine receptor.

[0083] In some embodiments, the present invention provides a method for generating cytotoxic innate lymphoid (iCIL) cells, the method comprising culturing a cell population comprising engineered iPSCs of any embodiment provided herein under conditions for differentiating iPSCs into cytotoxic innate lymphoid (iCIL) cells, wherein a non-physiological ligand for a synthetic cytokine receptor is added during at least part of the culturing process. In some embodiments, the culturing comprises: a) culturing a cell population comprising engineered iPSCs under conditions for forming aggregates; b) culturing the cells produced in a) under conditions for inducing mesoderm formation in a plurality of cells, wherein the start of culturing in b) is day 0; c) culturing the cells produced in b) under conditions for differentiating the cells into hematopoietic progenitor cells (HP); and d) culturing the cells produced in c) under conditions for producing iCIL cells, wherein at least a portion of one or more steps a)-d) is performed in the presence of a non-physiological ligand for a synthetic cytokine receptor.

[0084] Thus, in some embodiments, provided herein is a method for producing cytotoxic innate lymphoid (iCIL) cells, the method comprising: a) culturing a cell population comprising the engineered iPSCs of any provided embodiment under conditions in which aggregates are formed; b) culturing the cells produced in a) under conditions in which mesoderm is induced to form in a plurality of cells, wherein the start of culturing in b) is day 0; c) culturing the cells produced in b) under conditions in which the cells are differentiated into hematopoietic progenitor (HP) cells; and d) culturing the cells produced in c) under conditions in which iCIL cells are produced, wherein at least a portion of one or more of steps a)-d) is performed in the presence of a non-physiological ligand for a synthetic cytokine receptor.

[0085] In some embodiments, the culture is carried out in a container that has been treated to promote cell adhesion and growth. In some embodiments, the container is a basement membrane (Matrigel). In some embodiments, the culture is carried out in a non-adhesive culture container. In some embodiments, the non-adhesive culture container is an Aggrewell. TM In some embodiments of these methods, the aggregates in a) are embryoid bodies (EBs).

[0086] In some embodiments, the culture is carried out in suspension. In some embodiments, the culture is carried out in a culture vessel that has not been treated to promote cell adhesion and proliferation. In some of any such embodiments, the culture in step a) includes: (i) performing a first incubation, the first incubation comprising culturing a cell population of engineered stem cells under conditions that form a first aggregate; (ii) contacting the aggregate with a dissociation agent to form a dissociated cell population; and (iii) performing a second incubation, the second incubation comprising culturing the dissociated cell population under conditions that form a second aggregate. In some embodiments, the incubation is carried out in suspension.

[0087] In some or any embodiments of the method, the culturing in b) is carried out in a culture medium comprising one or more of BMP4, FGF2, VEGF, and a Rock inhibitor. In some embodiments, the Rock inhibitor is Y27632. In some embodiments, the culturing in b) is carried out in a culture medium comprising BMP4, FGF2, VEGF, and Y27632. In some embodiments, the culturing in b) is carried out in a culture medium comprising BMP4, FGF2, and VEGF. In some embodiments, the culturing in b) is carried out in a culture medium comprising a non-physiological ligand. In some embodiments, the culturing in b) is carried out in a culture medium comprising the non-physiological ligand without any additional growth factors. In some embodiments, the culturing in b) is carried out for 2-4 days. In some embodiments, the culturing in b) is carried out for 3 days or about 3 days.

[0088] In some embodiments, the culturing in c) is carried out in a culture medium comprising one or more of BMP4, FGF2, VEGF, TPO, SCF, and LDL. In some embodiments, the culturing in c) is carried out in a culture medium comprising one or more of BMP4, FGF2, VEGF, and LDL. In some embodiments, the culturing in c) is carried out in a culture medium comprising BMP4 and FGF2. In some embodiments, the culturing in c) using BMP4 and FGF2 is carried out on day 3 to day 15 or for days 3-15. In some embodiments, the culturing in c) comprises a PI3K inhibitor. In some embodiments, the PI3K inhibitor is LY2940002. In some embodiments, the PI3K inhibitor is added during part of the culturing in c). In some embodiments, the PI3K inhibitor is added from about day 6 to day 15.

[0089] In some embodiments, the culturing in c) is performed in a medium without SCF and TPO. In some embodiments, the culturing in c) is performed in a medium containing a non-physiological ligand. In some embodiments, the culturing in c) is performed in a medium containing the non-physiological ligand without any additional growth factors, cytokines, or both.

[0090] In some embodiments, the culture in c) is carried out on day 3-15 or for 3-15 days. In some embodiments, during at least a portion of the culture in c), the culture medium comprises an aryl hydrocarbon receptor (AHR) antagonist (such as StemRegenin-1), a pyrimidine-[4,5-b]-indole derivative (such as UM729), or both. In some embodiments, the portion of the culture is carried out on day 9-15 or about day 9-15, or for 9-15 days or about 9-15 days. In some embodiments, the AHR antagonist is StemRegenin1 (SR1). In some embodiments, the pyrimido-[4,5-b]-indole derivative is UM729. In some embodiments, SR1 and UM729 are added to the culture in c) starting on one of the days 6 to 9. In some embodiments, SR1 and UM729 are added to the culture in c) starting on about day 6.

[0091] In some embodiments, the culture in d) is carried out in a culture medium comprising one or more of FLT3L, IL-7, IL-12, IL-15, SR-1, and UM729. In some embodiments, the culture in d) is carried out in a culture medium containing a non-physiological ligand. In some embodiments, the culture in d) is carried out in a culture medium containing the non-physiological ligand without any additional growth factors, cytokines, or both. In some embodiments, the culture in d) is carried out on day 15 to day 40 or for a period of 15 to 40 days. In some embodiments, the culture in d) is carried out on day 15 to day 30 or for a period of 15 to 30 days.

[0092] In some aspects, provided herein is a method for generating cytotoxic innate lymphoid (iCIL) cells, the method comprising: a) culturing a cell population comprising the engineered iPSC of any one of claims 1 to 48 under conditions that form aggregates; b) culturing the cells produced in a) in a culture medium comprising one or more selected from the group consisting of BMP4, VEGF, FGF2, and ROCKi to induce the formation of mesoderm in a plurality of cells, wherein the start of culturing in b) is day 0; c) culturing the cells produced in b) in a culture medium comprising BMP4, FGF2, and LY2940002 to differentiate the cells into a population of hematopoietic progenitor (HP) cells, wherein the start of culturing in c) is day 3; d) culturing the cells produced in d) in a culture medium comprising SCF and IL-15 to generate iCIL cells, wherein the start of culturing in d) 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 for a synthetic cytokine receptor. In some embodiments, SR1 and UM729 are added to the culture in c) starting on one of day 6 to day 9. In some embodiments, SR1 and UM729 are added to the culture in c) starting on about day 6.

[0093] Also provided is a method for generating cytotoxic innate lymphoid (iCIL) cells, comprising contacting a cell population comprising an engineered stem cell of any one of the embodiments provided with a non-physiological ligand for a first time period sufficient to generate CLPs, and contacting CLPs with a differentiation medium for a second time period sufficient to generate iCILs. 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 time period is 1-15 days, and wherein the second time period is 1-15 days. In some embodiments, the method comprises contacting iCILs with a pre-activation medium comprising IL-7, IL-12, IL-15, IL-18, and IL-21 for a third time period sufficient to generate mature iCILs. In some embodiments, the pre-activation medium comprises a non-physiological ligand. In some embodiments, the third time period is 1-10 days. In some embodiments, mature iCIL expresses NKp46, NKG2D, LFA1, DNAM1, CD16, and CD56.

[0094] In some or any embodiments, the non-physiological ligand is rapamycin or a rapamycin analog. In some embodiments, the non-physiological ligand is a rapamycin analog (rapalog). In some embodiments, the non-physiological ligand is added to the culture medium at concentrations of 5nM-200nM, 5nM-150nM, 5nM-100nM, 5nM-50nM, 5nM-20nM, 5nM-10nM, 10nM-200nM, 10nM-150nM, 10nM-100nM, 10nM-50nM, 10nM-20nM, 20nM-200nM, 20nM-150nM, 20nM-100nM, 20nM-50nM, 50nM-200nM, 50nM-150nM, 50nM-100nM, 100nM-200nM, 100nM-150nM, and 150nM-200nM. In some embodiments, the non-physiological ligand is added to the culture medium at a concentration of 10 nM or about 10 nM. In some embodiments, the non-physiological ligand is added to the culture medium at a concentration of 100 nM or about 100 nM. In some embodiments, the non-physiological ligand is added to the culture medium at a concentration of 2.5 nM to 10 nM. In some embodiments, the non-physiological ligand is added to the culture medium at a concentration of 3 nM to 7 nM. In some embodiments, the non-physiological ligand is added to the culture medium at a concentration of 3.1 nM or about 3.1 nM.

[0095] In some embodiments, provided herein are hematopoietic progenitor (HP) cells produced by any of the methods provided herein. In some embodiments, 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 HP cells is 1 / 8, 1 / 7, 1 / 6, 1 / 5, 1 / 4, 1 / 3, 1 / 2, or 1-fold lower than that of CD34+ cord blood cells. In some embodiments, CD34+ cord blood cells comprise hematopoietic stem cells (HSCs).

[0096] In some or any embodiments, provided herein is a hematopoietic progenitor (HP) cell that has been differentiated from a pluripotent stem cell according to any of the methods provided herein, wherein the HP comprises a synthetic cytokine receptor.

[0097] In some or any embodiments, provided herein is a cell population of hematopoietic progenitor cells (HP) produced by any method provided herein. In some or any embodiments, the population of HP cells comprises lower expression of HLF, HOXA9 and / or CD133 compared to the population of CD34+ cord blood cells. In some embodiments, the expression of HLF, HOXA9 and / or CD133 in HP cells is 1 / 8, 1 / 7, 1 / 6, 1 / 5, 1 / 4, 1 / 3, 1 / 2 or 1 times compared to the population of CD34+ cord blood cells. In some or any embodiments, the population of CD34+ cord blood cells comprises hematopoietic stem cells (HSC).

[0098] Also provided herein is a cytotoxic innate lymphoid (iCIL) cell produced by any of the provided methods.

[0099] Also provided herein is a cytotoxic innate lymphoid (iCIL) cell that has been differentiated from any of the engineered stem cells provided, wherein the iCIL comprises a synthetic cytokine receptor.

[0100] Also provided herein are populations of cytotoxic innate lymphoid (iCIL) cells produced by any of the provided methods.

[0101] Also provided herein is a pharmaceutical composition comprising an iCIL or a population of iCILs according to any of the provided embodiments.

[0102] In some aspects, the present disclosure provides cell populations produced by the methods described herein.

[0103] In some aspects, the present disclosure provides pharmaceutical compositions comprising the cell populations described herein.

[0104] Also provided herein is a method for amplifying cytotoxic innate lymphoid cells (iCILs), the method comprising contacting an iCIL or iCIL population provided herein, or a pharmaceutical composition comprising an iCIL or iCIL population, with a non-physiological ligand for a synthetic cytokine receptor. Also provided herein is a method for killing cancer cells or inhibiting cancer cell proliferation, the method comprising contacting cancer cells with an iCIL or iCIL population provided herein, or a pharmaceutical composition comprising an iCIL or iCIL population provided herein, and a non-physiological ligand for a synthetic cytokine receptor. In some embodiments, the synthetic cytokine receptor has a first dimerization domain and a second dimerization domain, which are heterologous dimerization domains selected from a 12kD size FK506 binding protein (FKBP) and an FKBP12-rapamycin binding (FRB) domain. In some embodiments, the non-physiological ligand is rapamycin or an analog of rapamycin. In some embodiments, the non-physiological ligand is a rapamycin analog (rapalog).

[0105] In some embodiments, the method is performed in vitro or ex vivo. In some or any embodiments, the method is performed ex vivo in a subject, and the non-physiological ligand is contacted with stem cells (such as iPSCs) from the subject.

[0106] In some embodiments, the non-physiological ligand is contacted at a concentration of 2.5 nM-200 nM, 2.5 nM-150 nM, 2.5 nM-100 nM, 5 nM-200 nM, 5 nM-150 nM, 5 nM-100 nM, 5 nM-50 nM, 5 nM-20 nM, 5 nM-10 nM, 10 nM-200 nM, 10 nM-150 nM, 10 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.

[0107] In some embodiments, the method is performed in a subject, and a non-physiological ligand is administered to the subject.

[0108] In some aspects, the present disclosure provides a method of treating cancer in a subject, comprising administering to the subject an effective amount of a cell population or pharmaceutical composition described herein. In some embodiments, the cell population is a population of iCILs provided herein. In some embodiments, the method comprises administering to the subject a non-physiological ligand in an amount effective to induce expansion of iCILs in the subject.

[0109] In some or any embodiments, the subject has not been administered lymphodepleting therapy prior to administration of the iCILs, population of iCILs, or pharmaceutical compositions containing such cells.

[0110] In some or any embodiments, the iCIL expresses a CAR targeting a cancer cell of the subject. In some or any embodiments, the CAR is an anti-FITC CAR and the subject has been administered a FITC ligand to mark cancer cells in the subject, wherein the ligand specifically binds to a molecule expressed on the tumor. In some or any embodiments, the FITC ligand is FITC-folate or FITC-folate.

[0111] In some or any embodiments, the method comprises administering to the subject a non-physiological ligand for a synthetic cytokine receptor.

[0112] In some or any embodiments, the non-physiological ligand is rapamycin or a rapamycin analog. In some embodiments, the rapamycin analog is a rapamycin analog (rapalog).

[0113] In some or any embodiments, the non-physiological ligand is administered at a dose of 1 mg to 100 mg. In some or any embodiments, the non-physiological ligand is administered at a dose of 10-100 mg. In some or any embodiments, 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 above doses.

[0114] In some or any embodiments, multiple doses of a non-physiological ligand are administered to a subject. In some or any embodiments, the multiple doses are administered intermittently or periodically after administration of the iCIL or population or composition thereof to the subject. In some embodiments, the doses are administered over a predetermined time period. In some or any embodiments, two to eight doses of a non-physiological ligand are administered to a subject. In some embodiments, a single dose of a non-physiological ligand is administered to a subject.

[0115] In some or any embodiments, the iCIL population or composition thereof is administered at a dose of at or about 1 x 10 8iCIL cells up to 100x10 9 In some embodiments, the iCIL population or composition thereof is greater than or about 5 x 10 9 In some embodiments, the dose is from or about 5x10 9 iCIL cells are approximately 100 x 10 9 iCIL cells.

[0116] In some aspects, the present disclosure provides a kit comprising a cell population as described herein and instructions for administering the cell population to a subject in need thereof. In some embodiments, the kit comprises a container comprising 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 suffers from cancer.

[0117] In some aspects, the present invention provides a population of induced cytotoxic innate lymphoid (iCIL) cells, wherein the iCIL is a mature iCIL that expresses CD56 and LFA1, and wherein: at least 25% of the iCIL express a cytotoxic receptor; no more than 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 iCILs express NKG2D. In some embodiments, no more than 75% of the iCILs express a dysfunctional receptor. In some embodiments, the dysfunctional receptor is one or more of KLRG1, CD73, and CD38. In some embodiments, no more than 45%, no more than 40%, no more than 35%, no more than 30%, no more than 25%, no more than 20%, no more than 15%, no more than 10%, or no more than 5% of the iCILs express KLRG1. In some embodiments, no more than 10%, no more than 9%, no more than 8%, no more than 7%, no more than 6%, no more than 5%, no more than 4%, no more than 3%, no more than 2%, or no more than 1% of the iCILs express CD73. In some embodiments, no more than 75%, no more than 65%, no more than 55%, no more than 45%, no more than 35%, no more than 25%, no more than 15%, or no more than 5% of the iCILs express CD38.

[0118] 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- cells.

[0119] In some embodiments, the iCIL further comprises a synthetic cytokine receptor for a non-physiological ligand, wherein the cytokine receptor comprises: a synthetic γ chain polypeptide and a synthetic β chain polypeptide, wherein the synthetic γ chain polypeptide comprises a first dimerization domain, a first transmembrane domain, and an interleukin-2 receptor subunit γ (IL-2RG) intracellular domain; and the synthetic β chain polypeptide comprises a second dimerization domain, a second transmembrane domain, and an intracellular domain, wherein the intracellular domain is selected from the group consisting of an interleukin-2 receptor subunit β (IL-2RB) intracellular domain, an interleukin-7 receptor subunit β (IL-7RB) intracellular domain, and / or an interleukin-21 receptor subunit β (IL-21RB) intracellular domain. In some embodiments, the first dimerization domain and the second dimerization domain are extracellular domains.

[0120] In some embodiments, the synthetic γ chain polypeptide comprises, in N- to C-terminal order, a first dimerization domain, a first transmembrane domain, and an interleukin-2 receptor subunit γ (IL-2RG) intracellular domain, and the synthetic β chain polypeptide comprises, in N- to C-terminal order, a second dimerization domain, a second transmembrane domain, and an intracellular domain.

[0121] In some embodiments, the IL-2RG intracellular domain comprises a polypeptide sequence at least 95% identical to SEQ ID NO: 1, or a polypeptide sequence as 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 at least 95% identical to SEQ ID NO: 8 or 31, or a polypeptide sequence as set forth in SEQ ID NO: 8 or 31. In some embodiments, the β chain intracellular domain comprises the IL-2RB intracellular domain. In some embodiments, the IL-2RB intracellular domain comprises a polypeptide sequence at least 95% identical to SEQ ID NO: 2, or a polypeptide sequence as set forth in SEQ ID NO: 2. In some embodiments, the β chain intracellular domain comprises the IL-7RB intracellular domain. In some embodiments, the IL-7RB intracellular domain comprises a polypeptide sequence at least 95% identical to SEQ ID NO: 3, or a polypeptide sequence as set forth in SEQ ID NO: 3. In some embodiments, the β chain intracellular domain comprises the IL-21RB intracellular domain. In some embodiments, the IL-21RB intracellular domain comprises a polypeptide sequence at least 95% identical to SEQ ID NO: 4, or a polypeptide sequence as set forth in SEQ ID NO: 4. In some embodiments, the second transmembrane domain comprises a transmembrane domain from the same β chain intracellular domain. In some embodiments, wherein the second transmembrane domain is the intracellular domain of IL-2RB, comprising a polypeptide sequence at least 95% identical to SEQ ID NO: 35 or 36, or a polypeptide sequence as set forth in SEQ ID NO: 35 or 36. In some embodiments, the synthetic γ chain polypeptide comprises 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

[0122] The synthetic β chain polypeptide contains an IL-2RB TM domain comprising the sequence shown in SEQ ID NO: 35 or 36, and an IL-2RB intracellular domain comprising the sequence shown in SEQ ID NO: 2.

[0123] In some embodiments, the first dimerization domain and the second dimerization domain are heterologous dimerization domains selected from the group consisting of a 12 kD FK506 binding protein (FKBP) and a FKBP12-rapamycin binding (FRB) domain; and / or the non-physiological ligand is rapamycin or a rapamycin analog. In some embodiments, the FRB domain comprises a polypeptide sequence at least 95% identical to SEQ ID NO: 6 or SEQ ID NO: 7. In some embodiments, the FRB domain comprises a polypeptide sequence as shown in SEQ ID NO: 6 or SEQ ID NO: 7. In some embodiments, the first dimerization domain and the second dimerization domain are heterologous dimerization domains selected from the group consisting of a 12 kD FK506 binding protein (FKBP) and a calcineurin domain; and / or the non-physiological ligand is FK506 or an analog thereof.

[0124] 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 a polypeptide sequence as set forth in SEQ ID NO: 5 or SEQ ID NO: 30. In some embodiments, the synthetic gamma chain polypeptide has an amino acid sequence as 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 an amino acid sequence as 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 an amino acid sequence as set forth in SEQ ID NO: 28, and the synthetic beta chain polypeptide has an amino acid sequence as set forth in SEQ ID NO: 33.

[0125] In some embodiments, the first dimerization domain and the second dimerization domain are homologous dimerization domains selected from the group consisting of: i) FK506 binding protein (FKBP) of 12 kD; ii) cyclophilin A (CypA); or iii) gyrase B (CyrB); and the non-physiological ligand is: i) FK1012, AP1510, AP1903, or AP20187, or an analog thereof; ii) cyclosporine-A (CsA) or an analog thereof; or iii) kumarin or an analog thereof. In some embodiments, the population comprises approximately 1 x 10 6 to about 1x10 12 iCIL, about 1x10 6 to about 1x1010 iCIL, about 1x10 6 to about 1x10 8 iCIL, about 1x10 8 to about 1x10 12 iCIL, about 1x10 8 to about 1x10 10 iCIL or about 1x10 10 to about 1x10 12 In some embodiments, 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.

[0126] In some aspects, provided herein is a pharmaceutical composition comprising any population of iCILs according to any of the embodiments provided herein. In some embodiments, the pharmaceutical composition further comprises a cryoprotectant. In some aspects, provided herein is a cryopreservation composition comprising any population of iCILs according to any of the embodiments provided herein. In some embodiments, the composition comprises about 1 x 10 6 to about 1x10 12 iCIL, about 1x10 6 to about 1x10 10 iCIL, about 1x10 6 to about 1x10 8 iCIL, about 1x10 8 to about 1x10 12 iCIL, about 1x10 8 to about 1x10 10 iCIL or about 1x10 10 to about 1x10 12In some embodiments, 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.

[0127] In some aspects, provided herein is a method of killing a target cell or inhibiting the proliferation of a target cell, comprising contacting the target cell with a population of iCILs according to any one of the embodiments provided herein or a composition according to any one of the embodiments provided herein.

[0128] In some embodiments, the target cell is a cancer cell. In some embodiments, the iCIL further comprises a synthetic cytokine receptor for a non-physiological ligand, and the method comprises contacting the target cell with the non-physiological ligand for 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 an analog of rapamycin. In some embodiments, the analog of rapamycin is a rapamycin analog (rapalog). In some embodiments, the non-physiological ligand is contacted at a concentration of 5nM-200nM, 5nM-150nM, 5nM-100nM, 5nM-50nM, 5nM-20nM, 5nM-10nM, 10nM-200nM, 10nM-150nM, 10nM-100nM, 10nM-50nM, 10nM-20nM, 20nM-200nM, 20nM-150nM, 20nM-100nM, 20nM-50nM, 50nM-200nM, 50nM-150nM, 50nM-100nM, 100nM-200nM, 100nM-150nM, or 150nM-200nM. 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 culture medium at a concentration of 2.5 nM to 10 nM. In some embodiments, the non-physiological ligand is added to the culture medium at a concentration of 3 nM to 7 nM. In some embodiments, the method is performed in a subject, and the iCIL population or composition thereof is administered to the subject. In some embodiments, the iCIL further comprises a synthetic cytokine receptor for the non-physiological ligand, and the method comprises administering the non-physiological ligand to the subject.

[0129] In some aspects, provided herein is a method of inducing natural killer (NK) cell-mediated cell killing in a subject, comprising administering to the subject any effective amount of an iCIL population of any embodiment provided herein or any composition of any embodiment provided herein. In some aspects, provided herein is a method of treating cancer in a subject, comprising administering to the subject an effective amount of any iCIL population of any embodiment provided herein or any composition of any embodiment provided herein. In some embodiments, the subject has not been administered lymphodepletion therapy prior to administering the iCIL population or composition thereof.

[0130] In some embodiments, iCIL expresses a CAR targeting a subject's cancer cells. In some embodiments, CAR is an anti-FITC CAR, and the subject has been administered a FITC ligand to mark cancer cells in the subject's body, wherein the ligand specifically binds to molecules expressed on the tumor. In some embodiments, the FITC ligand is FITC-folic acid or FITC-folate. In some embodiments, iCIL also includes a synthetic cytokine receptor for a non-physiological ligand, and the method includes administering a non-physiological ligand for a synthetic cytokine receptor to the subject. In some embodiments, the non-physiological ligand is rapamycin or an analog of rapamycin. In some embodiments, the analog of rapamycin is a rapamycin analog (rapalog). In some embodiments, the non-physiological ligand is administered with the following dosage: 1 mg to 100 mg, optionally 10-100 mg, optionally for or about 10 mg, 20 mg, 25 mg, 30 mg, 40 mg, 50 mg or any value between any of the aforementioned doses. In some embodiments, multiple doses of non-physiological ligands are administered to the subject. In some embodiments, the multiple doses are administered intermittently or periodically, optionally for a predetermined period of time, after the iCIL population or composition thereof is administered to the subject. In some embodiments, two to eight 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.

[0131] In some embodiments, the iCIL population or composition thereof is at or about 1×10 8 iCIL cells are approximately 100 x 10 9 In some embodiments, the iCIL population or composition thereof is administered at a dose of greater than or about 5×10 9 administration of a dose of 5 x 10 iCIL cells, optionally wherein the dose is or is about 5 x 10 9 iCIL cells are approximately 100 × 10 9 In some embodiments, multiple doses of iCIL cells are administered to the subject. In some embodiments, multiple doses of iCIL cells are administered intermittently or periodically, optionally for a predetermined period of time.

[0132] In some embodiments, two to eight doses of iCIL cells are administered to a subject. In some embodiments, a single dose of iCIL cells is administered to a subject.

[0133] In some aspects, provided herein is a kit comprising any of the iCIL populations of any embodiment provided herein or any of the compositions of any embodiment provided herein, and instructions for administering the iCIL population or composition thereof to a subject in need thereof. In some embodiments, the iCIL further comprises a synthetic cytokine receptor for a non-physiological ligand, and the kit further comprises a container containing the non-physiological ligand, and instructions for administering the non-physiological ligand to a subject after administration of the iCIL population or composition thereof.

[0134] In some embodiments, the subject has cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0135] Figure 1A is a schematic diagram of an embodiment showing expansion of engineered cytotoxic innate lymphoid cells.

[0136] Figure 1B is a schematic diagram of an embodiment showing the differentiation of engineered stem or progenitor cells.

[0137] Figure 2A This figure depicts the RACR expansion of CD19 CAR hematogenous NK (bdNK) cells. The top panel shows the experimental timeline. The left panel shows the percentage of CAR expression in each group over time, and the right panel shows the total CAR+ NK cells over time.

[0138] Figure 2B Describes CAR expression analysis by flow cytometry. The upper panel shows CAR expression of bdNK cells expanded in the presence of IL-2 (1000 U / ml) or AP21967 (20 nM). The lower panel shows CD56 and CD16 expression of CAR+ cells.

[0139] Figure 2C The ability of RACR-expanded blood-derived NK cells to recognize and target tumor cells is depicted. The upper panel shows the timeline of the experiment. The upper right panel shows a flow cytometric plot at day 31 post-transduction, depicting CAR expression. 5 NK cells and 1x10 5Individual K562 cells, Nalm6 cells or Nalm6 CD19 knockout cells were incubated in 100 μl RPMI culture medium in 96-well plates in the presence of Brefeldin A, Monensin and anti-CD107 for 5 hours. AP21967 amplifies CD107A (left panel) and IFNγ (middle panel) expression levels on CAR+ cells (AP-20) or untransduced control groups. By comparing the total number of cells that survive in each well, the total number of cell trace purple+ cells and the total number of cells in the non-effector control wells, the death percentage (right panel) was calculated. NK effector cells were added with different effector cells: target cells (E: T) ratios.

[0140] Figure 3A Schematic diagram of the process of generating iPSC-derived CIL cells.

[0141] Figure 3B Graph depicting % CD34+ cells before and after cell selection and analysis on day 12 of the differentiation process.

[0142] Figure 3C Depicted is CD34+ selection by flow cytometry analysis at day 12 of the differentiation process.

[0143] Figure 3D The percentage of CD45+ cells among leukocytes and the percentage of leukocytes in progenitor cells and CIL cells are shown. The percentage of CD45+ cells is plotted as leukocyte %, CD7+ cells are plotted as progenitor cells, and CD56+ cells are plotted as CIL cells.

[0144] Figure 3E Flow cytometric analysis of differentiated leukocytes (CD45+), progenitor cells (CD45+ / CD5- / CD7+), and CIL cells (CD45+ / CD5- / CD7+ / CD56+) is described.

[0145] Figure 3F Depicted are the percentage of leukocytes or CIL cells at day 40 (left panel) and flow cytometric analysis of differentiated cells at day 40 (right panel).

[0146] Figure 3G Depicted are the percentages of iCIL cells harvested and immunophenotyped by flow cytometry for detection of the following markers: CD16, IL7R, KIR, NKp30, and NKp46 (left panel) and cytotoxicity as determined by % 4HLysis of K562 cells for different effector:target (E:T) ratios.

[0147] Figure 4A The timeline of the experiment is described.

[0148] Figure 4B Depicted are representative flow cytometric analyses of CD56+ cells and TagCAR (anti-fluorescein isothiocyanate (FITC) chimeric antigen receptor (CAR)) enrichment over the course of the experiment. The x-axis represents TagCAR detection via FITC-AF647, and the y-axis represents side scatter. Cells were analyzed on days 32, 35, 38, and 42 after "mock" transduction.

[0149] Figure 4C Depicted are the RACR enrichment (left panel), cell counts (middle panel), and percentages of cells expressing CD45 and CD56 (right panel) of the BXS cell line TagCAR cells over time.

[0150] Figure 4D Depicted are RACR enrichment (left panel), cell counts (middle panel), and the percentage of cells expressing CD45 and CD56 (right panel) of NHS cell line TagCAR cells over time.

[0151] Figure 5A A set of histograms depicts different activation markers in cytokine-differentiated mock CIL cells (BXS cell line) and RACR-iCIL cells (BXS and NH5 cell lines). Cells were gated based on CD45+CD7+CD5-, and then the activation markers CD56, CD16, NKp30, NKp40, and NKG2D were plotted.

[0152] Figure 5B The figure shows the cytotoxicity test of cytokine-differentiated simulated CIL cells (BXS cell line) and RACR-iCIL cells (BXS and NH5 cell lines) against MDA-mCherry tumor cell lines.

[0153] Figure 6A is a diagram describing the timeline of differentiation and expansion experiments, with experimental results such as Figures 6B-6E shown.

[0154] Figure 6B Depicted are the % VT103iCIL cells (left panel) and the total number of VT103iCIL cells (right panel) over time for cells treated with expansion medium containing IL-2, IL-15, IL-21, IL-18, IL-7 (cytokine cocktail) or with the A / C heterodimer AP21967 (AP 100 nM).

[0155] Figure 6C Figure 2 is a flow cytometry profile depicting cells stained as RACR-FRB positive as detected by mCherry.

[0156] Figure 6D is a set of graphs depicting the % FITC-CAR iCIL cells (upper panels) and the total number of FLCAR iCIL cells (lower panels) over time for cells transduced with viral constructs containing TagCAR-RACR-FRB (206) and FRB-RACR-TagCAR (205).

[0157] Figure 6E Figure 1 is a flow cytometry profile depicting cells that stain as FLCAR-positive.

[0158] Figure 7A Figure 2 is a flow cytometry profile depicting cells stained for CD56 and Tag-CAR.

[0159] Figure 7B Figure 2 is a set of histograms depicting different activation and cytotoxicity markers, including NKp30, NKp46, NKG2D, NKG2A, and CD57, in TagCAR+RACR-expanded iCIL cells, mock-IL2-expanded CIL cells, and unstained cells.

[0160] Figure 7C is a set of histograms depicting CD107a secretion by mock IL2-expanded CIL cells and TagCAR RACR-expanded iCIL cells in response to antigen.

[0161] Figure 8 is a schematic diagram of an exemplary CAR of the present disclosure, wherein the fusion protein is encoded by a lentiviral expression vector, and wherein "SP" is a signal peptide, the CAR is an anti-FITC CAR, the CD8α hinge is present, the transmembrane domain ("TM") is present, the costimulatory domain is 4-1BB, and the activation signaling domain is CD3ζ.

[0162] Figure 9: is a set of graphs that describe the differentiation factors involved along the timeline during the transition from iPSC to CIL cells. The differentiation factors involved in each differentiation stage are described in the drawn boxes; the bold font is a differentiation factor illustrating an embodiment of the present disclosure. In some embodiments, starting from week 4, cells are differentiated in a culture medium comprising IL7, IL15, SCF, FLT3L and UM729, optionally with or without rapamycin analogs. Starting from week 6, these cells are expanded in a culture medium comprising IL7, IL15, SCF, FLT3L, UM729 and CD2 / NKp46 beads, optionally with or without rapamycin analogs. In some embodiments, starting from week 4, cells are differentiated in a culture medium comprising SCF, FLT3L and UM729, optionally with or without rapamycin analogs. Starting from week 6, these cells are expanded in a culture medium comprising SCF, FLT3L, UM729 and CD2 / NKp46 beads, optionally with or without rapamycin analogs. In some embodiments, starting from week 4, cells are differentiated in a medium comprising IL7, IL15, and UM729, optionally with or without a rapamycin analogue. Starting from week 6, these cells are expanded in a medium comprising IL7, IL15, UM729, and CD2 / NKp46 beads, optionally with or without a rapamycin analogue. In some embodiments, starting from week 4, cells are differentiated in a medium comprising UM729, optionally with or without a rapamycin analogue. Starting from week 6, these cells are expanded in a medium comprising UM729 and CD2 / NKp46 beads, optionally with or without a rapamycin analogue.

[0163] Figure 10 It is the percentage of RACR CD19-CAR positive cells as a function of culture days. Hematogenous NK cells were transduced using viruses containing CD19-CAR-RACR (IL-2RG / IL-2RB; RACR2), CD19-CAR-RACR (IL-2RG / IL-7RB; RACR7) or CD19-CAR-RACR (IL-2RG / IL-21RB; RACR21). The cells were then amplified in complete medium at 100 IU / mL human IL-2 or 100 nM AP219667, with membrane-bound IL-21 (mbIL-21) 41BBL and K562 feeder cells added weekly. Among AP219667, RACR (IL-2RG / IL-2RB) "RACR2" supported the highest amplification, and RACR (IL-2RG / IL-7RB) "RACR7" and RACR (IL-2RG / IL-21RB) "RACR21" supported RACR amplification to a lower extent.

[0164] Figure 11 Depicted are the enrichment of RACR in CAR-expressing cells over time (left panel), the fold change in progenitor cells (0-4 weeks) and the fold change in RACR-iCIL cells (4-9 weeks) (middle panel), and the cytotoxicity measured by the % killing of K562 cells by iCIL and RACR-iCIL cells at different effector cell: target cell (E:T) ratios (right panel). The top of the figure depicts a schematic diagram of hematopoietic cell line differentiation and corresponding cell culture.

[0165] Figure 12A Depicted are the % of cells containing the markers CD45+CD5-, CD45+CD5-CD7+, or CD45+CD5-CD7+CD56+ for cells thawed on day 26 or analyzed on day 29 post-transduction.

[0166] Figure 12B Depicted are the % of CAR+ cells containing the markers CD45+CD5-, CD45+CD5-CD7+, or CD45+CD5-CD7+CD56+ in cells thawed and mock-transduced on day 26 or analyzed on day 29 after viral vector transduction.

[0167] Figure 13A Figure 2 is a graph depicting the percentage of CAR+ iCIL cells after analysis of cells placed in medium containing IL7, IL15, SCF, FLT3L, and UM729 (complete mix), IL7, IL15, and UM729 (IL7 / IL15), or UM729 (none). Each medium condition was treated with or without a rapamycin analog.

[0168] Figure 13B Figure 2 is a graph depicting the fold change (FC) in the percentage of CAR+iCIL cells analyzed in culture medium containing IL7, IL15, SCF, FLT3L, and UM729 (complete mix), IL7, IL15, and UM729 (IL7 / IL15), or UM729 (none). Each culture medium condition was treated with or without a rapamycin analog.

[0169] Figure 13C Figure 2 is a graph depicting the total number of CAR+ iCIL cells analyzed after culture in media containing IL7, IL15, SCF, FLT3L, and UM729 (complete mix), IL7, IL15, and UM729 (IL7 / IL15), or UM729 (none). Each culture medium condition was treated with or without a rapamycin analog.

[0170] Figure 13DFigure 2 is a graph depicting the fold change (FC) in the total number of CAR+iCIL cells after analysis of cells placed in medium containing IL7, IL15, SCF, FLT3L, and UM729 (complete mix), IL7, IL15, and UM729 (IL7 / IL15), or UM729 (none). Each medium condition was treated with or without a rapamycin analog.

[0171] Figure 14A Figure 1 is a set of histograms depicting the expression of various markers, including CD45, CD7, and CD56, in TagCAR+RACR-iCIL cells cultured in media containing IL7, IL15, SCF, FLT3L, and UM729 (complete mix), SCF, FLT3L, and UM729 (SCF / FLT3L), IL7, IL15, and UM729 (IL7 / IL15), UM729 (none), or unstained cells. Each media condition was treated with or without a rapamycin analog.

[0172] Figure 14B is a histogram depicting CD56 markers in progenitor cells analyzed on day 26 (CD56-) and cells analyzed on day 40 (CD56+) placed in medium containing IL7, IL15, SCF, FLT3L and UM729 (complete mix).

[0173] Figure 15A Schematic diagram showing CRISPR-Cas-mediated site-specific knock-in of a construct encoding RACR.

[0174] Figure 15B is a graph showing RACR mRNA detection after knock-in on different promoters, including EEF1A1 (locus 1), ACTB (locus 2), and B2M-EF1a.

[0175] Figure 15C Shown is a set of histograms depicting RACR protein detection after knock-in at different promoters.

[0176] Figure 15D is a graph showing RACR protein detection after knock-in at different promoters, including locus 1 (EEF1A1) and locus 2 (ACTB).

[0177] Figure 15E is a graph depicting the mismatch response of CD8 T cells after B2M gene knockout.

[0178] Figure 16A Schematic diagram showing the role of FKBP12 in rapamycin inhibition of proliferation via mTOR.

[0179] Figure 16B are graphs showing protection of a polyclonal FKBP12 knockout (KO) line from rapamycin-mediated inhibition of iPSC proliferation (left panel), and phase contrast images of morphology in wild-type and FKBP12 KO cells (right panel).

[0180] Figure 16C is a graph showing the confluence of wild-type iPSCs after four days of treatment with different doses of rapamycin.

[0181] Figure 16D is a graph showing the confluence of FKBP12 KO iPSC growth during four days of treatment with different doses of rapamycin.

[0182] Figure 16E is a graph showing the ratio of hematopoietic progenitor cells (HPs) to iPSCs in cloned FKBP12 KO iPSCs compared to control iPSCs.

[0183] Figure 16F is a graph showing the confluence of FKBP12 KO iPSCs grown during four days of treatment with 25 nM rapamycin.

[0184] Figure 17A is a graph showing the yield of hematopoietic progenitor cells obtained from iPSCs genetically engineered to express RACR after treatment with various differentiation factors relative to unmodified iPSCs.

[0185] Figure 17B Figure 2 is a flow cytometric graph depicting triple-positive HP cells derived from RACR-engineered iPSCs after treatment with or without SR-1.

[0186] Figure 17C HP yields in RACR-engineered FKBP12 KO iPSCs in the presence ("on") or absence ("off") of rapamycin, as well as in iPSCs derived by conventional techniques (Schemes 1 and 2), are described.

[0187] Figure 17D Shown are flow cytometric plots depicting the phenotypic analysis of HP cell markers CD43, CD45, CD34, and CD38. Also shown are unstained controls.

[0188] Figure 18A The graph shows the percentage of CD45+CD5-CD56+LFA1+ wild-type iNK cells and RACR-iCIL cells after 37 days of culture, with or without the addition of pre-activation medium.

[0189] Figure 18BShown is the immunophenotyping comparing iPSC-derived NK cells and RACR-iCIL.

[0190] Figure 18C Shown are line graphs depicting the fold increase in RACR-iCILs one to two months after harvest following rapamycin and cytokine stimulation.

[0191] Figure 18D Flow cytometry plots showing cell purity and upregulation of LFA-1+ / CD56+ cells and NKp46+ / NKGD+ cells one to two months after harvest.

[0192] Figure 18E Shown are killing curves of RACR-iCIL targeting MDA-MBA-231 cells one or two days after harvest.

[0193] Figure 19A Graph showing the number of tumor cells after incubation with untreated iCIL, cytokine-stimulated iCIL, or RACR-stimulated iCIL.

[0194] Figure 19B Graph showing the proliferation of RACR-iCILs at day 47 upon treatment with different cytokines or with rapamycin analogs.

[0195] Figure 19C Shown are graphs depicting cytotoxicity as measured, for example, by % 4HLysis of MDA tumor cells for different effector to target (E:T) ratios.

[0196] Figure 19D Figure 2 shows tumor cell growth after incubation of breast cancer cells with unstimulated iCIL ("RACR-iCIL+RACR-OFF"), cytokine-stimulated iCIL ("RACR-iCIL+IL2 / IL15"), or RACR-stimulated iCIL ("RACR-iCIL+RACR-ON"). Breast cancer cells not incubated with iCIL are represented by bold, black lines. Tumor cells were reintroduced (i.e., tumor rechallenge) as indicated by the arrows.

[0197] Figure 20A Figure 2 shows the growth of tumor cells after incubation of ovarian cancer cells with unstimulated NK cells, cytokine-stimulated NK cells, or RACR-stimulated NK cells. Ovarian cancer cells not incubated with NK cells are represented by bold, black lines. Tumor cells were reintroduced 50 hours later (i.e., tumor cell re-challenge).

[0198] Figure 20BFigure 2 shows a graph of tumor cell growth after incubation of bladder cancer cells with unstimulated NK cells, cytokine-stimulated NK cells, or RACR-stimulated NK cells. Bladder cancer cells not incubated with NK cells are represented by bold, black lines. Tumor cells were reintroduced at 50 and 100 hours (i.e., tumor cell re-challenge).

[0199] Figure 20C Figure 2 shows the growth of tumor cells after incubation of breast cancer cells with unstimulated NK cells, cytokine-stimulated NK cells or RACR-stimulated NK cells. Breast cancer cells not incubated with NK cells are represented by bold, black lines. Tumor cells were reintroduced 40 hours later (i.e., tumor cells were challenged again).

[0200] Figure 20D Graphs showing growth of RACR-NK cells after incubation without rapamycin or cytokines (unstimulated NK), with rapamycin (RACR-stimulated NK), or with cytokines (cytokine-stimulated NK).

[0201] Figure 21A Figures depicting tumor cell killing of tumor cells incubated with RACR-engineered iCILs and no CAR antigen, RACR-engineered iCILs and moderate CAR antigen expression, or RACR-engineered iCILs and high CAR antigen expression are shown. The CAR antigen is specific for FITC conjugated to folate, which binds to folate receptors on tumor cells.

[0202] Figure 21B Figure 3 depicts the function of iPSC-derived NK cells and CAR- and RACR-engineered iPSC-derived CILs. CAR-RACR-iCIL cells secrete CD107a in response to antigens recognized by the cells.

[0203] Figure 21C Graphs showing growth of RACR-CAR-NK cells after incubation without rapamycin or cytokines (unstimulated NK), with rapamycin (RACR-stimulated NK), or with cytokines (cytokine-stimulated NK).

[0204] Figure 22A The timeline of the in vivo mouse model of breast cancer is shown. Five days before the start of the experiment, MDA-231mCherry / luciferase cells were injected into mice. One day before the start of the experiment, FITC-folic acid (salt) was injected subcutaneously into mice for two weeks. On the first day (D0) of the experiment, RACR-TagCAR-NK-92 cells were injected into the peritoneum of mice, followed by treatment with rapamycin three times a week; or treated with IL2 / IL15 three times a week.

[0205] Figure 22B Shown are graphs depicting the quantification of luminescence associated with tumor growth in mice receiving various treatments over three weeks.

[0206] Figure 22C Shown are raw luminescence images of tumor growth in mice receiving various treatments over three weeks.

[0207] Figure 22D Described Figure 22C RACR-NK in the blood of mice.

[0208] Figure 22E Described Figure 22C RACR-NK detection in mouse tissues.

[0209] Figure 23 Depicted are the ratios of hematopoietic progenitor (HP) cells to iPSC cells in day 14 3D suspension cultures generated after differentiation of unedited iPSCs (no RACR) or RACR-edited iPSCs in the presence (+Rapa) or absence (-Rapa) of rapamycin.

[0210] Figure 24A Fold expansion of iCILs from RACR-engineered iPSCs compared to unmodified iPSCs generated using commercially available standard cell culture protocols in the presence ("on") or absence ("off") of rapamycin is depicted.

[0211] Figure 24B Phenotypic analysis of iCIL markers CD56, LFA1, NKG2D, NKp46, NKp30, and DNAM1 in iCILs generated from RACR-engineered iPSCs is described.

[0212] Figure 24C Depicted are the fold expansion of iCILs generated from RACR-engineered iPSCs with FKBP12 KO in the presence ("on") or absence ("off") of rapamycin compared to unmodified iPSCs generated using standard commercial cell culture protocols.

[0213] Figure 24D Depicted are the fold expansion of iCILs generated from RACR-engineered FKBP12KO iPSCs in the presence ("on") or absence ("off") of rapamycin compared to CILs generated using standard commercial cell culture protocols.

[0214] Figure 24E Phenotypic analysis of iCIL markers CD45, CD56, LFA1, and FSC-A at day 40 of differentiation is described.

[0215] Figure 25Figure 3. Tumor cell killing by RACR iCILs in vitro when co-cultured with MDA-MB-231 breast cancer cells. Breast cancer cells were cultured alone and maintained without, treated with, or cytokines or rapamycin analogs (left panel) or co-cultured with RACR iCILs and maintained without, treated with, or cytokines or rapamycin analogs (right panel).

[0216] Figure 26A A timeline of an in vivo mouse model of breast cancer is described. Five days prior to the start of the experiment, mice were injected with MDA-231 mCherry / luciferase cells. One day prior to the start of the experiment, mice were imaged, and blood was drawn weekly during the experiment. On the first day of the experiment (D0), mice were injected intraperitoneally with RACR-iCIL cells and subsequently treated with rapamycin three times per week or IL2 / IL15 three times per week.

[0217] Figure 26B Depicted are raw luminescence images of tumor growth in mice that received multiple treatments over three weeks.

[0218] Figure 27A A timeline of the in vivo mouse model of breast cancer is described. Five days before the start of the experiment, mice were injected with MDA-231 mCherry / luciferase cells. One day before the start of the experiment, mice were imaged weekly for the duration of the experiment. On day 1 (D0), mice were injected with RACR-iCIL cells and subsequently treated with rapamycin three times a week or IL2 / IL15 three times a week. On day 27, mice were injected with RACR-iCIL cells again.

[0219] Figure 27B Depicted are raw luminescence images of tumor growth in mice that received multiple treatments over six weeks.

[0220] Figure 27C Described Figure 27B Absolute quantification of raw luminescence as described in .

[0221] Figure 27D Described Figure 27B The percentage of mouse body weight loss is described in .

[0222] Figure 27E Shown Figure 27B Survival graph of mice described in .

[0223] Figure 28A Shown is a heat map of hematopoietic stem cell (HSC) genes differentially expressed in RACR-engineered FKBP12 KO iPSC-derived hematopoietic progenitor (HP) cells.

[0224] Figure 28BShown are flow cytometric plots depicting EPCR+ / CD90+ cells at day 9 of differentiation.

[0225] Figures 28C-28E Describes the differentiation day 9 ( Figure 28C ), Day 12( Figure 28D ) and day 15 ( Figure 28E ) Myeloid and erythroid potency of HP cells derived from RACR-engineered HSCs.

[0226] Figure 29A Shown are principal component analyses of RNA in RACR-engineered iPSC-derived iCILs relative to: RACR-engineered iPSC-derived hematopoietic progenitors (HP); peripheral blood-derived NK (bdNK) cells; cord blood-derived NK (cbNK) cells; iNK cells; cord blood CD34+ HSCs (cbCD34+); and the number of feeder cell stimulations (e.g., feeder 1 or feeder 2).

[0227] Figure 29B A heat map showing genes differentially expressed in RACR-engineered iPSC cell-derived iCIL cells compared to NK cells, including iPSC-derived NK cells (iNK), blood-derived NK cells (bdNK), and cord blood-derived NK cells (cbNK).

[0228] Figure 29C The expression of Fc receptors and genes associated with the CD3 complex in iCIL cells derived from RACR-engineered iPSCs compared to HP cells and NK cells, including blood-derived NK cells (bdNK). Each condition depicted on the x-axis is grouped from left to right as follows: hematopoietic progenitor cells (HP), iCIL, and blood-derived NK cells (BD NK).

[0229] Figure 29D Figure 2 shows the gene expression of cytokine receptors in iCIL cells derived from RACR-engineered iPSC cells compared to HP cells and NK cells, including blood-derived NK cells (bdNK). Each condition described on the x-axis is grouped from left to right as follows: hematopoietic progenitor cells (HP), iCIL, blood-derived NK cells (BD NK).

[0230] Figure 29E Figure 2 shows the gene expression of KIR receptors in iCIL cells derived from RACR-engineered iPSC cells compared to HP cells and NK cells, including blood-derived NK cells (bdNKs). Each condition is grouped from left to right on the x-axis as follows: hematopoietic progenitor cells (HP), iCIL, blood-derived NK cells (BD NK).

[0231] Figure 29F Figure 2 shows gene expression in iCIL cells derived from RACR-engineered iPSCs compared to HP cells and NK cells, including blood-derived NK cells (bdNK). Each condition depicted on the x-axis is grouped from left to right as follows: hematopoietic progenitor cells (HP), iCIL, blood-derived NK cells (BD NK).

[0232] Figure 30A The percentage of cells expressing cytotoxic receptors is shown. Specifically, the percentages of bdNK cells, iCIL cells, and iNK cells+feeder cells that are NKp30+ / CD56+ / LFA1+, NKp46+ / CD56+ / LFA1+, and NKG2D+ / CD56+ / LFA1+ are depicted.

[0233] Figure 30B The percentage of cells expressing dysfunctional receptors is shown. Specifically, the percentages of bdNK cells, iCIL cells, and iNK cells+feeder cells that are KLRG1+ / CD56+ / LFA1+, CD73+ / CD56+ / LFA1+, and CD38+ / CD56+ / LFA1+ are depicted.

[0234] Figure 30C Shown are the percentages of CD56+ / LFA1+bdNK cells, iCIL cells, and iNK+ feeder cells that are highly proliferative (CD56brightCD57-), transitional (CD56dimCD57-), and senescent (CD57+CD56dim).

[0235] Figure 30D Shown are histograms depicting different markers in iNK cells+feeder cells, iCILs, and bdNK cells, including cytotoxicity receptors (NKG2D, NKp30, NKG2C, NKp46), phenotypic markers (NKG2A, CD161, CD96), and dysfunction markers (KLGR1, CD73, CD57).

[0236] Figure 31A Figure 2 shows a 200-hour in vitro sequential killing assay of MDA-MB-231 breast cancer cells by iCIL, bdNK, and iNK+ feeder cells. Continuous killing was determined by the percentage of remaining cells. Arrows indicate the time period during which breast cancer cells were reintroduced.

[0237] Figure 31B A graph depicting the growth of effector cells (iCIL, bdNK, feeder-iNK) over 200 hours is shown. Arrows indicate the time period of reintroduction into breast cancer cells.

[0238] Figure 32A timeline describing the differentiation and expansion process starting from RACR-engineered iPSCs, differentiation into iCILs, and expansion of iCIL cells. Figure 33 Schematic diagram of the differentiation factors involved in the conversion of iPSCs to CIL cells in an example process ("Version 5") compared to a total control. The differentiation factors involved in each differentiation stage are marked with boxes and correspond to the timeline marked on the y-axis.

[0239] Figure 34 Schematic diagram depicting the temporal generation of hematopoietic progenitor cells (HP) and iCILs from RACR-engineered iPSCs.

[0240] Figure 35A Shown is a graph depicting the number of hematopoietic progenitor cells (HP) per iPSC cell in response to cell confluence.

[0241] Figure 35B The graph shows the relationship between the number of iCIL cells per iPSC cell and the cell confluence at the start of differentiation.

[0242] Figure 35C Graph showing the fold expansion of hematopoietic progenitor cells (HP) in wells seeded with iPSC cells at different densities.

[0243] Figure 36A Shown are graphs depicting the fold expansion of hematopoietic progenitor cells (HP) in various culture media.

[0244] Figure 36B Graph showing fold expansion of hematopoietic progenitor cells (HP) added with UM729 and SR1 on day 6 compared to day 9.

[0245] Figure 37A Shown are graphs depicting the fold expansion of hematopoietic progenitor (HP) cells upon removal of rapamycin at different stages.

[0246] Figure 37B Shown is a graph depicting the fold expansion of hematopoietic progenitor cells (HP) at varying rapamycin concentrations in culture.

[0247] Figure 38A Shown are graphs depicting the fold expansion of iCIL cells at day 33 from cultures deprived of various differentiation factors during the differentiation phase.

[0248] Figure 38B Shown is a graph depicting the fold expansion of hematopoietic progenitor (HP) cells at day 15 after deprivation of various differentiation factors in culture during the differentiation phase.

[0249] Figure 39AShown is a graph depicting the fold expansion at day 40 after iCIL cells were differentiated and expanded in a bioreactor at day 15.

[0250] Figure 39B Shown is a graph depicting the fold expansion of iCIL cells at different seeding densities in a bioreactor.

[0251] Figure 39C Shown is a graph depicting the fold expansion at day 35 when iCIL cells were differentiated and expanded in a vertical wheel bioreactor compared to a GRex bioreactor.

[0252] Figure 39D Shown is a graph depicting the fold expansion at day 35 of iCIL cells differentiated and expanded in a stirred tank bioreactor compared to a vertical wheel bioreactor.

[0253] Figure 40 Shown are graphs depicting the fold expansion of iCIL cells using different culture media during the iCIL differentiation stages.

[0254] Figure 41 Shown is a graph depicting the fold expansion of iCIL cells when different concentrations of rapamycin were used during the iCIL differentiation stage.

[0255] Figure 42A Shown is a graph depicting the fold expansion of iCIL cells in which IL7 and FLT3L were removed from the culture medium during the iCIL differentiation stage. Each condition depicted on the x-axis is grouped from left to right as follows: clone 3.1, clone 56.1.

[0256] Figure 42B Shown are graphs depicting the cytotoxicity of iCIL cells to tumor cells from which IL7 and FLT3L were depleted in the culture medium during the iCIL differentiation stage.

[0257] Figure 43 Shown is a graph depicting the fold expansion of iCIL cells supplemented with SCF and IL-15 in the culture medium at days 21-28 of the iCIL differentiation stage.

[0258] Figure 44 Shown is a graph depicting the fold expansion of iCIL cells supplemented with SCF and IL-15 in the culture medium at day 24-28 of the iCIL differentiation stage.

[0259] Figure 45 Shown are graphs depicting the fold expansion of iCIL cells after removal of various differentiation and expansion factors from the culture medium during HP differentiation and iCIL differentiation stages.

[0260] Figure 46Shown is a graph depicting the percentage of tumor remaining after in vitro co-culture of RACR iCILs with MDA-MB-231 breast cancer cells.

[0261] Figure 47A Flow cytometry showed the expression of LFA-1 and CD19 in RACR-expanded iCIL cells.

[0262] Figure 47B Shows the use of iCIL cells in a bioreactor Figure 32 and Figure 33 Figure 5 shows a graph of the fold-amplification following amplification using the exemplary protocol described in version 5.

[0263] Figure 48A Shown is a graph depicting the percentage of tumor remaining after in vitro co-culture of RACR iCILs with MDA-MB-231 breast cancer cells with or without cytokines and rapamycin.

[0264] Figure 48B Shown is a graph depicting the percentage of tumor remaining after in vitro co-culture of RACR iCILs with SW620 colorectal cancer cells with or without cytokines and rapamycin.

[0265] Figure 49A Shown is a graph depicting the percentage of tumor remaining after in vitro co-culture of RACR iCILs with Burkitt's lymphoma tumor cells with or without cytokines, rapamycin, and / or rituximab.

[0266] Figure 49B Shown is a graph depicting the percentage of tumor remaining after in vitro co-culture of RACR iCILs with non-Hodgkin's lymphoma tumor cells with or without cytokines, rapamycin, and / or rituximab.

[0267] Figure 50 Shown are graphs depicting the percentage of residual tumor after in vitro co-culture of RACR iCILs with ten tumor cell types with or without cytokines.

[0268] Figure 51 Shown are graphs depicting the percentage of residual tumor following in vitro co-culture of RACR iCILs with triple-negative breast cancer cells in a serial killing assay (left panel). Figure 51 , right panel shows raw luminescence images of tumor growth co-cultured with RACR iCILs.

[0269] Detailed description of the invention

[0270] Induced pluripotent stem cells (iPSCs) are a renewable, modifiable, and scalable source of material for cell therapy manufacturing. However, current approaches to iPSC-based cell therapies present significant challenges. Specifically, current approaches to differentiating iPSCs into therapeutic immune cell types require exogenous growth factors and, in some cases, the presence of feeder cells.

[0271] iPSCs can be made by reprogramming adult cells into a cell state similar to embryonic stem cells. iPSCs are believed to be able to differentiate into all cell types in the human body and have unlimited amplification capacity, which means they can reproduce and proliferate indefinitely, producing almost endless starting materials. In addition, iPSCs are amenable to precise multiple genome editing and can safely introduce multiple genetic modifications. Due to these characteristics, iPSCs provide consistent starting material derived from a single cell (clone), thereby maintaining consistent genomic integrity of processing intermediates and final cell products.

[0272] Current cell therapy manufacturing pathways involve engineering disease-targeted therapeutic cell products from autologous or allogeneic starting material. In some embodiments, allogeneic or "off-the-shelf" cell therapies have the potential to transform cell therapy from personalized medicine to conventional therapy. However, current "off-the-shelf" cell therapies struggle to demonstrate the same in vivo cell engraftment and persistence as approved autologous cell therapy products. This is due, in part, to the foreign nature of the engineered components of allogeneic or even autologous cells, which can be recognized and rejected by the host immune system. To date, cell therapy has been achieved by administering highly toxic chemotherapy regimens (known as lymphodepletion therapy (LD)) to patients prior to administering the cell therapy product. LD depletes the host immune system and offers numerous benefits to cell therapy products, primarily providing a readily available supply of "homeostatic cytokines" for the cell therapy product ex vivo and reducing the host immune response to the foreign graft. However, LD is only a temporary solution, and the host immune system rapidly reestablishes. Therefore, multiple rounds of LD and cell infusions are required to maintain allogeneic cell exposure. Alternatively, exogenous cytokines such as IL-2 can be administered, which offer shorter exposure times but are associated with high toxicity when used. Finding better ways to increase cell persistence is key to achieving sustained tumor remissions and has proven to be a challenge in the field of allogeneic cell therapy.

[0273] Allogeneic cells can be further subdivided into donor cells or iPSC-derived cells. Donor-derived cells are generally derived from the blood circulation or umbilical cord blood of healthy donors, and therapeutic cell types (such as natural killer cells or NK cells) are selected, subsequently harvested and amplified in a complex cell culture process, which generally includes a variety of cytokines, growth factors, genetic engineering and feeder cells to produce multiple doses. In addition, iPSC-derived cells also require a variety of complex cell culture conditions, which must be implemented in a step-by-step manner to promote the cells to undergo the necessary progenitor cell stage and ultimately obtain the desired final cell product (such as immune effector cells). For example, effective methods for clinical-scale CIL cell expansion are known to require exogenous cytokines, including IL-2, IL-15 and / or IL-7, as well as antigen molecules, co-stimulatory molecules and / or cell adhesion molecules. CIL cells are a type of cytotoxic lymphocyte characterized by the ability to distinguish between self and non-self cells by monitoring the expression of MHC class I molecules and releasing cytokines, and directly killing non-self cells or infected target cells. It is well known that CIL cells do not represent a homogeneous population. In contrast, CIL cells have many distinguishable subpopulations. In many studies, these exogenous factors are supplemented during the in vitro expansion of CIL cells and / or after the CIL cells are infused into the subject. Using currently known methods, the efficient expansion of CIL cells, which rely on a large number of different exogenous factors, often requires complex and expensive production processes. Another challenge in this field involves how to regulate the activity of endogenous CIL cells in vivo, especially considering that NK cell activity is significantly reduced in patients with cancer compared to healthy patients.

[0274] Furthermore, the gold standard in cell therapy is autologous chimeric antigen receptor (CAR) T-cell therapy. Beginning with the "first generation" of CAR T-cell therapies in the 1990s and culminating in the FDA approval of the first CAR T-cell therapies in 2017, decades of CAR T-cell therapy efforts have yielded success in treating B-cell malignancies, achieving long-term remissions in 30%-40% of certain patient populations. Importantly, the efficacy of CAR T cells requires lymphodepleting chemotherapy to eliminate the reservoir of survival factors such as IL-15. While CAR T-cell therapy has revolutionized the treatment of malignancies such as hematologic diseases, major limitations have hindered its widespread adoption. The field of allogeneic CAR T-cell therapy has shown promising early clinical results; however, compared to autologous CAR T-cell therapy, durable responses have generally been inferior, despite the use of increasingly intensive LD regimens. This may be due to limitations in the drug product cell type, manufacturing process, and anti-allogeneic transplant responses against the therapeutic cells. Therefore, despite the promising clinical efficacy of CAR T cells in hematologic malignancies, significant challenges remain, including patient accessibility, complex manufacturing processes, and high costs. The engineered CIL cells and related methods provided herein provide an "off-the-shelf" cancer therapy to overcome these challenges.

[0275] iPSCs can also be modified via CRISPR to express CARs, thereby overcoming challenges in targeting, for example, the heterogeneous microenvironment of solid tumors.

[0276] In general, iPSC-based cell therapies are generally inefficient in generating the necessary intermediate progenitor cells, resulting in low initial yields of therapeutic cell types such as cytotoxic innate lymphoid (CIL) cells, which then require feeder cell-driven expansion. This feeder cell-driven expansion significantly reduces the proliferation capacity of the final cell therapy product. Therefore, in order to achieve the necessary engraftment to have any therapeutic effect, high cell numbers (approximately 1 billion cells) and repeated doses are required in addition to repeated cycles of lymphodepleting chemotherapy.

[0277] The current approach to differentiating iPSCs into therapeutic immune effector cells, such as natural killer (NK) cells, requires complex growth factors and feeder cells to achieve sufficient yields. The synthetic receptor-activated differentiation (ShRED) provided herein is a directed differentiation and expansion process controlled by the rapamycin-activated cytokine receptor (RACR). RACR is activated by adding its synthetic ligand rapamycin, which induces JAK / STAT signaling, prompting cells to differentiate and expand into hematopoietic progenitor cells (HP), and then differentiate and expand into immune effector cells, known as RACR-induced cytotoxic innate lymphoid cells (RACR-iCIL). In addition, because rapamycin is a safe, effective and approved immunosuppressive therapeutic approach, RACR can also be engaged in vivo by rapamycin doses to increase the persistence of RACR-iCIL while protecting these cells from allogeneic rejection.

[0278] Provided herein is a platform for producing immune effector cells in the absence of exogenous cytokines and feeder cells by genetically modifying iPSC and iPSC-derived progenitor cells to express synthetic cytokine receptors. Synthetic small molecule ligands (such as rapamycin) activate receptors to drive the differentiation and amplification of immune effector cells. The compositions and methods provided herein include CIL cells expressing synthetic cytokine receptors. The non-limiting advantages of engineered CIL cells include: superior and controllable amplification effects when administered to a subject; similar cytotoxic activity compared to natural CIL cells; improved manufacturing processes for iPSC-derived cells and enhanced anti-tumor activity.

[0279] Improved cell manufacturing. In the aspects provided herein, the RACR engineering platform provided herein improves the manufacture of iPSC-derived cells by controlling cell production. Through the activation of rapamycin dosage and RACR, the reproducibility of the differentiation process is higher and the cell products are more homogeneous. The RACR engineering platform can also reduce manufacturing costs because it is free from the addition of expensive growth factors, cytokines and other raw materials after RACR activation. In certain embodiments, the method disclosed herein can further improve the amplification effect by the ability of the CIL cells described herein to amplify in the absence of exogenous factors or with less exogenous factors (such as without IL-2, IL-15 and / or IL-7). In some embodiments, compared with traditional processes, the method disclosed herein can further improve the differentiation and / or amplification ability by removing one or more exogenous factors to generate the iCIL cells described herein. For example, in some cases, the process described herein needs to reduce at least 7 exogenous factors. The RACR engineering platform increases the yield of high-purity intermediate and final cell products. The RACR engineering platform provided herein produces high-purity hematopoietic progenitor cells (HPs), intermediate progenitor cell populations, and high-purity and phenotypically mature results CIL. The RACR engineering platform improves cell compatibility for patients because the manufacturing process is completely feeder- and xeno-free. The RACR engineering platform is also compatible with suspension cells, increasing the scalability of cell production.

[0280] Among other aspects provided, the RACR engineering platform does not require additional physical manipulation of the differentiated progenitor cells. In conventional processes for progenitor cell differentiation, residual cell aggregates must be removed prior to blood cell differentiation. Physical manipulations include enzymatic digestion (e.g., collagenase or TrypLE TM In contrast, the RACR-engineered platform produced embryoid bodies that could be completely dissociated into pure HPs without the need for cell filtration.

[0281] Enhanced anti-tumor activity. In the aspects provided, the RACR engineering platform provided herein improves the anti-tumor activity of iPSC-derived cells by improving the transplantability, persistence, and effector function of the cells. The RACR engineering platform provided herein can also improve the anti-tumor activity of iPSC-derived cells by suppressing the host immune response through rapamycin, thereby further promoting the transplantation of cells (such as CIL). The RACR engineering platform provided herein can also activate the RACR system to selectively support RACR cell expansion and survival, thereby eliminating the need for the use of toxic LDs, thereby improving the anti-tumor activity of iPSC-derived cells.

[0282] In another aspect, the study results also demonstrated an unexpected finding: RACR-engineered iPSC-derived CIL (iCIL) cells expressed low levels of CD38, a target of certain therapeutic antibodies, such as daratumumab. A problem with many iPSC-derived cell therapies targeting certain cancer or tumor target antigens, such as CD38, is that iPSC-derived cells can express CD38. In fact, some have reported that NK cell compositions contain a large population of cells expressing a high proportion (e.g., greater than 90%) of CD38+ NK cells. CD38 expression in iPSC-derived cell therapies, such as NK cell therapies, can be a problem because when anti-CD38-targeting antibodies (e.g., daratumumab) bind to CD38, fratricidal killing occurs, resulting in ADCC leading to tumor and cell therapy elimination. In contrast, the results of the study demonstrated that the percentage of CD38+ cells in iCIL cells was significantly lower compared to traditional bdNK cells or iNK cells differentiated from feeder cells. These results support the utility of a combination therapy consisting of provided iCIL cells and anti-CD38 antibodies, which can enhance anti-tumor activity without causing iCIL cell cannibalism.

[0283] In some aspects, the RACR system on CIL, including engineered synthetic cytokine receptors and their activation with rapamycin or rapamycin analogs, has advantages including the ability to engineer an unlimited amount of starting material that can efficiently generate direct progenitor cells characterized by minimal expansion requirements for the final cell type; the ability to efficiently edit cells; the elimination of feeder cells, thereby minimizing complex, raw materials; the absence of lymphodepletion in subjects receiving RACR-engineered cells; low or no incidence of cytokine release syndrome (CRS) or immune effector cell-associated neurotoxicity syndrome (ICAN); and the use of rapamycin or rapamycin to promote cell engraftment, expansion, and persistence. In some embodiments, RACR-iCIL provides a cell source for allogeneic cell therapy, which in some aspects can be achieved while minimizing or eliminating low immunoengineering requirements.

[0284] The synthetic cytokine receptors provided herein can be used to support the derivation of cytotoxic innate lymphoid cells (CIL). CIL cells can be derived from stem cells or progenitor cells, and such cells are referred to herein as "induced cytotoxic innate lymphoid cells" (iCIL) cells. As described herein, iCIL cells share different cell surface markers and functional properties. As used herein, the term "induced cytotoxic innate lymphoid cells" or "iCIL" refers to CIL made by inducing the differentiation of progenitor cells. As disclosed herein, iCIL can be produced and / or amplified by expressing synthetic cytokine receptors in stem cells or progenitor cells and acting on the synthetic cytokine receptors through non-physiological ligands. This process may include differentiating progenitor cells that can express synthetic cytokine receptors by activating synthetic cytokine receptors. The process may also involve or alternatively involve amplifying progenitor cells or CIL by activating synthetic cytokine receptors.

[0285] In some embodiments, the present disclosure provides stem cells (such as iPSC) and CIL cells that have been engineered to express rapamycin-activated cytokine receptors (RACRs), i.e., synthetic cytokine receptors activated by small molecule rapamycin or rapamycin analogs. CIL cells comprising RACR and activated with rapamycin or rapamycin analogs are referred to herein as "RACR-iCIL" cells. Stem cells containing RACR and activated with rapamycin or rapamycin analogs are referred to herein as "RACR-SC". It has been shown that RACR can support the differentiation and / or expansion of RACR-SC and RACR-iCIL cells in a feeder-free manufacturing process. RACR-iCIL cells express a variety of innate tumor targeting receptors and are able to exert CAR-directed cytolytic activity after being modified to express chimeric antigen receptors (CARs). Therefore, RACR-iCIL cells provide a "ready-made" allogeneic cell therapy.

[0286] This disclosure relates, in part, to the unexpected discovery that stem cells engineered to express synthetic cytokine receptors differentiate into hematopoietic progenitor cells, CLPs, or CMPs, in response to the receptor's cognate abiotic ligand. CIL cells derived from the engineered stem cells retain the synthetic cytokine receptors and proliferate in response to the receptor's cognate abiotic ligand. The engineered CIL cells can be generated in large quantities and exhibit functional activity equal to or greater than that of CIL cells from other sources.

[0287] like Figure 1AAs shown in [ 1 ], isolated CIL cells can be transduced with a vector containing at least one polynucleotide encoding a synthetic cytokine receptor. When the transduced CIL cells are exposed to a non-physiological ligand, the extracellular domains of the cytokine receptors dimerize through mutual binding with the non-physiological ligand. This dimerization generates an amplification signal within the CIL cells, thereby generating a phenotypically enriched, functionally active engineered CIL cell population.

[0288] like Figure 1B As shown in , stem or progenitor cells can be transduced with a vector containing at least one polynucleotide encoding a synthetic cytokine receptor. When the transduced cells come into contact with a non-physiological ligand, the extracellular domain of the cytokine receptor dimerizes. This dimerization generates differentiation signals within the stem or progenitor cells, inducing their sequential differentiation into CIL cells.

[0289] CIL cells can be derived from iPSCs, common lymphoid progenitors (CLPs), or other stem or progenitor cells. Also provided herein are stem or progenitor cells that express synthetic cytokine receptors, and methods for differentiating engineered stem or progenitor cells into CIL cells by contacting the stem or progenitor cells with a cognate non-biological ligand for the cytokine receptor.

[0290] In some embodiments, provided herein are CIL cells generated in vitro. The engineered cells and related compositions described herein can be used for ligand-controlled ex vivo expansion immunotherapy. Also provided herein is a method for expanding CIL cells by contacting cells with cognate non-biological ligands of synthetic cytokine receptors. In addition, the engineered CIL cells disclosed herein can be further engineered to express chimeric antigen receptors (CARs), thereby targeting engineered CIL cells to express cells that are recognized by CARs or cells that are marked with antigens recognized by CARs.

[0291] In some embodiments, provided engineered CIL cells and methods improve immunotherapy compared to existing strategies.While chimeric antigen receptor (CAR) T cell therapy has revolutionized the treatment of hematological malignancies, its major limitations have hindered its widespread application.

[0292] In some embodiments, the participation of synthetic cytokine receptors can not only promote differentiation, but also increase cell growth and promote amplification by the participation of synthetic cytokine receptors on the provided engineering iCIL cells. In some aspects, the provided engineered iCIL cells and related methods can be used to increase cell growth and amplification of engineered cell therapy in vivo by administering rapamycin to the patient after the cell therapy product. In some embodiments, rapamycin amplifies and protects cells simultaneously. Amplification is achieved by activating JAK / STAT signals, and protection is achieved by suppressing the host's anti-graft response by rapamycin. In some embodiments, lympholysis and exogenous cytokine dosage are not required. In some embodiments, the administration and treatment methods of the provided engineered iCIL cells can be carried out without lympholysis (for example, without the need for lympholysis treatment, such as cyclophosphamide and / or fludarabine). In some embodiments, the administration and treatment methods of the provided engineered iCIL cells can be carried out without the administration of exogenous cytokines (for example, without the need for administration of IL-2 and / or IL-15).

[0293] In some embodiments, the provided methods can also include administering a non-physiological ligand for a synthetic cytokine receptor (e.g., rapamycin or a rapamycin analog) to the subject to expand or reactivate the engineered iCIL in the subject. Therefore, in some embodiments, since the cells can be expanded in vivo using a non-physiological ligand, there is no need to re-administer iCIL to the subject. However, since the hypoimmune engineering described herein enables allogeneic cell therapy, re-administration of iCIL is also possible. Furthermore, since the provided methods can be performed without lymphodepletion, this further provides advantages for promoting the expansion of metastatic cells and promoting host anti-tumor responses. This is because, in the absence of lymphodepletion, the host's immune system remains and is not depleted. Therefore, the immune response generated by the iCIL cells (e.g., the release of cytokines and other pro-inflammatory factors) can stimulate the host's existing immune system to fight the tumor. Furthermore, the exemplary non-physiological ligand rapamycin not only promotes the expansion of metastatic cells through the engagement of synthetic cytokine receptors, but also reactivates T cells and promotes the apoptosis of suppressive macrophages, similar to the transient mTOR inhibition achieved by rapamycin. Furthermore, although the nonphysiological ligand rapamycin or its analogs can suppress the host's anti-graft response, it is expected to be only transient, and once the administration of the nonphysiological ligand is stopped, the host's anti-tumor response will return to normal.

[0294] In some embodiments, the engineered cells herein are further modified to be resistant to the effects of rapamycin to inhibit or reduce cell growth and proliferation. In some embodiments, cells can be rendered "rapamycin-resistant" by providing free cytoplasmic FRB to the cells so that they can complex with rapamycin, thereby eliminating or reducing rapamycin-mediated growth inhibition of source cells or iCILs. In other embodiments, cells can be rendered "rapamycin-resistant" by disrupting FKBP12 in engineered cells, such as by inactivating or knocking it out. It was found herein that, in some cases, overexpressing FRB may not result in the generation of free FRB that can completely quench rapamycin. Therefore, in some cases, editing endogenous genes in cells, such as knocking out FKBP12, can render cells completely resistant to rapamycin.

[0295] Thus, the provided embodiments utilize a synthetic cytokine receptor system, such as a rapamycin-activated cytokine receptor (RACR), which can be activated by rapamycin or an analog (e.g., a rapamycin analog) to protect and expand cells in a single technique. Furthermore, the addition of gene disruption, such as knocking out certain immune genes (e.g., beta-2-microglobulin (B2M)), can also generate "stealth" cells, bringing further advantages to allogeneic cell therapy.

[0296] All publications, including patent documents, scientific articles, and databases, cited in this application are incorporated herein by reference in their entirety to the same extent as if each individual publication were individually incorporated by reference. In the event of any conflict or inconsistency between definitions set forth herein and definitions in patents, applications, published applications, and other publications cited herein, the definitions set forth herein shall control.

[0297] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. Those skilled in the art will recognize that there are numerous possibilities for implementation within the scope and spirit of this disclosure. The following description illustrates the disclosure and, of course, should not be construed as limiting the scope of the invention described herein in any way.

[0298] I. Definition

[0299] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0300] Unless the context dictates otherwise, the various features described herein may be used in any combination with any feature or combination of features described herein, and each feature may be excluded or omitted from the combination.

[0301] As used herein, the singular forms "a," "an," and "the" include the plural forms as well, unless the context clearly indicates otherwise. The conjunction "and / or" refers to all possible combinations of one or more of the listed elements.

[0302] As used herein, "subject" refers to a recipient of engineered CIL cells or other preparations. The term includes mammals, such as primates, mice, rats, dogs, cats, cows, horses, goats, camels, sheep, or pigs, preferably humans.

[0303] As used herein, "treatment" refers to any type of action or administration that produces a benefit to a subject suffering from a disease or disorder, including improvement in the patient's condition (i.e., amelioration, alleviation, or improvement of one or more symptoms, as well as partial or complete response to treatment).

[0304] The term "effective amount" refers to an amount effective to produce a desired biochemical, cellular or physiological response. The term "therapeutically effective amount" refers to a therapeutic amount, dose or therapeutic dosage regimen effective to produce a desired therapeutic effect.

[0305] As used herein, "polynucleotide" refers to a biopolymer composed of two or more nucleotide monomers covalently bound by an ester bond between a phosphate group of a nucleotide and a hydroxyl group of the sugar portion of the next nucleotide in the chain. DNA and RNA are non-limiting examples of polynucleotides.

[0306] As used herein, "polypeptide" refers to a polymer composed of amino acid residues linked by peptide bonds, which constitutes a portion (or all) of a protein.

[0307] Those skilled in the art will appreciate that, due to the degeneracy of the genetic code, many different polynucleotides and nucleic acids can encode the same polypeptide. Furthermore, those skilled in the art will appreciate that nucleotide substitutions can be made using conventional techniques to reflect the codon usage of any particular host organism to be expressed without affecting the polypeptide sequence encoded by the polynucleotides described herein.

[0308] Nucleic acids can include DNA or RNA. They can be single-stranded or double-stranded. They can also be polynucleotides containing synthetic or modified nucleotides. Many different types of oligonucleotide modifications are well known in the art. These include methylphosphate and thiophosphate backbones, and the addition of acridine or polylysine chains to the 3' and / or 5' ends of the molecule. For the purposes of the uses described herein, it should be understood that polynucleotides can be modified by any method available in the art. The purpose of such modifications can be to increase the activity or lifespan of the polynucleotide of interest in vivo.

[0309] The term "variant" refers to a polynucleotide or polypeptide having at least one substitution, insertion, or deletion in its sequence compared to a reference polynucleotide or polypeptide. A "functional variant" refers to a variant that retains one or more functions of a reference polynucleotide or polypeptide.

[0310] As used herein, the term "sequence identity" or "identity", with respect to polynucleotide or polypeptide sequences, refers to the degree of match between two optimally aligned polynucleotide or polypeptide sequences at each position in the alignment, over the entire length of a reference sequence. "Percent identity" is the number of matching positions in the optimal alignment, divided by the length of the reference sequence, plus the sum of the lengths of any gaps in the reference sequence in the alignment. Optimal alignment refers to the alignment that results in the greatest percentage of identity. Alignment of sequences can be accomplished by many well-known methods, including, for example, using mathematical algorithms such as the BLAST component or the algorithm in the ClustalOmega sequence analysis program. Unless otherwise indicated, the term "sequence identity" in the claims refers to sequence identity as calculated by BLAST version 2.12.0 using default parameters. Also, unless otherwise indicated, an alignment is an alignment of all or part of the polynucleotide or polypeptide sequence of interest over the full length of the reference sequence.

[0311] As used herein, "small molecule" refers to a low molecular weight (<1000 Dalton), organic compound. Small molecules can bind to specific biomacromolecules and have a variety of biological functions or applications, including but not limited to cell signaling molecules, drugs, secondary metabolites, or other various modes of action.

[0312] The term "analog," as it relates to small molecules, refers to a compound that is similar in structure and / or function to another compound but differs in some composition. Analogs may differ in one or more atoms, functional groups, or substructures, which are replaced by other atoms, functional groups, or substructures. Despite a high degree of structural and / or functional similarity, analogs may have different physical, chemical, physicochemical, biochemical, or pharmacological properties.

[0313] The term "rapamycin analogs" is a group of rapamycin analogs recognized in the art that are structurally and functionally similar to rapamycin. Some rapamycin analogs are known to possess some, but not all, of the functional properties of rapamycin. For example, some rapamycin analogs are suitable for use as non-physiological ligands because they promote dimerization but have essentially no immunosuppressive activity (e.g., AP21967, AP23102, or iRAP).

[0314] AP21967 is an exemplary rapamycin analog of the present disclosure.

[0315] AP23102 is an exemplary rapamycin analog of the present disclosure.

[0316] iRAP is an exemplary rapamycin analog of the present disclosure.

[0317] The term "cell population" refers to a mixture of cells suspended in solution, attached to a matrix, or stored in a container. By performing bulk measurements on a sample volume containing multiple cells, the overall characteristics of the cell population can be investigated. Flow cytometry can be used to reduce background fluorescence issues encountered in bulk cell population measurements.

[0318] As used herein, the term "cytotoxic innate lymphoid cells" or "CIL cells" refers to a type of cytotoxic lymphocyte that is a major component of the innate immune system. In humans, CILs 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, and NKp80. CILs generally do not express CD3, or express CD3 at lower levels than CD3+ T cells. CIL cells are cytotoxic and contain small granules in their cytoplasm that contain specialized proteins, such as perforin and proteases called granzymes. CIL cells can rapidly respond to virally infected cells and to transformed cells. When perforin is released near cells to be killed, it forms pores in the target cell's membrane, allowing granzymes and related molecules to enter the cell and induce apoptosis. CIL cells can be effector cells of lymphocyte colonies in anti-tumor 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 the sake of clarity, the term "CIL" cells do not include adaptive immune cells and their progenitor cells, nor do they include common lymphoid progenitor cells (CLP). However, CIL may be derived from CLP. In some embodiments, CIL is induced cytotoxic innate lymphoid cells (iCIL), wherein iPSC is induced to differentiate into cytotoxic innate lymphoid cells using the methods described herein. In some aspects, compared with traditional NK cells (such as bdNK cells or iPSC-NK cells), iCIL can exhibit one or more unique phenotypic or functional characteristics.

[0319] As used herein, the term "engineered" refers to cells that have been stably transduced with a heterologous polynucleotide, or cells that have been genetically edited to introduce, delete, or modify a polynucleotide in the cell, or cells that have been transiently transduced with a polynucleotide in a manner that results in a stable phenotypic change in the cell.

[0320] As used herein, the term "stem cell" refers to a cell with an undifferentiated phenotype, for example, capable of differentiating into a hematopoietic progenitor cell, a general lymphoid progenitor cell, a cytotoxic innate lymphoid cell, and / or a NK cell.

[0321] As used herein, the term "pluripotency" means that a stem cell is capable of forming essentially all differentiated cell types of an organism, at least in culture. For example, embryonic stem cells are a type of pluripotent stem cell that are capable of forming cells from each of the three germ layers: ectoderm, mesoderm, and endoderm.

[0322] As used herein, the terms "induced pluripotent stem cells" and "iPSCs" refer to cells of somatic origin that have been reprogrammed to a pluripotent state and are capable of proliferation, selective differentiation, and maturation. iPSCs are stem cells derived from differentiated adult, neonatal, or fetal cells that have been induced or modified, i.e., reprogrammed, to differentiate into tissues of all three germ layers or the dermis: mesoderm, endoderm, and ectoderm. Manufactured iPSCs do not refer to cells found in nature.

[0323] As used herein, the term "hematopoietic stem cell" refers to a stem cell capable of giving rise to mature myeloid and lymphoid cell types, including natural killer cells, T cells, and B cells. Hematopoietic stem cells are typically characterized by being CD34+.

[0324] The term "progenitor cell" refers to a cell that has partially differentiated into a desired cell type. Progenitor cells retain a certain degree of totipotency and can differentiate into a variety of cell types.

[0325] As used herein, the term "hematopoietic progenitor cell" refers to a cell that is an intermediate cell type capable of further differentiation into a blood cell lineage, wherein the hematopoietic progenitor cell can differentiate into a common myeloid progenitor cell or a common lymphoid progenitor cell. Hematopoietic progenitor cells are typically characterized by CD34+ and CD45+. CD38 is also considered a marker for hematopoietic progenitor cells. CD45 is considered a hematopoietic cell lineage marker.

[0326] As used herein, the term "lymphoid progenitor cell" or "lymphoblast" or "common lymphoid progenitor cell" refers to a precursor cell of a lymphocyte, such as a CIL and NK cell. Lymphoid progenitor cells are the first stage of differentiation of hematopoietic stem cells, following the differentiation of the lymphocyte lineage. As used herein, the term "lymphoid progenitor cell" refers to a cell that is capable of hematopoietic transformation into a hematopoietic cell type. Lymphoid progenitor cells may be characterized by CD45+CD7+CD5+ / loCD3-CD56-. Lymphoid progenitor cells may be characterized by CD45+CD5+ / loCD7+.

[0327] As used herein, "differentiation" or "differentiated" refers to the process and conditions by which undifferentiated or immature (e.g., unspecialized) cells acquire the characteristics of mature (specialized) cells, thereby acquiring specific morphology and functions. Stem cells (unspecialized) are typically exposed to different conditions (e.g., growth factors and morphogenetic factors) to induce the specific lineage commitment or differentiation of the stem cells.

[0328] As used herein, "expansion" or "expansion" refers to increasing the number and / or purity of a cell type in a cell population by mitosis of cells with limited proliferative capacity (eg, CIL cells).

[0329] As used herein, "activity," "activation," or "activation" refers to stimulation of activating receptors on cytotoxic innate lymphocytes, leading to cell division, cytokine secretion (e.g., IFNγ and / or TNFα ) and / or the release of cytolytic granules to modulate or assist the immune response.

[0330] II. Source Cells and Methods of Differentiating Them into Cytokine-Producing Innate Lymphoid (CIL) Cells

[0331] Provided herein are stem cells or progenitor cells containing synthetic cytokine receptors. In some embodiments, the synthetic cytokine receptor is any of those described in Section II. B. In some embodiments, the synthetic cytokine receptor comprises a common γ chain intracellular signaling domain (such as interleukin-2 receptor subunit γ, IL-2RG) and an intracellular domain from interleukin-2 receptor subunit β (IL-2RB), interleukin-7 receptor subunit β (IL-7RB) or interleukin-21 receptor subunit β (IL-21RB). In some embodiments, the synthetic cytokine receptor further comprises an extracellular domain that can bind to a non-physiological ligand (such as rapamycin or the like). In this way, the binding of non-physiological ligands to the extracellular domain of the synthetic cytokine receptor activates cytokine receptor-mediated signal transduction, including JAK / STAT signal transduction, which is an important pathway for stem cells such as iPSC or other pluripotent stem cells to differentiate into downstream cell lineages, such as CIL. Thus, in the presence of non-physiological ligands (such as rapamycin), synthetic cytokine receptors can participate in cell differentiation processes without the need for endogenous receptors or exogenous growth factors. In some embodiments, this enhances the control of JAK / STAT signaling during cell differentiation and reduces its variability, thereby effectively generating inducible CILs (iCILs).

[0332] As described above, provided herein are stem cells or progenitor cells that can be differentiated into lymphocytes using synthetic cytokine receptor complexes activated by non-physiological ligands, and differentiated cells are produced by these stem cells or progenitor cells for medical treatment. Differentiated cells can be, but are not limited to, iCIL cells. As a non-limiting illustration of the compositions and methods described herein, cytotoxic innate lymphoid cells can be produced from pluripotent stem cells, such as induced pluripotent stem cells, on the basis of or in place of exogenous cytokines, and the stem cells are engineered to express synthetic cytokine receptors, which can be activated by the non-physiological ligands (such as rapamycin) to induce differentiation. In some embodiments, the synthetic cytokine receptor is a rapamycin-activated cytokine receptor (RACR), which is induced to differentiate using rapamycin or rapamycin analogs, to supplement or replace exogenous cytokines. The advantages of the embodiments may include the ability to produce effector cells expressing synthetic cytokine receptor complexes activated by non-physiological ligands from abundant cell sources (such as induced pluripotent stem cells), thereby controlling the proliferation of effector cells in the patient's body by administering or ceasing to administer non-physiological ligands. Other advantages of embodiments of the present invention include, but are not limited to, the ability to generate effector cells from source cells in a culture medium that is substantially free of cytokines used in the art for CIL cell differentiation (eg, IL-2, IL-7, and / or IL-15).

[0333] A. Source cells

[0334] CIL cells can be generated from a variety of sources, including iPSCs, PBMCs, or UCBs. In some embodiments, the CIL-derived cells are autologous. In some embodiments, the CIL-derived cells are allogeneic. In some embodiments, the CIL-derived cells are heterologous. For example, when a subject treated with the compositions of the present disclosure has received high-dose chemotherapy or radiation therapy to disrupt the subject's immune system, allogeneic cells may be used.

[0335] As used herein, the term "peripheral blood cells" refers to cells derived from circulating blood and includes hematopoietic stem cells capable of proliferation, selective differentiation, and maturation. Therefore, peripheral blood NK cells may also be referred to as differentiated blood-derived NK cells (bdNK).

[0336] In some embodiments, the lymphocytes used to generate engineered stem cells or CIL cells can be obtained from a donor or subject (for autologous therapy) by various methods known in the art. For example, lymphocytes can be obtained by collecting peripheral blood from a patient, performing Ficoll density gradient centrifugation and / or leukocyte removal on the blood, and then using a separation kit to separate a lymphocyte population from the peripheral blood. In an exemplary embodiment, the lymphocyte population does not need to be a pure selected cell type and may also include other cell types, such as T cells, monocytes, macrophages, natural killer cells, and B cells. In some embodiments, the collected cell population includes 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.

[0337] In certain embodiments, CIL cells are generated from a stem cell source.

[0338] In some embodiments, the source cells include hematopoietic stem cells characterized by CD34+ and / or CD45+; common lymphoid progenitor cells characterized by CD45+CD7+CD56-; CIL progenitor cells characterized by CD45+CD5-CD7+; and / or CIL cells characterized by CD45+CD56+CD3-, and optionally CD5- and / or CD7+.

[0339] In some embodiments, the stem cells are pluripotent stem cells. The method can use pluripotent stem cells from various sources, including embryonic stem cells (ES) and induced pluripotent stem cells (iPSC). The method can use pluripotent stem cells from various sources, including embryonic stem cells (ES) and induced pluripotent stem cells (iPSC). In some embodiments, the pluripotent stem cells are induced pluripotent stem cells (iPSCs) artificially derived from non-pluripotent cells. In some aspects, non-pluripotent cells are cells with lower self-renewal and differentiation capabilities than pluripotent stem cells. iPSCs can be produced by a process called reprogramming, in which non-pluripotent cells are effectively "dedifferentiated" into a state similar to embryonic stem cells by engineering them to express genes such as OCT4, SOX2, and KLF4. Takahashi and Yamanaka, Cell (2006) 126: 663-76.

[0340] In some embodiments, the source cells can be human embryonic stem cells (hESC) or induced pluripotent stem cells (iPSC).In immunotherapy, the source cells can be allogeneic or autologous, meaning from a donor or subject, respectively.

[0341] In some embodiments, CIL cells can be generated from induced pluripotent stem cells (iPSC). iPSC is a pluripotent stem cell derived from adult somatic cells, which is genetically reprogrammed to a state similar to embryonic stem cells by forced expression of genes and factors important for maintaining the defining characteristics of embryonic stem cells. Induced pluripotent stem cells can be generated from tissues containing somatic cells, including but not limited to skin, dental tissue, peripheral blood and urine. To generate iPSC, somatic cells can be reprogrammed by methods including but not limited to transient expression of reprogramming factors, virus-free methods, adenovirus, plasmid, minicirculation vector, exon vector, Sendai virus, synthetic mRNA, self-replicating RNA, retrovirus, lentivirus, PhiC31 integrase, excisable transposon, CRISPR-based gene editing or recombinant protein.

[0342] While the cell therapy industry has demonstrated the transformative potential of using genetically engineered patient-derived cells to treat specific disease indications, the use of patient-derived materials faces many challenges, including limited expansion and scalability, manufacturing complexity, high costs, patient-to-patient variability, and patient accessibility. In contrast, iPSCs are pluripotent stem cells, which are theoretically capable of differentiating into any other cell type, including cells used in cancer treatment. Natural killer(NK) cells. Using iPSCs, it is possible to provide anti-target cell (e.g., anti-cancer) cell therapies, such as scalable and simplified production of NK cells, thereby reducing costs and improving patient accessibility to cell therapies. iPSCs have unlimited expansion capabilities, which means that they can reproduce and proliferate indefinitely, potentially producing almost endless differentiated immune cells for treatment, such as for cancer treatment. iPSCs can also undergo precise multiple genome editing, allowing the introduction of multiple genetic modifications to enhance the disease targeting ability and safety of the immune cells they eventually become. Similarly, iPSCs can be engineered to protect them from (allogeneic) rejection by the patient's own immune system, thereby improving their initial expansion and transplantation time. In addition, blood materials from patients or donors are not consistent, while iPSCs provide consistent starting materials derived from single cell clones, which can ensure the genomic consistency and integrity of the final cell product.

[0343] In some embodiments, PSC (such as iPSC) is an autologous cell of the subject to be treated, that is, the PSC comes from the same subject to which the differentiated cells are administered. In some embodiments, as described herein, before differentiating into CIL, non-pluripotent cells (such as fibroblasts) from the patient to be treated are reprogrammed to become iPSC. In certain embodiments, fibroblasts can be reprogrammed into iPSC by transforming fibroblasts with genes (OCT4, SOX2, NANOG, LIN28 and KLF4) cloned into a plasmid (e.g., see, Yu, et al., Science DOI: 10.1126 / science.1172482). In some embodiments, before differentiating into CIL, non-pluripotent fibroblasts from the patient are first reprogrammed into iPSC, for example, using a non-integrating Sendai virus to reprogram the cells (e.g., using CTS TM CytoTune TM -iPS2.1 Sendai Reprogramming Kit). In some embodiments, the differentiated cells thus generated are administered to a patient as an autologous cell therapy source.

[0344] In some embodiments, the PSCs (e.g., iPSCs) are allogeneic to the subject to be treated, i.e., the PSCs are derived from an individual different from the subject to whom the differentiated cells will be administered. In some embodiments, non-pluripotent cells (e.g., fibroblasts) from another individual (e.g., an individual who does not have a disease or condition to be treated, such as a healthy individual) are reprogrammed into iPSCs prior to differentiation into CILs. In some embodiments, at least part of the reprogramming is achieved by reprogramming the cells using a non-integrating Sendai virus (e.g., using a CTS virus). TM CytoTune TM-iPS 2.1 Sendai Reprogramming Kit). In some embodiments, the differentiated cells thus generated are administered to an individual from whom the differentiated cells were derived (e.g., allogeneic cell therapy or allogeneic cell transplantation). In such embodiments, the PSCs described herein (e.g., allogeneic cells) can be genetically engineered to be low immunogenic cells. Methods for reducing immunogenicity are known, including ablation of the expression of polymorphic HLA-A / -B / -C and HLA class II molecules. Exemplary methods for reducing one or more HLA molecules include disrupting the beta-2 microglobulin (B2M) gene, as described herein.

[0345] In some embodiments, iPSCs are gene-edited using lentivirus. In some embodiments, iPSCs are gene-edited using CRISPR.

[0346] In some embodiments, HSC genes are edited using lentivirus. In some embodiments, HSC genes are edited using CRISPR. In some embodiments, blood progenitor cell genes are edited using lentivirus. In some embodiments, blood progenitor cell genes are edited using CRISPR. In some embodiments, common lymphoid progenitor cell genes are edited using lentivirus. In some embodiments, common lymphoid progenitor cell genes are edited using CRISPR. In some embodiments, common lymphoid progenitor cell genes are edited using lentivirus. In some embodiments, common lymphoid progenitor cell (CLP) cell genes are edited using lentivirus. In some embodiments, common lymphoid progenitor cell (CLP) cell genes are edited using CRISPR. Exemplary methods for gene editing are shown in Section III.

[0347] In some embodiments, stem cells can be engineered to express synthetic cytokine receptors.

[0348] In some embodiments, iPSCs can be engineered to express synthetic cytokine receptors.

[0349] In some embodiments, hematopoietic stem cells can be engineered to express synthetic cytokine receptors. In some embodiments, blood progenitor cells (leukocytes) can be engineered to express synthetic cytokine receptors. In some embodiments, common lymphoid progenitor cells can be engineered to express synthetic cytokine receptors. In some embodiments, CIL cells can be engineered to express synthetic cytokine receptors.

[0350] The method for producing CIL cells may include an in vitro culture process, wherein the CIL cells are differentiated from non-terminal differentiated cells. In some embodiments, non-terminal differentiated cells are stem cells. In some embodiments, non-terminal differentiated cells are iPSC cells. In some embodiments, non-terminal differentiated cells are progenitor cells. In embodiments, non-terminal differentiated cells (such as stem cells, such as iPSC) express synthetic cytokine receptors. In one aspect, the present disclosure provides a method for producing CIL cells, including providing stem cells or progenitor cells, and differentiating cells into CIL cells by controlled activation of synthetic cytokine receptors or not activating synthetic cytokine receptors. In some embodiments, differentiation is carried out by activating synthetic cytokine receptors, without the need for any additional cytokines (for example, without the need for one or more of IL-2, IL-15 and IL-7). In some embodiments, differentiation is achieved by activating synthetic cytokine receptors by one or more additional cytokines. In some embodiments, differentiation can also be carried out with cytokines without activating synthetic cytokine receptors.

[0351] In any of the provided embodiments, the synthetic cytokine receptor can be any of the receptors 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), which can be activated by rapamycin or a rapamycin analog. In the provided embodiments, activation of the synthetic cytokine receptor can induce differentiation in addition to or in place of an exogenous cytokine.

[0352] In some embodiments, non-physiological ligands can induce differentiation in addition to or in place of exogenous cytokines. In some embodiments, non-physiological ligands can induce differentiation in one or more of mesoderm formation, hematopoietic cell differentiation, lymphoid progenitor cell differentiation, and CIL cell differentiation.

[0353] In some embodiments, a non-physiological ligand may be contacted with cells at a stage of differentiation requiring IL-7 and / or IL-15 signaling.

[0354] In some embodiments, a non-physiological ligand may be contacted with cells that are in an expansion phase requiring an IL-2 signal.

[0355] In some embodiments, cells are engineered to express synthetic cytokine receptors and to activate the receptors with non-physiological ligands, allowing for the generation of CIL cells that can differentiate and / or expand without the use of exogenous factors.

[0356] In some embodiments, iPSCs are engineered to express RACR, and activation of the receptor with rapamycin or a rapamycin analog allows for the generation of CIL cells that can differentiate without the use of exogenous factors (eg, without the use of IL-15 and / or IL-7).

[0357] In some embodiments, iPSCs are engineered to express RACR, and activation of the receptor with rapamycin or a rapamycin analog allows for the generation of CIL cells that can be expanded without the use of exogenous factors (eg, without the use of IL-2 and / or IL-7).

[0358] Because 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, it is surprising and unexpected that the engineered CIL cells described herein can differentiate and / or expand in the absence of one, two or all of IL-2, IL-15 and IL-7.

[0359] In some embodiments, the engineered common lymphoid progenitor cells are differentiated into CIL cells in a differentiation medium that does not contain IL-15.

[0360] In some embodiments, the engineered common lymphoid progenitor cells are differentiated into CIL cells in a differentiation medium that does not contain IL-7.

[0361] In some embodiments, the engineered common lymphoid progenitor cells are differentiated into CIL cells in a differentiation medium that does not contain IL-15 or IL-7.

[0362] In some embodiments, common lymphoid progenitor cells are engineered to express a synthetic cytokine receptor, such as RACR, and differentiated into CIL cells in a differentiation medium containing a rapamycin analog without IL-15 or IL-7.

[0363] B. Synthetic cytokine receptor complexes

[0364] The synthetic cytokine receptors disclosed herein comprise a synthetic gamma chain and a synthetic beta chain, each chain comprising a dimerization domain. In the presence of non-physiological ligands, the dimerization domain can control dimerization, thereby activating the signaling of the synthetic cytokine receptor.

[0365] The synthetic gamma chain polypeptide comprises a first dimerization domain, a first transmembrane domain, and the intracellular domain of interleukin-2 receptor subunit gamma (IL-2RG). The dimerization domain can be the extracellular domain (N-terminal to the transmembrane domain) or the intracellular domain (C-terminal to the transmembrane domain and N-terminal or C-terminal to the IL-2G intracellular domain).

[0366] The synthetic beta chain polypeptide comprises 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), or the intracellular domain of interleukin-21 receptor subunit beta (IL-21RB). The dimerization domain can be an extracellular domain (N-terminal to the transmembrane domain) or an intracellular domain (C-terminal to the transmembrane domain and N-terminal or C-terminal to the IL-2RB or IL-7RB intracellular domain).

[0367] In some embodiments, the synthesized gamma chain polypeptide is encoded by a nucleic acid sequence encoding a signal peptide. In some embodiments, the synthesized beta chain polypeptide is encoded by a nucleic acid sequence encoding a signal peptide. Those skilled in the art will readily be familiar with signal peptides, which can provide a signal for transporting a nascent protein within a cell. Any of a variety of signal peptides can be used.

[0368] In some embodiments, the signal peptide is the CD8a signal sequence shown in SEQ ID NO: 12: MALPVTALLLPLALLLHAARP.

[0369] In some embodiments, the signal peptide is the signal sequence shown in SEQ ID NO: 29: MPLGLLWLGLALLGALHAQA.

[0370] In some embodiments, a non-physiological ligand activates a synthetic cytokine receptor in a cytotoxic innate lymphoid cell to induce expansion and / or activation of the engineered cytotoxic innate lymphoid cell. In some embodiments, the non-physiological ligand is rapamycin or a rapamycin receptor, and this synthetic cytokine receptor is referred to as a rapamycin-activated cytokine receptor (RACR).

[0371] In some embodiments, a non-physiological ligand activates a synthetic cytokine receptor in CIL cells to induce expansion of the CIL cells. In some embodiments, activation of the synthetic cytokine receptor results in an increase in the number of CIL cells of 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 compared to uninduced cells. In some embodiments, activation of the synthetic cytokine receptor results in an increase in the number of CIL cells of 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 compared to uninduced cells.

[0372] In some embodiments, the CIL cells increase is 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 are increased by about 4,000-fold to about 6,000-fold, about 5,000-fold to about 7,000-fold, about 6,000-fold to about 8,000-fold, about 7,000-fold to about 9,000-fold, about 8,000-fold to about 10,000-fold, about 9,000-fold to about 50,000-fold, about 10,000-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.

[0373] In some embodiments, the non-physiological ligand activates a synthetic cytokine receptor in the stem cell to induce differentiation. In some embodiments, the non-physiological ligand is rapamycin or a rapamycin receptor, and this synthetic cytokine receptor is called a rapamycin-activated cytokine receptor (RACR).

[0374] In some embodiments, a non-physiological ligand activates a synthetic cytokine receptor in a stem cell to induce expansion of hematopoietic progenitor cells or CLPs differentiated from the stem cell. In some embodiments, activation of the synthetic cytokine receptor results in an increase in the number of hematopoietic progenitor cells or CLPs by 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 compared to unengineered cells.

[0375] In some embodiments, the increase in hematopoietic progenitor cells or CLPs is 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.

[0376] 1. Intracellular domain

[0377] In some embodiments, the intracellular signaling domain of the first transmembrane receptor protein comprises an interleukin-2 receptor subunit gamma (IL2Rg) domain. In some embodiments, the IL2Rg domain comprises the sequence set forth in SEQ ID NO: 1. In some embodiments, the IL2Rg 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 with SEQ ID NO: 1.

[0378] The sequence of the IL2RG common gamma chain intracellular domain is shown in SEQ ID NO: 1: ERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGAL GEGPGASPCNQHSPYWAPPCYTLKPET.

[0379] In some embodiments, the synthetic cytokine receptor comprises a first transmembrane receptor protein comprising the IL-2RG intracellular domain, a first dimerization domain, a second transmembrane receptor protein comprising the IL-2RB intracellular domain, and a second dimerization domain.

[0380] In some embodiments, the synthetic beta chain comprises the intracellular domain of the interleukin-2 receptor subunit beta (IL2RB). IL2RB is also known as IL15RB or CD122. Therefore, when IL2RB is mentioned herein, IL15RB may also be referred to. That is, in this disclosure, these terms can be used interchangeably. 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 with SEQ ID NO:2.

[0381] The sequence of IL2RB intracellular domain is as SEQ ID NO:2 shows: NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSS PQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV

[0382] In some embodiments, the synthetic cytokine receptor comprises a first transmembrane receptor protein comprising the IL-2RG intracellular domain, a first dimerization domain, a second transmembrane receptor protein comprising the IL-7RB intracellular domain, and a second dimerization domain.

[0383] In some embodiments, the synthetic beta chain comprises the 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 with SEQ ID NO: 3.

[0384] The sequence of the IL7RB intracellular domain is shown in SEQ ID NO: 3: KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSSFYQNQ

[0385] In some embodiments, the synthetic cytokine receptor comprises a first transmembrane receptor protein comprising the IL-2RG intracellular domain, a first dimerization domain, a second transmembrane receptor protein comprising the IL-21RB intracellular domain, and a second dimerization domain.

[0386] In some embodiments, the synthetic beta chain comprises the interleukin-21 receptor subunit beta (IL21RB) intracellular domain. 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 with SEQ ID NO: 4.

[0387] The sequence of IL21RB intracellular domain is as SEQ ID Shown in NO:4: SLKTHPLWRLWKKIWAVPSPERFFMPLYKGCSGDFKKWVGAPFTSSLELGPWSPEVPSTLEVYSCHPPSPAKRLQLTELQEPAELVESDGVPKPSFWPTAQNSGGSAYSEERDRPYGLVSIDTVTVLDAEGPCTWPC SCEDDGYPALDLDAGLEEPSPGLEDPLLDAGTTVLSCGCVSAGSPGLGGPLGSLLDRLKPPLADGEDWAGGLPWGGRSPGGVSESEAGSPLAGLDMDTFDSGFVGSDCSSPVECDFTSPGDEGPPRSYLRQWVVIPPPLSSPGPQAS

[0388] 2. Dimerization domain

[0389] The dimerization domain may be a heterologous dimerization domain, including but not limited to a 12 kD FK506 binding protein (FKBP) and a FKBP12-rapamycin binding (FRB) domain, which dimerize in the presence of rapamycin or a rapamycin analog, as known in the art. In some embodiments, the FRB domain may 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. In some embodiments, the FKBP domain may 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: 5. In some embodiments, the FKBP 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: 49. In some embodiments, the FKBP 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: 30.

[0390] In some embodiments, the sequence of an exemplary FKBP domain is set forth in SEQ ID NO:5:

[0391] GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQE VIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE

[0392] In some embodiments, the sequence of an exemplary FKBP domain is set forth in SEQ ID NO:49:

[0393] GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQE VIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKL

[0394] In some embodiments, the sequence of an exemplary FKBP domain is set forth in SEQ ID NO:30:

[0395] GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQE VIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLGE

[0396] In some embodiments, the sequence of an exemplary FRB domain is shown in SEQ ID NO: 6: ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAY GRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISK

[0397] In some embodiments, the sequence of the variant FRB domain (FRB mutant domain) is as shown in SEQ ID NO:7:

[0398] ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQA YGRRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK

[0399] In some embodiments, the first dimerization domain is set forth as SEQ ID NO:5 and the second dimerization domain is set forth as SEQ ID NO:6.

[0400] In some embodiments, the first dimerization domain is set forth as SEQ ID NO:49 and the second dimerization domain is set forth as SEQ ID NO:6.

[0401] In some embodiments, the first dimerization domain is set forth as SEQ ID NO:30 and the second dimerization domain is set forth as SEQ ID NO:6.

[0402] In some embodiments, the first dimerization domain is set forth as SEQ ID NO:5 and the second dimerization domain is set forth as SEQ ID NO:7.

[0403] In some embodiments, the first dimerization domain is set forth as SEQ ID NO:49 and the second dimerization domain is set forth as SEQ ID NO:7.

[0404] In some embodiments, the first dimerization domain is set forth as SEQ ID NO:30 and the second dimerization domain is set forth as SEQ ID NO:7.

[0405] Alternatively, the first dimerization domain and the second dimerization domain may be the 12 kD FK506 binding protein (FKBP) and calcineurin domains, which are known in the art to dimerize in the presence of FK506 or an analog thereof.

[0406] In some embodiments, the dimerization domain is a homodimerization domain selected from the group consisting of:

[0407] i) FK506 binding protein (FKBP) with a size of 12 kD;

[0408] ii) Cyclophilin A (CypA); or

[0409] iii) gyrase B (CyrB);

[0410] The corresponding non-physiological ligands are

[0411] i) FK1012, AP1510, AP1903 or AP20187;

[0412] ii) cyclosporine-A (CsA); or

[0413] iii) Coumarin or its analogues.

[0414] In some embodiments, the first and second dimerization domains of the transmembrane receptor protein are a FKBP domain and a cyclophilin domain.

[0415] In some embodiments, the first and second dimerization domains of the transmembrane receptor protein are a FKBP domain and a bacterial dihydrofolate reductase (DHFR) domain.

[0416] In some embodiments, the first and second dimerization domains of the transmembrane receptor protein are a calcineurin domain and a cyclophilin domain.

[0417] 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).

[0418] 3. Transmembrane domain

[0419] The transmembrane domain is a sequence that synthesizes the cytokine receptor across the membrane. The transmembrane domain may comprise a hydrophobic alpha helix. In some embodiments, the transmembrane domain is derived from a human protein.

[0420] The sequence of the transmembrane (TM) domain is shown in SEQ ID NO: 8: VVISVGSMGLIISLLCVYFWL

[0421] The sequence of the TM domain is shown in SEQ ID NO: 9: VAVAGCVFLLISVLLLSGL

[0422] The sequence of the TM domain is shown in SEQ ID NO: 10: PILLTISILSFFSVALLVILACVLW

[0423] The sequence of the TM domain is shown in SEQ ID NO: 11: GWNPHLLLLLLLVIVFIPAFW

[0424] The sequence of the TM domain is shown in SEQ ID NO: 36: IPWLGHLLVGLSGAFGFIILVYLLI.

[0425] In some embodiments, the TM domain and the intracellular signaling domain are from the same cytokine receptor. In some embodiments, the synthetic gamma chain polypeptide comprises an IL-2RG TM domain and an IL-2RG intracellular domain. In some embodiments, the synthetic beta chain polypeptide comprises an IL-2RB TM domain and an IL-2RB intracellular domain. In some embodiments, the synthetic beta chain polypeptide comprises an IL-7RB TM domain and an IL-7RB intracellular domain. In some embodiments, the synthetic beta chain polypeptide comprises an IL-21RB TM domain and an IL-21RB intracellular domain.

[0426] In some embodiments, one or more additional contiguous amino acids of an ectodomain directly adjacent to the TM domain of a cytokine receptor can also be included as part of a synthetic cytokine receptor chain polypeptide sequence. In some embodiments, 1-20 contiguous amino acids of an ectodomain adjacent to the TM domain of a cytokine receptor are included as part of a synthetic cytokine receptor chain polypeptide sequence. The portion of the ectodomain 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 directly adjacent to the TM sequence (e.g., at its N-terminus).

[0427] In some embodiments, the synthetic gamma chain polypeptide comprises 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 comprises 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.

[0428] In some embodiments, the synthetic beta chain polypeptide comprises an IL-2RB TM domain comprising the sequence set forth in SEQ ID NO: 36 and an IL-2RG intracellular domain comprising the sequence set forth in SEQ ID NO: 2. In some embodiments, the synthetic beta chain polypeptide comprises an IL-2RB TM domain comprising the sequence set forth in SEQ ID NO: 35 and an IL-2RG intracellular domain comprising the sequence set forth in SEQ ID NO: 2.

[0429] In some embodiments, the synthetic cytokine receptor comprises a synthetic gamma chain polypeptide comprising the FKBP12 dimerization domain and the IL-2RG intracellular domain, and a synthetic beta chain polypeptide comprising the FRB dimerization domain and the 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.

[0430] In some embodiments, the synthetic cytokine receptor consists 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.

[0431] In some embodiments, the synthetic cytokine receptor is capable of binding to 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, wherein binding of the non-physiological ligand to the dimerization domain of the synthetic cytokine receptor induces cytokine receptor-mediated signaling in the cell, such as through the JAK / STAT pathway.

[0432] 4. Non-physiological ligands

[0433] In various embodiments of the compositions and methods of the present disclosure, the system comprises a non-physiological ligand. Exemplary small molecules that can be used as ligands include, but are not limited to, rapamycin, fluorescein, fluorescein isothiocyanate (FITC), 4-[(6-methylpyrazin-2-yl)oxy]benzoic acid (aMPOB), folic acid, rhodamine, acetazolamide, and CA9 ligand.

[0434] In some embodiments, the synthetic cytokine receptor is activated by a ligand. In some embodiments, the ligand is a non-physiological ligand.

[0435] In some embodiments, the non-physiological ligand is a rapamycin analog.

[0436] In some embodiments, the non-physiological ligand is rapamycin.

[0437] In some embodiments, the non-physiological ligand is AP21967.

[0438] In some embodiments, the non-physiological ligand is FK506.

[0439] In some embodiments, the non-physiological ligand is FK1012. In some embodiments, the non-physiological ligand is AP1510. In some embodiments, the non-physiological ligand is AP1903. In some embodiments, the non-physiological ligand is AP20187. In some embodiments, the non-physiological ligand is cyclosporine-A (CsA). In some embodiments, the non-physiological ligand is coumarins.

[0440] In some embodiments, the synthetic cytokine receptor complex is activated by folic acid, fluorescein, aMPOB, acetazolamide, CA9 ligand, tacrolimus, rapamycin, a rapamycin analog (analog of rapamycin), CD28 ligand, a poly(his) tag, a Strep tag, a FLAG tag, a VS tag, a Myc tag, a HA tag, a NE tag, biotin, digoxigenin, dinitrophenol, or a derivative thereof.

[0441] In some embodiments, the non-physiological ligand can be an inorganic or organic compound less than 1000 Daltons.

[0442] In some embodiments, the ligand can be rapamycin or an analog of rapamycin (rapamycin analog). In some embodiments, rapamycin analogs include rapamycin variants with one or more modifications relative to rapamycin: demethylation, elimination or substitution of the methoxy group at C7, C42 and / or C29; elimination, derivatization or substitution of the hydroxyl group at C13, C43 and / or C28; reduction, elimination or derivatization of the ketone at C14, C24 and / or C30; substitution of the 6-membered pipecolate ring with a 5-membered prolyl ring; and substitution of the cyclohexyl ring with an alternative cyclopentyl ring.

[0443] Thus, in some embodiments, the rapamycin analog is everolimus, novolimus, pimecrolimus, redalimus, tacrolimus, temsicolimus, umicolimus, zotarolimus, temsicolimus (CCI-779), C20-methylrapamycin, C16-(S)-3-methylindorapamycin, C16-(S)-3-methylindorapamycin (C16-iRap), AP21967 (A / C heterodimer, ), mycophenolate sodium, benidipine hydrochloride, rapamycin, AP23573 (lidafenib), AP1903 (Rimiducid) or their metabolites, derivatives and / or combinations.

[0444] In some embodiments, the ligand comprises FK1012 (a semisynthetic dimer of K506), tacrolimus (FK506), FKCsA (a mixture of FK506 and cyclosporine), rapamycin, kumarin, gibberellin, HaXS dimerizer (a chemical dimerizer for Halo tags and SNAP tags), TmP-H tag (trimethoprim halogenase protein dimerizer), or ABT-737 or a functional derivative thereof.

[0445] In some embodiments, the non-physiological ligand is present or provided in an amount of 0 nM to 1000 nM, for example, 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, 5 5.0 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 foregoing amounts.

[0446] 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.

[0447] 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.

[0448] In some embodiments, the non-physiological ligand is a rapamycin analog and is present or provided at 1 nM. In some embodiments, the non-physiological ligand is a rapamycin analog and is present or provided at 10 nM. In some embodiments, the non-physiological ligand is a rapamycin analog and is present or provided at 20 nM. In some embodiments, the non-physiological ligand is a rapamycin analog and is present or provided at 50 nM. In some embodiments, the non-physiological ligand is a rapamycin analog and is present or provided at 100 nM.

[0449] In some embodiments, the non-physiological ligand is present or provided at 1 nM.

[0450] In some embodiments, the non-physiological ligand is present or provided at 10 nM.

[0451] In some embodiments, the non-physiological ligand is present or provided at 100 nM.

[0452] In some embodiments, the non-physiological ligand is present or provided at 1000 nM.

[0453] C. Cytoplasmic FRB

[0454] In some embodiments, engineered cells, such as stem cells or iCIL cells, can be exposed to free cytoplasmic FRBs. As described in more detail elsewhere herein, rapamycin typically binds to FBP12, and the FKBP12-rapamycin complex then binds to the FRB subunit of mTOR and blocks mTOR signaling. Thus, in certain circumstances, contacting cells with rapamycin can inhibit or reduce cell growth and proliferation. In some embodiments, cells can be rendered "rapamycin-resistant" by providing them with free cytoplasmic FRBs to form a complex with rapamycin, thereby eliminating or reducing rapamycin-mediated growth inhibition of source cells or iCILs.

[0455] 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 cell culture medium to eliminate or reduce rapamycin-mediated growth inhibition.

[0456] In one embodiment of microinjecting soluble FRB 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 one embodiment of microinjecting soluble FRB into stem cells or NK cells, the soluble FRB is injected at a concentration of 1 μM.

[0457] 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 in the cell. Methods for gene insertion or knock-in are known, including any of the methods described in Section III. In some embodiments, insertion of the FRB encoding construct is performed by homology-directed repair, for example using a CRISPR-Cas system. In some embodiments, engineered cells expressing FRB at an endogenous locus are capable of expressing free cytoplasmic FRB in the cell.

[0458] The FRB domain is a roughly 100-amino acid domain derived from the mTOR protein kinase. It is expressed in the cytoplasm as a freely diffusible, soluble protein. Advantageously, the FRB domain reduces the inhibitory effects of rapamycin on mTOR in engineered cells and promotes sustained activation of engineered cells, resulting in a proliferation advantage over native cells.

[0459] In some embodiments, the synthetic cytokine receptor complex comprises a cytoplasmic polypeptide bound to a ligand or a complex comprising a ligand.

[0460] In some embodiments, the cellular polypeptide comprises a FRB domain. In some embodiments, the cellular polypeptide comprises a FRB domain and the ligand is rapamycin. The cytoplasmic FRB domain may 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 may comprise a naked FRB domain consisting essentially of a polypeptide 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.

[0461] In some embodiments, the cell is contacted with a FRB domain protein having the sequence set forth in SEQ ID NO:6.

[0462] In some embodiments, the cell is contacted with a FRB domain protein having the sequence set forth in SEQ ID NO:7.

[0463] Advantageously, cytoplasmic FRBs confer resistance to the immunosuppressive effects of non-physiological ligands, such as rapamycin or rapamycin analogs.

[0464] D. Engineered stem cells

[0465] In some embodiments, the present disclosure provides engineered stem cells that transiently or stably express a synthetic cytokine receptor complex. In some embodiments, the present disclosure provides engineered stem cells that stably express a synthetic cytokine receptor complex.

[0466] In some embodiments, the engineered stem cell comprises 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, TRAC and / or SIRPA locus. In some embodiments, the genome further comprises a disrupted FKBP12 locus. In some embodiments, the locus of the gene is destroyed by gene editing technology (such as the CRISPR-Cas system). In some embodiments, the destroyed locus inactivates the gene in the cell. In some embodiments, the destroyed locus relates to the knockout of the gene in the cell. In some embodiments, the destroyed locus includes an insertion / deletion (indel) in the endogenous gene or a deletion of a continuous segment of the endogenous gene genomic DNA. In some embodiments, the insertion / deletion is a frameshift mutation or a deletion of a continuous segment of the genomic DNA of the gene. In some embodiments, the insertion / deletion is in two alleles of the gene (indel / indel). Gene editing and engineering methods are known, including the methods described in Section III. Any of these methods can be used to generate an engineered stem cell further comprising a synthetic cytokine receptor complex as described herein.

[0467] In some embodiments, the engineered stem cell comprises a genome comprising (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 cell comprises a genome comprising: (i) a nucleotide sequence encoding a synthetic cytokine receptor complex, as described in Section II.B; (ii) a disrupted B2M locus; (iii) a disrupted FKBP12 locus. In some embodiments, the engineered stem cell comprises a genome comprising: (i) a nucleotide sequence encoding a synthetic cytokine receptor complex; (ii) a disrupted TRAC locus; (iii) a disrupted FKBP12 locus. In some embodiments, the engineered stem cell comprises a genome comprising: (i) a nucleotide sequence encoding a synthetic cytokine receptor complex; (ii) a disrupted SIRPA locus; (iii) a disrupted FKBP12 locus. In some embodiments, the engineered stem cell comprises a genome comprising: (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 cell comprises a genome comprising: (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 cell comprises a genome comprising: (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.

[0468] In some cases, the engineered stem cell further comprises a polynucleotide encoding a chimeric antigen receptor (CAR), thereby producing an engineered stem cell expressing CAR. Sections III and IV describe exemplary CARs and methods for engineering cells with CARs.

[0469] In some cases, the engineered stem cell further comprises a polynucleotide encoding a FRB, thereby generating an engineered stem cell that expresses a cytoplasmic FRB. The FRB may have a sequence as described in Section II.C. Methods for engineering cells, such as methods using exogenous FRBs, are known and include any of the methods described in Section III.

[0470] In some embodiments, the engineered stem cells are iPSCs. In some embodiments, engineered iPSCs are sequentially differentiated into hematopoietic progenitor cells (HPCs); HPCs differentiate into common lymphoid progenitor cells (CLPs); and CLPs then differentiate into CIL cells—referred to as "iCIL" cells. In one variation, CIL cells can be derived from HPCs by sequentially differentiating HPCs into CLPs, and then differentiating CLPs into iCIL cells. In another variation, CIL cells can be derived by differentiating CLPs into iCIL cells.

[0471] E. Cell differentiation

[0472] Provided herein is a method for differentiating pluripotent stem cells, such as iPSC, into CIL cells using synthetic cytokine receptors. In some embodiments, the differentiation pathway of iPSC includes differentiation into hematopoietic progenitor cells (HP) and common lymphoid progenitor cells (CLP). In some embodiments, differentiation is at least partially directed by the signal induced by synthetic cytokine receptors. In the embodiment of the provided method, the engagement of synthetic cytokine receptors (such as RACR) and their homologous non-physiological ligands (such as rapamycin) can deliver cytokine signals to cells, induce JAK / STAT pathways and drive differentiation. In some aspects, the need for further providing growth factors or cytokines to drive the differentiation of one or more different steps in the process is reduced or eliminated, thereby providing directional and consistent differentiation.

[0473] In some embodiments, the directed differentiation of stem cells, such as pluripotent stem cells (such as iPSC) into hematopoietic progenitor cells (HP) and common lymphoid progenitor cells (CLP) can be achieved by binding synthetic cytokine receptors to non-physiological ligands. In some embodiments, the engagement of synthetic cytokine receptors (such as RACR) with non-physiological ligands (such as rapamycin or the like) drives differentiation by inducing STAT5 signaling through 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 indicate that the engagement of synthetic cytokine receptors (such as RACR) engineered on iPSC with non-physiological ligands (such as rapamycin) is sufficient to drive iPSC to become CLP. This result is surprising because traditional cell differentiation pathways require the use of complex culture medium additives to achieve differentiation into CLP or HP.

[0474] Despite the promise of iPSC-derived cell therapies, the current process for deriving immune cells from iPSCs is complex, variable, and costly. Current approaches to deriving downstream cell types from iPSCs employ the use of exogenous factors to “trick” the iPSCs into engaging endogenous receptors in sequential steps of the differentiation pathway. In some embodiments, this process can require long culture times, expensive protein materials, and high variability due to reliance on constantly changing expression patterns of endogenous genes and receptors. In the provided embodiments, iPSCs are engineered with synthetic cytokine receptors to promote differentiation through highly controlled synthetic receptors, which has the potential to reduce the variability of cell differentiation and reduce the manufacturing cost of these cells by replacing expensive growth factors and cytokines with small molecule binders such as rapamycin.

[0475] In some embodiments, synthetic cytokine receptors (such as RACRs) are engineered into stem cells, such as iPSCs, providing an opportunity to obtain cells through cytokine receptor signals that mimic normal signals during cell differentiation. In particular, synthetic cytokine receptors induce JAK / STAT signaling, which is an essential downstream signaling pathway in the development of blood. Current protocols use a variety of growth factors to induce hematopoiesis in iPSCs, such as thrombopoietin (TPO), stem cell factor (SCF), bone morphogenetic protein (BMP4), and fibroblast activation protein (FGF2)—all of which induce JAK / STAT signaling in combination with other signaling pathways that help drive blood development. Therefore, replacing or supplementing these endogenous receptor pathways with non-physiological stimuli of synthetic cytokine receptors (such as through rapamycin or its analogs) to drive JAK / STAT signals has the potential to improve the blood development process and reduce the need for multiple exogenous protein signals. After blood cells appear during differentiation, blood progenitor cells can further differentiate into lymphoid progenitor cells, including CLPs. In some aspects, common gamma chain cytokines such as IL-7, IL-15, IL-2, and IL-21 are commonly used to drive CD34+ (HP) progenitor cells into differentiated immune cells. However, because synthetic cytokine receptors are designed based on common gamma chain cytokine signaling, the provided embodiments and methods can also be performed with reduced or no need for additional cytokine support and, therefore, can replace the need for these cytokine cocktails during cell differentiation, reducing the cost, variability, and complexity of cell generation.

[0476] In addition, the efficiency of generating HP and CLP from iPSC using traditional methods is usually very low, and is a bottleneck of the current manufacturing effector immune cell therapy. In some cases, only about 1X HP yield can be obtained from iPSC (1 HP is produced per 1 iPSC). In some embodiments, the method provided herein can increase the HP yield from iPSC to or increase by about 10 times (10X), 20 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 150 times, 200 times, 250 times, 300 times. In some embodiments, the method provided herein can increase the HP yield from iPSC to or increase by about 350 times, 400 times, 450 times, 500 times, 550 times, 600 times, 650 times, 1,000 times, 1,500 times, 2,000 times, 2,500 times, 3,000 times, 3,500 times. In some embodiments, the methods provided herein can increase the HP production from iPSC to or by about 630 times. In some embodiments, the methods provided herein can increase the HP production from iPSC to or by about 3,000 times. Compared to other methods for differentiating iPSC into HP, this result represents a substantial improvement. In addition, the generation of iCILs from HP using the provided embodiments results in highly potent iCILs, as demonstrated by killing tumor target cells.

[0477] In some embodiments, the provided methods can increase the yield of iCILs from iPSCs to or about 1×10 6 times (1,000,000X), 1.5x10 6 times, 2x10 6 times, 2.5x10 6 times, 3x10 6 times, 3.5x10 6 times, 4x10 6 times, 4.5×10 6 times, 5×10 6 times, 5.5×10 6 times, 6×10 6 times, 6.5×10 6 times, 7×10 6 times, 7.5×10 6 times, 8×10 6 times, 8.5×10 6 times, 9×10 6 times, 9.5×10 6 times, 10×10 6 In some embodiments, the provided methods can increase the yield of iCILs from iPSCs to or about 9x10 6 times.

[0478] In some embodiments, the cell selected for differentiation is pluripotent stem cell (PSC), such as iPSC, and these cells have been engineered to have synthetic cytokine receptors, as described in Section II.B. In some embodiments, the cell selected for differentiation is pluripotent stem cell (PSC), such as iPSC, and these cells are further destroyed the gene encoding B2M, for example, to reduce expression or knock out the gene encoding B2M. In some embodiments, as by HDR or other methods, engineered synthetic cytokine receptors are integrated into destroyed B2M locus. In some embodiments, the cell selected for differentiation is pluripotent stem cell (PSC), such as iPSC, and these cells are further destroyed the gene encoding FBP12, for example, to reduce expression or knock out the gene encoding FBP12. In some embodiments, the cell selected for differentiation is pluripotent stem cell (PSC), such as iPSC, which is any engineered cell described in Section ID.

[0479] In some embodiments, the provided methods include culturing engineered PSCs (such as iPSCs) under conditions that allow the stem cells to differentiate into iCIL cells or their progenitor cells (such as HP or CLP) by incubating with a non-physiological ligand for a synthetic cytokine receptor (such as rapamycin or its analogs). In some embodiments, the methods can include an additional incubation in which a different molecule is added to the culture medium. In some embodiments, the methods can include replacing the culture medium to supplement or add any one or more molecules to the culture medium.

[0480] In some embodiments, the method comprises first incubating the cells with a non-physiological ligand for a synthetic cytokine receptor (such as rapamycin or an analogue) under conditions that induce differentiation of the cells into hematopoietic progenitor (HP) cells. In some embodiments, one or more additional molecules (such as small molecules) may be added to further promote or induce differentiation into hematopoietic progenitor (HP) cells.

[0481] In some embodiments, one or more steps of producing HP cells may include adding a non-physiological ligand (such as rapamycin or an analogue) of a synthetic cytokine receptor to the culture medium to induce differentiation. In some embodiments, the non-physiological ligand is rapamycin or an analogue of rapamycin. In some embodiments, the analogue of rapamycin is a rapamycin analogue (rapalog). In some embodiments, the non-physiological ligand (such as rapamycin or a rapamycin analogue) is added to the culture medium at a concentration of 2.5nM-200nM, 2.5nM-150nM, 2.5nM-100nM, 2.5nM-50nM, 2.5nM-20nM, or 2.5nM-10nM. In some embodiments, a non-physiological ligand (such as rapamycin or a rapamycin analog) is added at a concentration of 5nM-200nM, 5nM-150nM, 5nM-100nM, 5nM-50nM, 5nM-20nM, 5nM-10nM, 10nM-200nM, 10nM-150nM, 10nM-100nM, 10nM-50nM, 1 Concentrations of 0 nM-20 nM, 20 nM-200 nM, 20 nM-150 nM, 20 nM-100 nM, 20 nM-50 nM, 50 nM-200 nM, 50 nM-150 nM, 50 nM-100 nM, 100 nM-200 nM, 100 nM-150 nM, and 150 nM-200 nM were added to the culture medium.

[0482] In some embodiments, a non-physiological ligand (such as rapamycin or a rapamycin analog) is added to the culture medium at a concentration of 10 nM or about 10 nM. In some embodiments, a non-physiological ligand (such as rapamycin or a rapamycin analog) is added to the culture medium at a concentration of 100 nM or about 100 nM. In some embodiments, rapamycin is added to the culture medium at a concentration of 100 nM or about 100 nM. In some embodiments, a rapamycin analog is added to the culture medium at a concentration of 100 nM or about 100 nM.

[0483] In some embodiments, it is surprising to find that low concentrations of non-physiological ligands (such as rapamycin or rapamycin analogs) can support differentiation and / or amplification. In some embodiments, non-physiological ligands (such as rapamycin or rapamycin analogs) are added to culture medium at a concentration of 10nM or less than 10nM. In some embodiments, non-physiological ligands (such as rapamycin or rapamycin analogs) are added to culture medium at a concentration of 2.5nM to 10nM, such as 3nM to 7nM. In some embodiments, non-physiological ligands (such as rapamycin or rapamycin analogs) are added to culture medium at a concentration of at or about 3nM, at or about 4nM, at or about 5nM, at or about 6nM, at or about 7nM, at or about 8nM, at or about 9nM or at or about 10nM, or any value between any of the above concentrations. In some embodiments, a non-physiological ligand (such as rapamycin or a rapamycin analog) is added to the culture medium at a concentration of 100 nM or about 3.1 nM. In some embodiments, a non-physiological ligand (such as rapamycin or a rapamycin analog) is added to the culture medium at a concentration of 100 nM or about 6.2 nM. In some embodiments, rapamycin is added to the culture medium at a concentration of 100 nM or about 3.1 nM. In some embodiments, rapamycin is added to the culture medium at a concentration of 100 nM or about 6.2 nM. In some embodiments, rapamycin is added to the culture medium at a concentration of 100 nM or about 3.1 nM. In some embodiments, rapamycin is added to the culture medium at a concentration of 100 nM or about 6.2 nM.

[0484] In some embodiments, provided methods include culturing engineered stem cells, e.g., stem cells engineered with synthetic cytokine receptors, with a non-physiological ligand, culturing the engineered stem cells with the non-physiological ligand for a first period of time sufficient to generate CLPs, and contacting the CLPs with a differentiation medium for a second period of time sufficient to generate iCILs.

[0485] In some embodiments, conditions other than or in addition to activation with synthetic cytokine receptors can be used in methods of differentiating engineered stem cells into CILs.

[0486] In some embodiments, the stem cells, such as iPSCs, engineered with synthetic cytokine receptors provided herein can be differentiated, instead or alternatively, by any other known method for differentiating CILs. In some embodiments, in addition to non-physiological ligand engagement of synthetic cytokine receptors, one or more growth factors or cytokines typically used in connection with the differentiation of CILs can also be used in the provided methods.

[0487] Various differentiation protocols for CIL cells are well known in the art.

[0488] In some embodiments, stem cells are suitable for feeder-free culture. As used herein, a "feeder-free" (FF) environment refers to an environment, such as culture conditions, cell culture, or culture medium that is substantially free of feeder cells or stromal cells, and / or an environment that has not been preconditioned by feeder cell culture. "Preconditioned" culture medium refers to culture medium that has been harvested after feeder cells have been cultured in the culture medium for a period of time, such as at least one day. Preconditioned culture medium contains a variety of mediators, including growth factors and cytokines, secreted by the feeder cells cultured in the culture medium.

[0489] In a broad sense, cell differentiation technology involves using approaches based on polynucleotides, polypeptides and / or small molecules to directly or indirectly regulate specific cell pathways. For example, the developmental efficacy of cells can be regulated by contacting the cells with one or more regulators. In some embodiments, cells are cultured in the presence of one or more agents (such as small molecules, proteins, peptides, etc.) that induce cell differentiation. In some embodiments, one or more differentiation agents are introduced into cells during in vitro culture. Cells can be maintained in a culture medium containing one or more agents for a sufficiently long time so that the cells reach the desired differentiation phenotype.

[0490] In some embodiments, the culture platform comprises one or more of the following: nutrients, extracts, growth factors, hormones, cytokines, and culture medium additives. Exemplary nutrients and extracts may include, for example, DMEM / F-12 (Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12), a widely used basal medium for supporting the growth of a variety of different mammalian cells; KOSR (Knockout Serum Replacement); L-glut; NEAA (non-essential amino acids). Culture medium additives may include, but are not limited to, MTG, ITS, (ME, antioxidants (e.g., ascorbic acid).

[0491] In some embodiments, the differentiation medium comprises supplements such as serum, extracts, growth factors, hormones, cytokines, and the like.

[0492] In some embodiments, the culture medium of the present invention comprises one or more of the following cytokines or growth factors: 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).

[0493] These cytokines are commercially available and can be natural or recombinant. In some other embodiments, the culture medium of the present disclosure includes 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 factor (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, growth factors, mitogens and cytokines are stage and / or cell type specific, and their concentrations can be determined empirically or under the guidance of established cytokine prior art. WO2020 / 124256 provides examples of exogenous cell culture medium additives and supplements and cell selection kit components, and all of its disclosures are incorporated herein by reference.

[0494] In some other embodiments, the culture medium of the present invention comprises Roswell Park Memorial Institute (RPMI) culture medium, cRPMI1640 culture medium, fetal bovine serum (FBS), Glutamax, penicillin, streptomycin, rosuvastatin, BX795, sulfatamine, 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 invention comprises AIM V culture medium, fetal bovine serum (FBS), Glutamax, penicillin, streptomycin, rosuvastatin, BX795, sulfatamine, 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 invention comprises AIM V medium STEMdiff APEL 2 culture medium (STEMCELL Technologies).

[0495] Examples of methods for differentiating stem cells (such as iPSCs) into multipotent 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.

[0496] In some embodiments, the method of producing CIL cells disclosed herein comprises: forming embryoid bodies (EBs) 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 yield EBs that are completely dissociated into pure hematopoietic progenitor cells or hematopoietic stem cells without the need for additional purification steps.

[0497] In some embodiments, a method of generating cytotoxic innate lymphoid cells (iCILs) is provided, comprising culturing a cell population comprising engineered iPSCs under conditions that differentiate the iPSCs into cytotoxic innate lymphoid cells (iCILs), wherein a non-physiological ligand for a synthetic cytokine receptor is added during at least a portion of the culturing process.

[0498] In some embodiments, one or more of the steps of producing CIL cells described above may include adding a non-physiological ligand for a synthetic cytokine receptor (such as rapamycin or an analogue) to the culture medium to induce differentiation.

[0499] In some embodiments, the non-physiological ligand is rapamycin or an analog of rapamycin. In some embodiments, the analog of rapamycin is a rapamycin analog (rapalog). In some embodiments, the non-physiological ligand (such as rapamycin or a rapamycin analog) is added to the culture medium at a concentration of 2.5nM-200nM, 2.5nM-150nM, 2.5nM-100nM, 2.5nM-50nM, 2.5nM-20nM, or 2.5nM-10nM. In some embodiments, a non-physiological ligand (such as rapamycin or a rapamycin analog) is added at a concentration of 5nM-200nM, 5nM-150nM, 5nM-100nM, 5nM-50nM, 5nM-20nM, 5nM-10nM, 10nM-200nM, 10nM-150nM, 10nM-100nM, 10nM-50nM, Concentrations of 10 nM-20 nM, 20 nM-200 nM, 20 nM-150 nM, 20 nM-100 nM, 20 nM-50 nM, 50 nM-200 nM, 50 nM-150 nM, 50 nM-100 nM, 100 nM-200 nM, 100 nM-150 nM, and 150 nM-200 nM were added to the culture medium.

[0500] In some embodiments, a non-physiological ligand (such as rapamycin or a rapamycin analog) is added to the culture medium at a concentration of 10 nM or about 10 nM. In some embodiments, a non-physiological ligand (such as rapamycin or a rapamycin analog) is added to the culture medium at a concentration of 100 nM or about 100 nM. In some embodiments, rapamycin is added to the culture medium at a concentration of 100 nM or about 100 nM. In some embodiments, a rapamycin analog is added to the culture medium at a concentration of 100 nM or about 100 nM.

[0501] In some embodiments, it is surprising to find that low concentrations of non-physiological ligands (such as rapamycin or rapamycin analogs) can support differentiation and / or amplification. In some embodiments, non-physiological ligands (such as rapamycin or rapamycin analogs) are added to culture medium at a concentration of 10 nM or less than 10 nM. In some embodiments, non-physiological ligands (such as rapamycin or rapamycin analogs) are added to culture medium at a concentration of 2.5 nM to 10 nM, such as 3 nM to 7 nM. In some embodiments, non-physiological ligands (such as rapamycin or rapamycin analogs) are added to culture medium at a concentration of at or about 3 nM, at or about 4 nM, at or about 5 nM, at or about 6 nM, at or about 7 nM, at or about 8 nM, at or about 9 nM or at or about 10 nM, or any value between any of the above concentrations. In some embodiments, a non-physiological ligand (such as rapamycin or a rapamycin analog) is added to the culture medium at a concentration of 100 nM or about 3.1 nM. In some embodiments, a non-physiological ligand (such as rapamycin or a rapamycin analog) is added to the culture medium at a concentration of 100 nM or about 6.2 nM. In some embodiments, rapamycin is added to the culture medium at a concentration of 100 nM or about 3.1 nM. In some embodiments, rapamycin is added to the culture medium at a concentration of 100 nM or about 6.2 nM. In some embodiments, rapamycin is added to the culture medium at a concentration of 100 nM or about 3.1 nM. In some embodiments, rapamycin is added to the culture medium at a concentration of 100 nM or about 6.2 nM.

[0502] In some embodiments, the method of producing cytotoxic innate lymphoid cells (iCIL) comprises a) culturing a cell population comprising the engineered iPSCs under conditions in which aggregates are formed; b) culturing the cells produced in a) under conditions in which a plurality of cells are induced to form mesoderm, wherein the start of culturing in b) is day 0; c) culturing the cells produced in b) under conditions in which the cells are differentiated into a hematopoietic progenitor (HP) population; and d) culturing the cells produced in c) under conditions in which iCIL cells are produced, wherein at least a portion of one or more of steps a)-d) is performed in the presence of a non-physiological ligand for a synthetic cytokine receptor.

[0503] In some embodiments, to promote the formation of aggregates, the engineered iPSCs are transferred to a suitable container. The container can be a two-dimensional or three-dimensional container. Two-dimensional containers suitable for culturing source cells include any culture dish or culture dish commonly used to culture cells in the laboratory. The culture container can be coated with a suitable culture medium, such as an extracellular medium for culturing cell attachment and / or differentiation. In some embodiments, the blood vessels are treated to promote cell adhesion and growth. An example of a culture medium suitable for the method of the present invention is MATRIGEL TMMembrane matrix (BD Biosciences, Franklin Lakes, NJ).

[0504] In some embodiments, the container is suitable for three-dimensional (3D) culture. Without being bound by a particular theory or mechanism, it is believed that three-dimensional culture may be more effective than two-dimensional (2D) culture in providing a scaffold for cell differentiation. Suitable three-dimensional culture systems may include, for example, hanging drop three-dimensional culture (such as hanging drop plates), three-dimensional microwell culture (such as ultra-low attachment multiwell plates), three-dimensional culture on hydrophobic surfaces, rotary culture, static three-dimensional suspension culture or bioreactors. Hanging drop plates are commercially available, for example, the PERFECTA 3D hanging drop plates from Biospherix, Parish, NY. Ultra-low attachment multiwell plates (also referred to as non-adhesive culture vessels in some cases) are also commercially available, for example, the AGGREWELL available from Stemcell Technologies, Vancouver, Canada. TM Ultra-low attachment multi-well plates.

[0505] 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 do not substantially adhere during culture. In some embodiments, the cells are cultured in suspension.

[0506] 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.

[0507] In some embodiments, the container is a bioreactor. In some embodiments, the bioreactor is used for the process of generating and proliferating iCILs after EB development. Bioreactors allow for optimization of cell culture conditions to achieve optimal hydrogen production and process robustness. Environmental conditions that can be adjusted or monitored in a bioreactor include gas composition (e.g., air, oxygen, nitrogen, carbon dioxide), gas flow rate, temperature, pH, dissolved oxygen level, and agitation speed / circulation rate within the cell culture. Any type of bioreactor known in the art can be used for cell culture to differentiate and expand cultured iCIL cells, including but not limited to stirred tank bioreactors, pneumatic bioreactors (e.g., bubble columns or airlift bioreactors), membrane bioreactors, hollow fiber bioreactors, wave bioreactors, vertical wheel bioreactors, gas permeation rapid expansion (G-Rex) bioreactors, or disposable bioreactors. In some embodiments, the bioreactor is a gas permeation rapid expansion (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.

[0508] iCIL cells can be expanded in any desired volume to suit a variety of purposes. For example, for medium- and high-throughput screening across a variety of culture conditions, the process can be scaled up in a microbial reactor (e.g., about 15 mL to about 500 mL) or a benchtop bioreactor (e.g., about 0.5 L to about 15 L). Alternatively, the process can be scaled up to a pilot-scale bioreactor (e.g., about 15 L to about 15,000 L) or a production-scale bioreactor (e.g., about 15,000 L to about 75,000 L or larger).

[0509] In some embodiments, multipotent aggregates can be formed in a bioreactor by suspending engineered iPSC in a bioreactor. In some embodiments, iPSC can spontaneously aggregate into spheroids directly in a bioreactor. In some embodiments, the size of the aggregates or spheroids formed directly in the bioreactor can be roughly the same as the spheroids formed in other 3D culture systems (such as ultra-low attachment microplates). In one embodiment, the method may include culturing source cells in a bioreactor with a xeno-free culture medium, and directly culturing the source cells into spheroids in the bioreactor without forming EBs. Spheroids may include undifferentiated iPSCs, such as engineered iPSCs.

[0510] In some embodiments, the aggregates in a) are embryoid bodies (EBs).

[0511] In some embodiments, cell differentiation to generate CIL cells requires changing the culture system, such as changing the stimulant in the culture medium or the physical state of the cells. The traditional strategy is to use the formation of embryoid bodies as a common and key intermediate to initiate lineage-specific differentiation. Embryoid bodies are aggregates of stem cells that are induced to differentiate through changes in environmental stimuli (such as contact and / or removal of specific molecules / chemical factors; and / or contact / interaction with three-dimensional structures). The formation of embryoid bodies promotes the specific differentiation of cells into the mesoderm.

[0512] Hematopoietic cells can be produced in the embryoid bodies that pluripotent cells obtain. As a part of differentiation process, pluripotent cells can be formed into embryoid bodies or aggregates. In order to induce differentiation and form " embryoid bodies " (EB) or growth cell clusters, it is usually necessary to aggregate human pluripotent stem cells into EB in vitro, and allow human pluripotent stem cells to spontaneously and randomly differentiate into the various tissue types representing endoderm, ectoderm and mesoderm origin. If there is no specific culture condition, EB may need about two weeks to differentiate to any one layer in the three germinal layers, and the differentiation process is carried out in a random pattern. In the embodiment provided, certain growth factors or cytokines can be added in culture condition to guide or strengthen EB to differentiate into hematopoietic lineage, which is formed by mesoderm pedigree. The EB cells differentiated into hematopoietic lineages can be identified by major hematopoietic lineage markers, for example any one or more of CD34, CD43, CD45, CD41, C235 and CD90.

[0513] In some embodiments, a single cell suspension of engineered iPSCs is cultured in an appropriate container to form EBs.

[0514] In some embodiments, pluripotent stem cell aggregates are transferred to a differentiation medium that provides induction cues for lineage selection (e.g., lymphoid lineage). In some embodiments, once EBs have formed, they are dissociated and then cultured in a culture medium to induce mesodermal specificity of the cells.

[0515] In some embodiments, suspension polymers can be produced by culturing in an untreated container or under suspension culture conditions. For example, the container is an untreated container and cannot promote cell adhesion and proliferation. In some embodiments, the step a) of culturing a cell colony comprising engineered iPSC under conditions of forming a polymer involves: (i) performing a first incubation, wherein the first incubation includes culturing a cell colony of engineered stem cells under conditions of forming a first aggregate; (ii) contacting the aggregate with a dissociating agent to form a dissociated cell colony; and (iii) performing a second incubation, wherein the second incubation includes culturing the dissociated cell colony under conditions of 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, dissociation is performed using a gentle cell dissociation reagent (GDCR; Stem Cell Technologies). In some embodiments, EDTA is used for dissociation.

[0516] In some aspects of the present invention, the above-described embryoid body derivation and differentiation into hematopoietic progenitor cells platform can be performed under serum-free conditions. Commercially available serum-free culture media suitable for cell attachment and / or induction include mTeSR from Stem Cell Technologies (Vancouver, Canada). TM 1. STEMdiff APEL 2 medium or TeSR TM 2. Primate ES / iPS cell culture medium from ReproCELL (Boston, MA), primate ES / iPS cell culture medium from Invitrogen (Carlsbad, CA), -34, from Invitrogen hESC SFM and X-VIVO from Lonza (Basel, Switzerland) TM .

[0517] In some embodiments, the culture medium for inducing the mesoderm specificity 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), and basic fibroblast growth factor (bFGF also known as FGF2). In some embodiments, the culture medium for inducing the mesoderm specificity 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), and basic fibroblast growth factor (bFGF also known as FGF2) and / or a ROCK inhibitor. In some embodiments, the culture medium for inducing the mesoderm specificity 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), and basic fibroblast growth factor (bFGF also known as FGF2) and / or a ROCK inhibitor. In some embodiments, the method comprises differentiating mesodermal-specific cells into hematopoietic stem cells in a first differentiation medium.

[0518] In some embodiments, the hematopoietic stem cells in the first differentiation medium include 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), TGF-β inhibitor, PI3K inhibitor, or any combination thereof.

[0519] 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.

[0520] In some embodiments, stem cells are differentiated into the mesodermal lineage in a differentiation medium comprising BMP4, FGF2, and VEGF. In some embodiments, stem cells are differentiated into the mesodermal lineage in a differentiation medium comprising BMP4, FGF2, VEGF, and a ROCK inhibitor. In some embodiments, stem cells are differentiated into the mesodermal lineage in a differentiation medium comprising BMP4, FGF2, VEGF, and Y27632.

[0521] In some embodiments, the culturing in b) is carried out in a culture medium comprising one or more of BMP4, FGF2, VEGF, and a Rock inhibitor. In some embodiments, the ROCK inhibitor is Y27632. In some embodiments, the culturing in b) is carried out in a culture medium comprising BMP4, FGF2, VEGF, and Y27632. In some embodiments, the culturing in b) is carried out in a culture medium comprising BMP4, FGF2, and VEGF. In some embodiments, the culturing in b) is carried out in a culture medium containing a non-physiological ligand. In some embodiments, the culturing in b) is carried out in a culture medium containing the non-physiological ligand without any additional growth factors.

[0522] In some embodiments, the culturing in b) lasts for 2-4 days. In some embodiments, the cells are cultured in the differentiation medium for 2 to 4 days. In some cases, the culture is for or about 2 days, for or about 3 days, or for or about 4 days. In some embodiments, the culture is for or about 3 days. In some embodiments, the concentration of BMP4 in the culture 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. In some embodiments, the concentration of BMP4 in the culture medium is at least about 0.5 ng / mL, 2.5 ng / mL, 50 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 30 ng / mL, or 50 ng / mL, inclusive. In some embodiments, the concentration of BMP4 in the culture medium is at least about 0.5 ng / mL, 2.5 ng / mL, 50 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 30 ng / mL, or 50 ng / mL, inclusive. In some embodiments, the concentration of BMP4 in the culture medium is about 10 ng / mL.

[0523] In some embodiments, the concentration of FGF2 in the culture medium is about 0.5 ng / mL-2.5 ng / mL, 0.5 ng / mL-5 ng / mL, 0.5 ng / mL-10 ng / mL, 0.5 ng / mL-15 ng / mL, 0.5 ng / mL-20 ng / mL, 0.5 ng / mL-30 ng / mL, 0.5 ng / mL-50 ng / mL, 2.5 ng / mL-5 ng / mL, 2.5 ng / mL-10 ng / mL, 2.5 ng / mL-15 ng / mL, 2. In some embodiments, the concentration of FGF2 in the culture medium is at least about 0.5 ng / mL, 2.5 ng / mL, 50 ng / mL, 5 ng / mL-10 ng / mL, 5 ng / mL-15 ng / mL, 5 ng / mL-20 ng / mL, 5 ng / mL-30 ng / mL, 5 ng / mL-50 ng / mL, 10 ng / mL-15 ng / mL, 10 ng / mL-20 ng / mL, 10 ng / mL-30 ng / mL, 10 ng / mL-50 ng / mL, inclusive. In some embodiments, the concentration of FGF2 in the culture 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 culture medium is about 10 ng / mL.

[0524] In some embodiments, the VEGF concentration in the culture medium is about 0.5 ng / mL-2.5 ng / mL, 0.5 ng / mL-5 ng / mL, 0.5 ng / mL-10 ng / mL, 0.5 ng / mL-15 ng / mL, 0.5 ng / mL-20 ng / mL, 0.5 ng / mL-30 ng / mL, 0.5 ng / mL-50 ng / mL, 0.5 ng / mL-100 ng / mL, 2.5 ng / mL-5 ng / mL, 2.5 ng / mL-10 ng / mL, 2.5 ng / mL-15 ng / mL, 2.5 ng / mL-20 ng / mL, 2 .5ng / mL-30ng / mL, 2.5ng / mL-50ng / mL, 2.5ng / mL-100ng / mL, 5ng / mL-10ng / mL, 5ng / mL-15ng / mL, 5ng / mL-20ng / mL, 5ng / mL-30ng / mL, 5ng / mL-50ng / mL, 5ng / mL-100ng / mL, 10ng / mL-15ng / mL, 10ng / mL-20ng / mL, 10ng / mL-30ng / mL, 10ng / mL-50ng / mL, 10ng / mL-100ng / mL, inclusive. In some embodiments, the concentration of VEGF in the culture 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 culture medium is about 50 ng / mL.

[0525] In some embodiments, the concentration of Y27632 in the culture medium is about 0.5 μM-2.5 μM, 0.5 μM-5 μM, 0.5 μM-10 μM, 0.5 μM-15 μM, 0.5 μM-20 μM, 0.5 μM-30 μM, 0.5 μM-50 μM, 2.5 μM-5 μM, 2.5 μM-10 μM, 2.5 μM- In some embodiments, the concentration of Y27632 in the culture 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 culture medium is about 10 μM.

[0526] In some embodiments, mesodermal cells are differentiated into hematopoietic stem cells in a differentiation medium comprising BMP4, FGF2, VEGF and SCF. In some embodiments, mesodermal cells are differentiated into hematopoietic stem cells in a differentiation medium comprising BMP4, FGF2, VEGF, SCF, TPO and LDL. In some embodiments, mesodermal cells are differentiated into hematopoietic stem cells in a differentiation medium comprising BMP4, FGF2 and VEGF. In some embodiments, the differentiation medium comprises a non-physiological ligand for a synthetic cytokine receptor (such as rapamycin or an analogue).

[0527] In some embodiments, hematopoietic stem cells are differentiated into lymphoid progenitor cells in a differentiation medium comprising BMP4, FGF2, VEGF and SCF. In some embodiments, hematopoietic stem cells are differentiated into lymphoid progenitor cells in a differentiation medium comprising BMP4, FGF2 and VEGF. In some embodiments, hematopoietic stem cells are differentiated into lymphoid progenitor cells in a differentiation medium comprising BMP4, FGF2, VEGF, SCF, TPO and LDL components. In some embodiments, the differentiation medium comprises a non-physiological ligand for a synthetic cytokine receptor (such as rapamycin or an analogue).

[0528] In some embodiments, the culturing in c) is carried out in a culture medium comprising one or more of BMP4, FGF2, VEGF, TPO, SCF, and LDL. In some embodiments, the culturing in c) is carried out in a culture medium comprising one or more of BMP4, FGF2, VEGF, and LDL. In some embodiments, the culturing in c) is carried out in a culture medium without SCF and TPO. In some embodiments, the culturing in c) is carried out in a culture medium comprising one or more of BMP4, FGF2, and a PI3K inhibitor. In some embodiments, the PI3K inhibitor is LY2940002. In some embodiments, the culturing in c) is carried out in a culture medium without LDL, VEGF, SCF, and / or TPO. In some embodiments, the culturing in c) is carried out in a culture medium containing a non-physiological ligand. In some embodiments, the culturing in c) is carried out in a culture medium containing the non-physiological ligand, the culture medium being free of any additional growth factors, cytokines, or both.

[0529] In some embodiments, the culture in b) lasts for 3-15 days. In some embodiments, during at least a portion of the culture in c), the culture medium comprises an aryl hydrocarbon receptor (AHR) antagonist (such as StemRegenin-1), a pyrimidine-[4,5-b]-indole derivative (such as UM729), or both. In some embodiments, the portion of the culture is carried out on day 9-15 or about day 9-15, or for day 9-15 or about day 9-15. In some embodiments, the portion of the culture is carried out on day 6-15 or about day 9-15, or for day 6-15 or about day 6-15. In some embodiments, the culture in c) is carried out on day 6. In some embodiments, the culture in c) is carried out on day 9.

[0530] In some embodiments, the concentration of BMP4 in the culture medium is about 0.5 ng / mL-2.5 ng / mL, 0.5 ng / mL-5 ng / mL, 0.5 ng / mL-10 ng / mL, 0.5 ng / mL-15 ng / mL, 0.5 ng / mL-20 ng / mL, 0.5 ng / mL-30 ng / mL, 0.5 ng / mL-50 ng / mL, 2.5 ng / mL-5 ng / mL, 2.5 ng / mL-10 ng / mL, 2.5 ng / mL-15 ng / mL, 2. In some embodiments, the concentration of BMP4 in the culture medium is at least about 0.5 ng / mL, 2.5 ng / mL, 50 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 30 ng / mL, or 50 ng / mL, inclusive. In some embodiments, the concentration of BMP4 in the culture medium is at least about 0.5 ng / mL, 2.5 ng / mL, 50 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 30 ng / mL, or 50 ng / mL, inclusive. In some embodiments, the concentration of BMP4 in the culture medium is about 10 ng / mL.

[0531] In some embodiments, the concentration of FGF2 in the culture medium is about 0.5 ng / mL-2.5 ng / mL, 0.5 ng / mL-5 ng / mL, 0.5 ng / mL-10 ng / mL, 0.5 ng / mL-15 ng / mL, 0.5 ng / mL-20 ng / mL, 0.5 ng / mL-30 ng / mL, 0.5 ng / mL-50 ng / mL, 2.5 ng / mL-5 ng / mL, 2.5 ng / mL-10 ng / mL, 2.5 ng / mL-15 ng / mL, 2. In some embodiments, the concentration of FGF2 in the culture medium is at least about 0.5 ng / mL, 2.5 ng / mL, 50 ng / mL, 5 ng / mL-10 ng / mL, 5 ng / mL-15 ng / mL, 5 ng / mL-20 ng / mL, 5 ng / mL-30 ng / mL, 5 ng / mL-50 ng / mL, 10 ng / mL-15 ng / mL, 10 ng / mL-20 ng / mL, 10 ng / mL-30 ng / mL, 10 ng / mL-50 ng / mL, inclusive. In some embodiments, the concentration of FGF2 in the culture 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 culture medium is about 10 ng / mL.

[0532] In some embodiments, the concentration of SCF in the culture medium is about 0.5 ng / mL-2.5 ng / mL, 0.5 ng / mL-5 ng / mL, 0.5 ng / mL-10 ng / mL, 0.5 ng / mL-15 ng / mL, 0.5 ng / mL-20 ng / mL, 0.5 ng / mL-30 ng / mL, 0.5 ng / mL-50 ng / mL, 0.5 ng / mL-100 ng / mL, 2.5 ng / mL-5 ng / mL, 2.5 ng / mL-10 ng / mL, 2.5 ng / mL-15 ng / mL, 2.5 ng / mL-20 ng / mL, 2. 5ng / mL-30ng / mL, 2.5ng / mL-50ng / mL, 2.5ng / mL-100ng / mL, 5ng / mL-10ng / mL, 5ng / mL-15ng / mL, 5ng / mL-20ng / mL, 5ng / mL-30ng / mL, 5ng / mL-50ng / mL, 5ng / mL-100ng / mL, 10ng / mL-15ng / mL, 10ng / mL-20ng / mL, 10ng / mL-30ng / mL, 10ng / mL-50ng / mL, 10ng / mL-100ng / mL, inclusive. In some embodiments, the concentration of SCF in the culture 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. In some embodiments, the concentration of SCF in the culture medium is about 50 ng / mL.

[0533] In some embodiments, the concentration of UM729 in the culture medium is about 0.5 μM-1 μM, 0.5 μM-5 μM, 0.5 μM-10 μM, 0.5 μM-15 μM, 0.5 μM-20 μM, 0.5 μM-30 μM, 0.5 μM-50 μM, 1 μM-5 μM, 1 μM-10 μM, 1 μM-15 μM, 1 μM-20 μM, 1 μM-30 μM, 1 μM-50 μM, 5 μM-10 μM, 5 μM-15 μM, 5 μM-20 μM, 5 μM-30 μM, 5 μM-50 μM, 10 μM-15 μM, 10 μM-20 μM, 10 μM-30 μM, 10 μM-50 μM, inclusive. In some embodiments, the concentration of UM729 in the culture medium is at least about 0.5 μM, 1 μM, 5 μM, 10 μM, 15 μM, 20 μM, 30 μM, or 50 μM. In some embodiments, the concentration of UM729 in the culture medium is about 1 μM.

[0534] In some embodiments, the concentration of SR1 in the culture medium is about or below about 0.5 μM-1 μM, 0.5 μM-5 μM, 0.5 μM-10 μM, 0.5 μM-15 μM, 0.5 μM-20 μM, 0.5 μM-30 μM, 0.5 μM-50 μM, 1 μM-5 μM, 1 μM-10 μM, 1 μM-15 μM, 1 μM-20 μM, 1 μM-30 μM, 1 μM-50 μM, 5 μM-10 μM, 5 μM-15 μM, 5 μM-20 μM, 5 μM-30 μM, 5 μM-50 μM, 10 μM-15 μM, 10 μM-20 μM, 10 μM-30 μM, 10 μM-50 μM, inclusive. The concentration of SR1 in the culture medium is at least about 0.5 μM, 1 μM, 5 μM, 10 μM, 15 μM, 20 μM, 30 μM, or 50 μM. In some embodiments, the concentration of SR1 in the culture medium is about 1 μM.

[0535] In some embodiments, lymphoid progenitor cells are differentiated into cytotoxic innate lymphoid cells in a differentiation medium comprising a non-physiological ligand, SCF, FLT3L, and UM729. In some embodiments, lymphoid progenitor cells are differentiated into cytotoxic innate lymphoid cells in a differentiation medium comprising a non-physiological ligand, SCF, and UM729. In some embodiments, the non-physiological ligand is a rapamycin analog. In some embodiments, the non-physiological ligand is rapamycin.

[0536] In some embodiments, the mesoderm-specific cells are differentiated into hematopoietic stem cells in the first differentiation medium for a time sufficient to allow the mesoderm-specific cells to become CD34+ hematopoietic stem cells.

[0537] In some embodiments, the method comprises differentiating the hematopoietic stem cells into lymphoid progenitor cells in a second differentiation medium.

[0538] In some embodiments, the lymphoid progenitor differentiation medium includes 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.

[0539] In some embodiments, the hematopoietic stem cells are differentiated into lymphoid progenitor cells in the second differentiation medium for a period of time sufficient to allow the hematopoietic stem cells to become Lin-CD34+CD38- / loCD45RA+CD90- lymphoid progenitor cells.

[0540] 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.

[0541] In some embodiments, the first differentiation medium and / or the second differentiation medium is substantially free of at least one cytokine (eg, IL-2, IL-15, and / or IL-7).

[0542] 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), a pyrimidine-indole derivative, or any combination thereof. The pyrimidine-indole derivative is UM729 (pyrimidine-[4,5-b]-indole derivative).

[0543] In some embodiments, the culturing in d) is performed in a culture 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 performed in a culture medium comprising one or more of IL-15, SCR, SR-1, and UM729. In some embodiments, the culturing in d) is performed in a culture medium without FLT3L, IL-7, and / or IL-12. In some embodiments, the culturing in d) is performed in a culture medium containing a non-physiological ligand. In some embodiments, the culturing in d) is performed in a culture medium containing the non-physiological ligand without any additional growth factors, cytokines, or both. In some embodiments, the culturing in d) is performed between day 15 and day 40, or for a period of 15-40 days. In some embodiments, the culturing in d) is performed between day 15 and day 35, or for a period of 15-35 days. In some embodiments, the culturing in d) is performed between day 15 and day 30, or for a period of 15-30 days.

[0544] In some embodiments, the lymphoid progenitor cells are differentiated into CIL cells in a differentiation medium for a time sufficient to allow 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, the lymphoid progenitor cells are differentiated into CIL cells in a differentiation medium for a time sufficient to allow the lymphoid progenitor cells to become CD45+CD56+. In some embodiments, the lymphoid progenitor cells are differentiated into CIL cells in a differentiation medium for a time sufficient to allow the lymphoid progenitor cells to become CD45+CD5-CD7+CD56+.

[0545] In some embodiments, the lymphoid progenitor cells are differentiated into CIL cells in the differentiation medium for about 8 to about 18 days. In some embodiments, the lymphoid progenitor cells are differentiated into CIL cells in the differentiation medium for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 days.

[0546] 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.

[0547] In some embodiments, after differentiation, CIL cells are expanded in a medium containing non-physiological ligands and CD2 / NKp46 stimulation. In some embodiments, the CIL cell expansion medium includes activation beads containing conjugated anti-CD2 and anti-NKp46 antibodies that can stimulate and activate CIL cells.

[0548] In some embodiments, after differentiation, RACR-iCIL cells are expanded in medium containing a non-physiological ligand, membrane-bound IL-21 (mbIL21), and 41BBL K562 primed feeder cells.

[0549] F. Induced Cytotoxic Innate Lymphoid Cells (iCIL) and Expansion of iCIL Cells

[0550] In some embodiments, CIL cells can be derived from iPSCs by sequentially differentiating iPSCs into hematopoietic progenitor cells (HPCs); differentiating HPCs into common lymphoid progenitor cells (CLPs); and then differentiating CLPs into CIL cells, and are referred to as "iCIL" cells. In one variation, CIL cells can be obtained from HPCs by sequentially differentiating HPCs into CLPs, and then differentiating CLPs into iCIL cells. In another variation, CIL cells can be obtained by differentiating CLPs into iCIL cells. The engineering of cells expressing synthetic cytokine receptors can be performed in the iPSC, HPC, CLP, or iCIL cell steps of the differentiation process.

[0551] In some embodiments, the iCIL cells are characterized as CD3-CD56+CD45+CD94+CD122+ / IL-2Rβ+CD127 / IL-7Rα-FcγRIII / CD16+KIR+NKG2A+NKG2D+NKp30+NKp44+NKP46+NKp80+. In some embodiments, the iCIL cells are characterized as CD3- / CD56+ / CD45+ cells. In some embodiments, the iCIL cells are characterized as CD3-CD56+ cells. In some embodiments, the 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 a combination thereof. iCIL cells can be CD45+; CD45+CD5-; or CD45+CD5-CD56+.

[0552] In some embodiments, iCIL cells are characterized by being CD45+ / CD7+ / CD56 + / lo .

[0553] In some embodiments, after iCIL cells are transduced with CAR, the cells are cultured under conditions that promote cell activation.

[0554] Culture conditions can be such that the cells can be administered to a patient regardless of their reactivity to culture medium components. For example, the culture conditions can omit bovine serum products, such as bovine serum albumin. In one exemplary aspect, activation can be achieved by introducing a known activator into the culture medium. In one aspect, the iCIL cell population can be cultured for approximately 1 to 4 days under conditions that promote activation. In one embodiment, the appropriate level of activation can be determined by cell size, proliferation rate, or activation markers measured by flow cytometry. In some embodiments, any of the culture methods disclosed herein can be used to promote activation of iCIL cells.

[0555] In some embodiments, the present invention provides a method for producing and / or expanding an engineered cell population comprising a synthetic cytokine receptor for a non-physiological ligand. In some embodiments, the method comprises:

[0556] providing an engineered cell comprising a synthetic cytokine receptor, optionally introducing a polynucleotide encoding the synthetic cytokine receptor into the source cell, and

[0557] The engineered cells are incubated in culture medium containing a non-physiological ligand.

[0558] In this embodiment, the cytokine receptor comprises a synthetic γ chain polypeptide and a synthetic β chain polypeptide, wherein the synthetic γ chain polypeptide comprises a first dimerization domain, a first transmembrane domain and an interleukin-2 receptor subunit γ (IL-2RG) intracellular domain; and the synthetic β chain polypeptide comprises a second dimerization domain, a second transmembrane domain and an intracellular domain, wherein the intracellular domain is selected from the group consisting of the interleukin-2 receptor subunit β (IL-2RB) intracellular domain, the interleukin-7 receptor subunit β (IL-7RB) intracellular domain and / or the interleukin-21 receptor subunit β (IL-21RB) intracellular domain; wherein the non-physiological ligand activates the synthetic cytokine receptor in the engineered cells to induce expansion and / or activation of the engineered cells.

[0559] In some embodiments, the expansion medium of the engineered CIL cells does not contain IL-2.

[0560] In some embodiments, CIL cells are engineered to express a synthetic cytokine receptor, such as RACR, and are expanded using a rapamycin analog in the expansion medium rather than using a recombinant cytokine in the expansion medium.

[0561] In some embodiments, the CIL cell expansion step is performed in a cell culture medium that is substantially free of recombinant cytokines.

[0562] In some embodiments, the expanding step is performed in feeder-free culture.

[0563] In some embodiments, the CIL cell expansion step is performed in a culture vessel that is not coated with a recombinant ligand for CIL cell expansion.

[0564] In some embodiments, a method is provided of administering an effective amount of a non-physiological ligand to a subject, wherein the non-physiological ligand causes iPSC cells to differentiate into iCIL cells according to any of the preceding embodiments.

[0565] In some embodiments, a method is provided of administering to a subject an effective amount of a non-physiological ligand, wherein the non-physiological ligand causes common lymphoid progenitor cells to differentiate into CIL cells according to any of the preceding embodiments.

[0566] In some embodiments, methods are provided for differentiating and expanding iCIL cells by culturing in a suitable container. The container can be a two-dimensional or three-dimensional container. Two-dimensional containers suitable for culturing source cells include any culture dish or dish commonly used to culture cells in the laboratory. The culture container can be coated with a suitable culture medium, such as an extracellular medium for culturing cell attachment and / or differentiation. In some embodiments, blood vessels are treated to promote cell adhesion and growth. An example of a culture medium suitable for the methods of the present invention is MATRIGEL TM Membrane matrix (BD Biosciences, Franklin Lakes, NJ).

[0567] In some embodiments, the container is suitable for three-dimensional (3D) culture. Without being bound by a particular theory or mechanism, it is believed that three-dimensional culture may be more effective than two-dimensional (2D) culture in providing a scaffold for cell differentiation. Suitable three-dimensional culture systems may include, for example, hanging drop three-dimensional culture (such as hanging drop plates), three-dimensional microwell culture (such as ultra-low adhesion multi-well plates), hydrophobic surface three-dimensional culture, rotary culture, static three-dimensional suspension culture or bioreactors. Hanging drop plates are commercially available, such as the PERFECTA3D hanging drop plates from Biospherix, Parish, NY. Ultra-low attachment multi-well plates (also referred to as non-adhesive culture dishes in some cases) are also commercially available, for example, the AGGREWELL from Stemcell Technologies, Vancouver, Canada. TM Ultra-low attachment multi-well plates.

[0568] 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 do not substantially adhere during culture. In some embodiments, the cells are cultured in suspension.

[0569] 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.

[0570] In some embodiments, the container is a bioreactor. In some embodiments, the bioreactor is used for the process of generating and proliferating iCILs after EB development. Bioreactors allow for optimization of cell culture conditions to achieve optimal hydrogen production and process robustness. Environmental conditions that can be adjusted or monitored in a bioreactor include gas composition (e.g., air, oxygen, nitrogen, carbon dioxide), gas flow rate, temperature, pH, dissolved oxygen level, and agitation speed / circulation rate within the cell culture. Any type of bioreactor known in the art can be used for cell culture to differentiate and expand cultured iCIL cells, including but not limited to stirred tank bioreactors, pneumatic bioreactors (e.g., bubble columns or airlift bioreactors), membrane bioreactors, hollow fiber bioreactors, wave bioreactors, vertical wheel bioreactors, gas permeation rapid expansion (G-Rex) bioreactors, or disposable bioreactors. In some embodiments, the bioreactor is a gas permeation rapid expansion (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.

[0571] iCIL cells can be expanded in any desired volume to suit a variety of purposes. For example, for medium- and high-throughput screening in various culture conditions, the process can be scaled up in microbioreactors (e.g., about 15 mL to about 500 mL) or benchtop bioreactors (e.g., about 0.5 L to about 15 L). Alternatively, the process can be scaled up to pilot-scale bioreactors (e.g., about 15 L to about 15,000 L) or production-scale bioreactors (e.g., about 15,000 L to about 75,000 L or larger).

[0572] In some embodiments, one or more containers can be used for iCIL expansion. In some embodiments, cells can be grown from day 0-3, day 0-10, day 0-15, day 0-20, day 0-25, day 0-30, day 0-35, day 0-40, day 0-50, day 0-60, day 0-100, day 3-10, day 3-15, day 3-20, day 3-25, day 3-30, day 3-35, day 0-40, day 0-50, day 0-60, day 0-100, day 3-10, day 3-15, day 3-20, day 3-25, day 3-30, day 3-35, day 0-40 3-40 days, 3-50 days, 3-60 days, 3-100 days, 10-20 days, 10-25 days, 10-30 days, 10-35 days, 10-40 days, 10-50 days, 10-60 days, 10-100 days, 15-20 days, 15-25 days, 15-30 days, 15-35 days, 15-40 days, Day 15-50, Day 15-60, Day 15-100, Day 20-25, Day 20-30, Day 20-35, Day 20-40, Day 20-50, Day 20-60, Day 20-100, Day 25-30, Day 25-35, Day 25-40, Day 25-50, Day 25-60, Day 25-100, Day 2 In some embodiments, cells may be cultured in a container from day 0-35. In some embodiments, cells may be cultured in a container from day 3-35. In some embodiments, the container is a bioreactor. In some embodiments, one or more bioreactors may be used for iCIL expansion. In some embodiments, one or more bioreactors are bioreactors of different types. In some embodiments, one or more bioreactors are bioreactors of different sizes.

[0573] G. Characteristics of Engineered Stem Cells and iCIL Cells

[0574] In some embodiments, the CIL cells are CD3-CD5-, CD16+, CD56+, CD57+, NKp30+, NKp46+, NKG2A+ and / or NKG2D+.

[0575] In some embodiments, the engineered cell population is 40% to 60% CD16+, 50% to 70% CD16+, 60% to 80% CD16+, 70% to 90% CD16+, 80% to 100% CD16+, or any percentage within the range defined by any two of the above values.

[0576] In some embodiments, the engineered cell population is 60% to 80% CD56+, 65% to 85% CD56+, 70% to 90% CD56+, 75% to 95% CD56+, 80% to 99% CD56+, or any percentage within the range defined by any two of the above values.

[0577] In some embodiments, the engineered CIL cell population is CD56lo. In some embodiments, the engineered CIL cell population is CD56high.

[0578] In some embodiments, the engineered CIL cell population is 60% to 80% CD16+CD56+, 65% to 85% CD16+CD56+, 70% to 90% CD16+CD56+, 75% to 95% CD16+CD56+, 80% to 99% CD16+CD56+, or any percentage within the range defined by any two of the above values.

[0579] In some embodiments, the engineered CIL cell population 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+.

[0580] In some embodiments, the engineered CIL cell population is at least 80% CD56+, at least 85% CD56+, at least 90% CD56+, at least 95% CD56+, or 100% CD56+.

[0581] In some embodiments, the engineered CIL cell population 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+.

[0582] In some embodiments, the CIL cells are characterized as CD45+CD56+.

[0583] In some embodiments, the engineered CIL cell population is 40% to 60% CD45+, 50% to 70% CD45+, 60% to 80% CD45+, 70% to 90% CD45+, 80% to 100% CD45+, or any percentage within the range defined by any two of the foregoing values.

[0584] In some embodiments, the engineered CIL cell population is 60% to 80% CD45+CD56+, 65% to 85% CD45+CD56+, 70% to 90% CD45+CD56+, 75% to 95% CD45+CD56+, 80% to 99% CD45+CD56+, or any percentage within the range defined by any two of the above values.

[0585] In some embodiments, the engineered CIL cell population 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+.

[0586] In some embodiments, the engineered CIL cell population 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+.

[0587] K562 cells are highly sensitive targets for in vitro cytotoxic innate lymphoid cell cytotoxicity activity detection. In the assay, CIL cells are co-cultured with K562 target tumor cells known to be sensitive to CIL cell-mediated cytotoxicity in different proportions. Target cells (K562) are pre-labeled with a fluorescent dye to distinguish them from effector cells (CIL cells). After the culture period, the killed target cells are identified by a nucleic acid stain that specifically penetrates into dead cells. The percentage of death is calculated by comparing the total number of surviving cells in each experimental test well with the total number of surviving cells in the non-effect control well. As used herein, the term "activity" refers to a measurement of the cytotoxic ability of CIL cells against target cells.

[0588] In some embodiments, the engineered CIL cells secrete CD107a in response to an antigen recognized by the engineered CIL cells.

[0589] Stimulation of innate CIL cell receptors leads to the secretion of CD107a by CIL cells. CD107a expression is associated with cytokine secretion and CIL cell-mediated target cell lysis. Therefore, CD107a secretion is a marker of CIL cell functional activity.

[0590] In some embodiments, the 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.

[0591] Upon activation, CIL cells secrete IFNγ and TNF-α, which synergistically enhance CIL cell cytotoxicity. In engineered CIL cells, the expression of IFNγ and / or TNF-α is a hallmark of CIL cell activity.

[0592] In some embodiments, the engineered CIL cells can be fresh or frozen. In some embodiments, the engineered CIL cells are fresh. In some embodiments, the engineered CIL cells are frozen. In some embodiments, the frozen CIL cells retain viability and cytotoxic function after thawing compared to fresh CIL cells. In some embodiments, the frozen / thawed CIL cells control tumors as well as fresh CIL cells.

[0593] In some embodiments, cells can be frozen using cryopreservation methods. In some embodiments, iCILs are cryopreserved after differentiation according to the provided methods. In some embodiments, iCILs are cryopreserved after engineering. In some embodiments, engineered iCILs produced according to the provided methods are cryopreserved. In some embodiments, the methods include cryopreserving cells in the presence of a cryoprotectant, thereby producing a cryopreservation composition. In some aspects, any of a variety of known freezing solutions and parameters can be used. In some embodiments, the cryoprotectant is DMSO. In some embodiments, cells are frozen, such as cryopreserved, in a solution having a final concentration of 1%-15%, 6%-12%, 5%-10%, or 6%-8% DMSO. In some embodiments, the cryopreservation solution is at or about 5% to at or about 10% DMSO (v / v). In some embodiments, the cryopreservation solution comprises one or more additional excipients, such as plasma protein A or human serum albumin (HSA). In some embodiments, the solution used for cryopreservation may also include human serum albumin (HSA). In certain embodiments, cells are frozen, such as 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 comprises a commercially available cryopreservation solution (CryoStor TM CS10 or CS5). CryoStor TM CS10 is a cryopreservation medium containing 10% dimethyl sulfoxide (DMSO). TMCS5 is a cryopreservation medium containing 5% dimethyl sulfoxide (DMSO). In some embodiments, the cells are then frozen to -80°C, typically at or about 1° per minute, and stored in the vapor phase of a liquid nitrogen storage tank. In some aspects, the engineered iCILs are thawed prior to use, as in connection with the therapeutic methods described herein. In some embodiments, after thawing the cells, the method includes washing the cryopreserved composition under conditions that reduce or remove the cell protectant.

[0594] III. Gene Editing and Engineering

[0595] In some embodiments, pluripotent stem cells (such as iPSC) or iCILs can be modified by gene editing. In some embodiments, pluripotent stem cells (such as iPSC) or iCILs can be modified by genetic engineering, such as introducing exogenous nucleic acids encoding transgenes, such as chimeric antigen receptors (CARs). In some embodiments, the gene-edited iPSCs can be used as source cells for differentiation into iCILs.

[0596] Genome editing generally refers to the process of editing or changing the nucleotide sequence of a genome, preferably in a precise, desired, and / or predetermined manner. Examples of genome editing compositions, systems, and methods described herein use site-directed nucleases to cut or cleave DNA at precise target sites in the genome, thereby generating double-strand breaks (DSBs) in the DNA. Such breaks can be repaired by endogenous DNA repair pathways, such as homology-directed repair (HDR) and / or non-homologous end joining (NHEJ) repair (see, e.g., Cox et al., (2015) Nature Medicine 21(2): 121-31).

[0597] In some embodiments, cells as described herein (such as stem cells, CIL) are genetically modified. In some embodiments, the modification involves knocking out one or more endogenous genes using a DNA targeting protein and a nuclease or RNA-guided nuclease, and / or knocking in one or more exogenous related genes of interest. In some embodiments, the gene of interest is knocked into 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 cytokine receptor (RACR) activated by rapamycin. In some embodiments, RACR is knocked into the locus of interest. In some embodiments, the gene of interest is a chimeric antigen receptor.

[0598] 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 zinc finger proteins (ZFPs), clustered regularly interspaced short palindromic nucleic acids (CRISPRs), or TAL-effector nucleases (TALENs). In some embodiments, CRISPR-Cas9 is used. In some embodiments, CRISPR-Mad7 is used.

[0599] Rejection reactions of cell therapies (such as CART cells) are at least due to human leukocyte antigen (HLA) mismatch between the donor and the recipient. A recently determined solution is to destroy the expression of genes involved in this rejection reaction, such as T cell receptor alpha constant (TRAC), beta-2-microglobulin (B2M) and signal regulatory protein alpha (SIRPA). Therefore, in some embodiments, the cells described herein (such as iPSC, CIL) are genetically engineered to knock out the B2M locus, TRAC locus and / or SIRPA locus. In some embodiments, the cells described herein are genetically engineered to knock out the B2M locus. In some embodiments, the cells described herein are genetically engineered to knock out the TRAC locus. In some embodiments, the cells described herein are genetically engineered to knock out the SIRPA locus.

[0600] 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, which is essential for cell growth and expansion. Therefore, in some cases, contact of cells with rapamycin can inhibit or reduce cell growth and expansion. In some embodiments, in order to eliminate or reduce rapamycin-mediated growth inhibition of source cells or CILs using the provided methods, the provided cells are disrupted with endogenous genes involved in rapamycin function, thereby rendering these cells "rapamycin-resistant". It should be understood that "rapamycin-resistant" cells refer to the ability of the cell's endogenous mTOR pathway to be unaffected by rapamycin or rapamycin analogs. However, it should be further understood that "rapamycin-resistant" cells may also respond to rapamycin through pathways that do not involve mTOR, for example due to activation of the synthetic RACR described herein.

[0601] In some embodiments, cells are genetically engineered to destroy genes associated with rapamycin recognition. In some embodiments, cells are genetically engineered to destroy 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 important binder of rapamycin and is necessary for rapamycin to work. In some embodiments, cells are genetically engineered to destroy the FKBP12 gene. In some embodiments, cells are genetically engineered to knock out the FKB12 ​​gene to induce rapamycin resistance. In some embodiments, the endogenous FKBP12 gene of the source stem cell (such as iPSC) is knocked out by the CRISPR-Cas system. In normal cells where the FKBP12 gene is not destroyed, FKBP12 is the main binder of rapamycin, and the FKBP12-rapamycin complex then binds to the FRB subunit of mTOR, blocking mTOR signaling. Disrupting FKBP12 gene expression through methods such as FKBP12 gene knockout has been shown in this study to successfully inhibit the activity of rapamycin, as rapamycin cannot function without first complexing with FKBP1A. Therefore, genetically disrupting FKBP12 through methods such as gene knockout can render stem cells (such as iPSCs) highly resistant to rapamycin-mediated mTOR inhibition, allowing them to grow robustly even in the presence of high doses of rapamycin. In some embodiments, the ability to render cells resistant to rapamycin growth inhibition allows rapamycin to engage the RACR during cell differentiation without adverse effects. Furthermore, knocking out FKBP12 can prevent competition between FKBP12 and RACR for rapamycin binding. Therefore, in some embodiments, the ability to render cells resistant to rapamycin growth through knockout of FKBP12 also allows for the in vivo activation of cells containing the RACR and suppresses potential allogeneic-versus-graft reactions through mTOR inhibition of the host immune system.

[0602] In some embodiments, the cells described herein are genetically engineered to include a nucleotide sequence encoding a synthetic cytokine receptor in an endogenous gene. In some embodiments, the synthetic cytokine receptor is engineered into a gene so as not to disrupt the expression of the endogenous gene. In some embodiments, the cells described herein are genetically engineered to include a nucleotide sequence encoding a synthetic cytokine receptor complex in a disrupted gene, such as a gene that has been inactivated or knocked out in the cell.

[0603] In some embodiments, the cells described herein are genetically engineered to include a nucleotide sequence encoding a synthetic cytokine receptor in a target endogenous gene. In some embodiments, the synthetic cytokine receptor is engineered as 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 1α (EEF1A), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ubiquitin C (UBC), or actin β (ACTB). In some embodiments, the target endogenous gene is B2M, TRAC, or SIRPα.

[0604] 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 the specific locus is used.

[0605] In some embodiments, an exogenous promoter is operably linked to a gene encoding a synthetic cytokine receptor complex to drive 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 so that two or more promoters drive expression of the exogenous gene of interest. In some embodiments, the two or more promoters may be the same or different. In some embodiments, the promoters are dual promoters, wherein the synthetic cytokine receptor is under the operative control of two promoters. In some embodiments, the dual promoters are dual EF1α promoters.

[0606] For example, in some embodiments, the cells comprise a disrupted B2M gene and a nucleotide sequence within the disrupted B2M gene encoding a synthetic cytokine receptor.

[0607] In some embodiments, the cells described herein (eg, iPSCs, CILs) comprise (i) a disrupted B2M locus, and (ii) a nucleotide sequence encoding a synthetic cytokine receptor complex (eg, RACR) under the control of an endogenous B2M promoter and an EEF1A promoter.

[0608] In some embodiments, the cells described herein (e.g., iPSCs, CILs) contain: (i) a disrupted B2M locus; (ii) a nucleotide sequence encoding a synthetic cytokine receptor complex (e.g., RACR), which is inserted into the endogenous B2M gene and controlled by the endogenous B2M promoter and the EEF1A promoter.

[0609] In some embodiments, a cell comprising (i) a disrupted B2M locus and (ii) a nucleotide sequence encoding a synthetic cytokine receptor complex (eg, RACR) is generated by any of the following methods.

[0610] A. Genome Editing System

[0611] In some embodiments, the cell editing system described herein includes a site-directed nuclease, such as a CRISPR / Cas system, and optional gRNA. In some embodiments, the system includes an engineered nuclease. In some embodiments, the system includes a site-directed nuclease. In some embodiments, the site-directed nuclease comprises 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 comprising the CRISPR / Cas system is a single guide RNA (sgRNA).

[0612] The sequences herein illustrate exemplary gRNA targeting sequences. In some embodiments, the gRNA targeting sequence may comprise one or more thymines in the complementary portion sequence that are replaced by uracils. It will be understood by those skilled in the art that both uracil and thymine may be represented by "t," rather than "u" for uracil and "t" for thymine; in the case of RNA, "t" represents uracil unless otherwise indicated.

[0613] 1. Nuclease

[0614] a. CRISPR / Cas nuclease system

[0615] The naturally occurring CRISPR / Cas system is a genetic defense system that can provide acquired immunity for prokaryotes. CRISPR is the abbreviation of Clustered Regularly Interspaced Short Palindromic Repeats, a family of DNA sequences found in bacterial and archaeal genomes, wherein the DNA fragments (interval DNA) contained therein are similar to foreign DNA previously exposed to the cell, such as viruses that infect or attack prokaryotes. Prokaryotes use these DNA fragments to detect and destroy similar foreign DNA that is reintroduced, for example, foreign DNA introduced from similar viruses in subsequent attacks. The transcription of the CRISPR locus forms an RNA molecule containing an intervening subsequence, which is bound to a Cas (CRISPR-associated) protein that can recognize and cut foreign DNA and use it as a target. Many types and classifications of CRISPR / Cas systems have been described (see, for example, Koonin et al., (2017) Curr Opin Microbiol 37: 67-78).

[0616] Various engineered versions of the CRISPR / Cas system have been developed for mutating or editing the genomic DNA of cells from other species. General approaches to using the CRISPR / Cas system include heterologous expression or introduction of site-directed nucleases (such as Cas nucleases) into cells in combination with guide RNA (gRNA), resulting in DNA breakage events (such as the formation of single-strand or double-strand breaks (SSBs or DSBs)) at precisely targetable sites on the cellular genomic DNA backbone. The way cells repair DNA breakage events provides an opportunity to edit the genome by adding, removing, or modifying (replacing) DNA nucleotides or sequences (such as genes).

[0617] In some embodiments, the system for cell editing described herein includes a nuclease that can induce DNA breaks in endogenous target genes in cells. In some embodiments, DNA breaks include double-strand breaks (DSBs), which are induced by nucleases that can induce DSBs by cleaving two chains of double-stranded DNA at the cleavage site. In some embodiments, DNA breaks include single-strand breaks (SSBs) at the sense or antisense chain cleavage sites of the endogenous target gene. In some embodiments, DNA breaks include SSBs at the cleavage site of the sense chain and SSBs at the cleavage site of the antisense chain, thereby forming DSBs. In some embodiments, DSBs are induced by a pair of recombinant nucleases, such as nickases, each of which can induce single-strand breaks (SSBs) on opposite DNA chains at different cleavage sites, such as cleavage sites upstream of a gene variant on one chain of the target gene and cleavage sites downstream of a gene variant on the other chain. In some embodiments, a first nickase in a pair of nickases forms a complex with a first guide RNA, such as a first sgRNA, for targeted cleavage of one strand, such as the sense strand, while a second nickase in a pair of nickases forms a complex with a second guide RNA, such as a second sgRNA, for targeted cleavage of the other strand, such as the antisense strand. In some embodiments, DSBs are induced by SSBs on each opposing strand, i.e., the sense strand and the antisense strand, of an endogenous target gene in a cell.

[0618] Generally, genes are located in double-stranded DNA, which consists of a sense strand and an antisense strand, which are complementary. 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.

[0619] i. Guide RNA (gRNA)

[0620] The engineered CRISPR / Cas system consists of at least two parts: 1) guide RNA (gRNA) molecules and 2) Cas nucleases, which interact to form gRNA / Cas nuclease complexes. gRNA contains at least one custom targeting domain, called a "spacer", comprising 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 in a target nucleic acid (such as a genomic DNA molecule) by generating a gRNA, wherein the gRNA comprises a spacer with a nucleotide sequence that can bind to a 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)). Therefore, the spacer provides the targeting function of the gRNA / Cas nuclease complex.

[0621] In the naturally occurring type II CRISPR / Cas system, "gRNA" consists of two RNA chains: 1) CRISPR RNA (crRNA) containing a spacer sequence and a CRISPR repeat sequence; 2) trans-activating CRISPR RNA (tracrRNA). In the type II CRISPR / Cas system, the crRNA portion containing the CRISPR repeat sequence hybridizes with the tracrRNA portion to form a crRNA:tracrRNA duplex, which interacts with the Cas nuclease (such as Cas9). As used herein, the term "split gRNA" or "modular gRNA" refers to a gRNA molecule comprising two RNA chains, wherein the first RNA chain comprises crRNA function and / or structure, the second RNA chain comprises tracrRNA function and / or structure, and wherein the first and second RNA chains partially hybridize.

[0622] Therefore, in some embodiments, gRNA comprises two RNA molecules. In some embodiments, gRNA comprises CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA). In some embodiments, gRNA is split gRNA. In some embodiments, gRNA is modular gRNA. In some embodiments, split gRNA comprises a first chain, which (from 5' to 3') comprises a spacer and a first complementary region; and a second chain, which (from 5' to 3') comprises a second complementary region and an optional tail domain.

[0623] In some embodiments, crRNA includes 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 (such as a genomic DNA molecule). In some embodiments, crRNA includes a region that is complementary to and hybridizes with a portion of tracrRNA.

[0624] In some embodiments, the target nucleic acid (such as an endogenous gene) is B2M. In some embodiments, the crRNA comprises the nucleotide sequence shown in SEQ ID NO: 18, or a nucleotide sequence having at least 80%, 85%, 90% or 95% sequence identity to the nucleotide sequence shown in SEQ ID NO: 18. In some embodiments, the crRNA comprises the sequence shown in SEQ ID NO: 18.

[0625] In some embodiments, the target nucleic acid (such as an endogenous gene) is FKBP12. In some embodiments, the crRNA comprises the nucleotide sequence shown in SEQ ID NO: 19, 20 and 21, or a nucleotide sequence having at least 80%, 85%, 90% or 95% sequence identity with the nucleotide sequence shown in SEQ ID NO: 19, 20 and 21. In some embodiments, the crRNA comprises the sequence shown in SEQ ID NO: 19, or a nucleotide sequence having at least 80%, 85%, 90% or 95% sequence identity with the nucleotide sequence shown in SEQ ID NO: 19. In some embodiments, the crRNA comprises the nucleotide sequence shown in SEQ ID NO: 19. In some embodiments, the crRNA comprises the nucleotide sequence shown in SEQ ID NO: 20, or a nucleotide sequence having at least 80%, 85%, 90% or 95% sequence identity with the nucleotide sequence shown in SEQ ID NO: 20. In some embodiments, the crRNA comprises the sequence shown 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%, 85%, 90%, or 95% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 21. In some embodiments, the crRNA comprises the sequence set forth in SEQ ID NO: 21.

[0626] In some embodiments, tracrRNA may comprise all or part of a wild-type tracrRNA sequence from a naturally occurring CRISPR / Cas system. In some embodiments, tracrRNA may comprise a truncated or modified variant of a wild-type tracrRNA. The length of tracrRNA may depend on the CRISPR / Cas system used. In some embodiments, the length of tracrRNA may comprise 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 some embodiments, the length of tracrRNA is at least 26 nucleotides. In some embodiments, the length of tracrRNA is at least 40 nucleotides. In some embodiments, tracrRNA may comprise certain secondary structures, such as one or more hairpins or stem-loop structures, or one or more protruding structures.

[0627] Single guide RNA (sgRNA)

[0628] The engineered CRISPR / Cas nuclease system is usually combined into one RNA molecule by adding a joint between crRNA and tracrRNA, referred to herein as "single guide RNA" (sgRNA). Without being bound by theory, similar to double-stranded crRNA and tracrRNA, sgRNA forms a complex with Cas nuclease (such as Cas9), guides Cas nuclease to the target sequence and activates Cas nuclease to cleave target nucleic acid (such as genomic DNA). Therefore, in some embodiments, gRNA may include operably connected crRNA and tracrRNA. In some embodiments, sgRNA may include crRNA covalently linked to tracrRNA. In some embodiments, crRNA and tracrRNA are covalently linked via a joint. In some embodiments, sgRNA may include a stem-loop structure formed by base pairing between crRNA and tracrRNA. In some embodiments, sgRNA includes a spacer, a complementary first region, a connecting domain, a complementary second region, and an optional tail domain from 5' to 3'.

[0629] The sgRNA can be unmodified or modified. For example, a modified sgRNA can include one or more 2'-O-methyl phosphorothioate nucleotides.

[0630] As described herein and in the art, as an example, the guide RNA or other smaller RNA used in the CRISPR / Cas system can be easily synthesized by chemical methods. Although chemical synthesis procedures are constantly expanding, as the length of polynucleotides increases significantly, after more than 100 nucleotides, it often becomes more challenging to purify such RNA by procedures such as high performance liquid chromatography (HPLC, which can avoid the use of gels such as PAGE). One way to generate longer RNA is to connect two or more molecules together. Longer RNA, such as RNA encoding Cas9 endonuclease, is more easily generated by enzymatic methods. Various types of RNA modifications can be introduced during or after the chemical synthesis and / or enzymatic generation of RNA, for example, as described in the art, to enhance stability, reduce the likelihood or extent of innate immune response and / or enhance modifications of other properties.

[0631] spacer

[0632] In some embodiments, the gRNA comprises a spacer sequence. A spacer sequence is a sequence that defines a target site in a target nucleic acid (e.g., DNA). The target nucleic acid is a double-stranded molecule: one strand contains the target sequence adjacent to the PAM sequence, referred to as the "PAM strand"; the second strand, referred to as the "non-PAM strand," is complementary to both 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 with the complementary strand (e.g., the non-PAM strand of the target nucleic acid / target site). In some embodiments, the spacer has sufficient complementarity with 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%, 85%, 90%, or 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 1 nucleotide that is not complementary to the non-PAM strand of the target nucleic acid. In some embodiments, the spacer comprises 2 nucleotides that are not complementary to the non-PAM strand of the target nucleic acid.

[0633] In some embodiments, the 5'-most nucleotide of the gRNA comprises the 5'-most 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 length of the spacer is about 15-50, about 20-45, about 25-40, or about 30-35 nucleotides. In some embodiments, the length of the spacer is about 19-22 nucleotides. In some embodiments, the length of the spacer is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, the length of the spacer is 19 nucleotides. In some embodiments, the length of the spacer is 20 nucleotides, and in some embodiments, the length of the spacer is 21 nucleotides.

[0634] In some embodiments, the nucleotide sequence of the spacer is designed or selected by a computer program. The computer program can use various variables, such as predicted melting temperature, secondary structure formation, predicted annealing temperature, sequence identity, genomic context, chromatin accessibility, GC%, genomic frequency (such as identical or similar but different sequences at one or more sites due to mismatches, insertions or deletions), methylation status and / or the presence of SNPs.

[0635] In some embodiments, the spacer comprises at least one or more modified nucleotides, such as nucleotides described herein. The gRNA molecules provided herein comprise spacers that may contain the nucleobase uracil (U), and any DNA encoding a gRNA comprising a spacer consisting of the nucleobase uracil (U) will comprise the nucleobase thymine (T) at the corresponding site.

[0636] ii. Method for preparing gRNA

[0637] Methods for making gRNA are well known to those skilled in the art and include, but are not limited to, in vitro transcription (IVT), synthesis and / or chemical synthesis methods, or combinations thereof. Enzymatic methods (IVT), solid phase methods, liquid phase methods, combinatorial synthesis methods, small area synthesis methods, and ligation methods are utilized. In one embodiment, the gRNA is synthesized using IVT enzymatic methods. Methods for making polynucleotides by IVT are known in the art and are described in International Application PCT / US2013 / 30062. Therefore, the present disclosure also includes polynucleotides (such as DNA), constructs, and vectors for in vitro transcription of the gRNA described herein.

[0638] In some embodiments, non-natural modified nucleobases are introduced into polynucleotides (such as gRNA) during or after synthesis. In some embodiments, modifications are made to internucleoside linkages, purine or pyrimidine bases, or sugars. In some embodiments, modifications are introduced at the ends of polynucleotides by chemical synthesis or polymerases. PCT application No. PCT / US2012 / 058519 discloses examples of modified nucleic acids and their synthesis. The synthesis of modified polynucleotides is also described in Verma and Eckstein, Annual Review of Biochemistry, vol. 76, 99-134 (1998).

[0639] In some embodiments, enzymatic or chemical ligation methods are used to conjugate polynucleotides or regions thereof to various functional moieties, such as targeting or delivery agents, fluorescent tags, liquids, nanoparticles, etc. Conjugates of polynucleotides and modified polynucleotides are reviewed by Goodchild, Bioconjugate Chemistry, vol. 1(3), 165-187 (1990).

[0640] In some embodiments, the present disclosure provides nucleic acids encoding gRNA described herein, such as vectors. In some embodiments, the nucleic acid is a DNA molecule. In some embodiments, the nucleic acid is an RNA molecule. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding crRNA. In some embodiments, the nucleotide sequence encoding crRNA comprises a spacer sequence, both sides of which are all or part of the repeat sequences from the naturally occurring CRISPR / Cas system. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding tracrRNA. In some embodiments, crRNA and tracrRNA are encoded by two separate nucleic acids. In other embodiments, crRNA and tracrRNA are encoded by a single nucleic acid. In some embodiments, crRNA and tracrRNA are encoded by opposite chains of a single nucleic acid. In other embodiments, crRNA and tracrRNA are encoded by the same chain of a single nucleic acid.

[0641] In some embodiments, the gRNA provided herein is chemically synthesized by any method described in the art (see, for example, WO / 2005 / 01248). Although chemical synthesis procedures are constantly expanding, as the length of polynucleotides increases significantly, after exceeding about 100 nucleotides, it often becomes more challenging to purify such RNAs by procedures such as high performance liquid chromatography (HPLC, which can avoid the use of gels such as PAGE). One way to generate longer RNAs is to connect two or more molecules together.

[0642] In some embodiments, the CRISPR / Cas nuclease system can use more than one guide RNA. Each guide RNA can contain different targeting sequences, so that the CRISPR / Cas system can cut more than one target nucleic acid. In some embodiments, one or more guide RNAs can have the same or different properties, such as activity or stability in the Cas9RNP complex. When more than one guide RNA is used, each guide RNA can be encoded on the same or different vectors. The promoters used to drive the expression of more than one guide RNA are the same or different.

[0643] The guide RNA can target any sequence of interest through the targeting sequence of the crRNA (e.g., a spacer sequence). In some embodiments, the degree of complementarity between the targeting sequence of the guide RNA and the targeting sequence on the target nucleic acid molecule is about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%. In some embodiments, the targeting sequence of the guide RNA is 100% complementary to the targeting sequence on the target nucleic acid molecule. In other embodiments, the targeting sequence of the guide RNA and the targeting sequence on the target nucleic acid molecule may contain at least one mismatch. For example, the targeting sequence of the guide RNA and the targeting sequence on the target nucleic acid molecule may 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 targeting sequence on the target nucleic acid molecule may contain 1-6 mismatches. In some embodiments, the targeting sequence of the guide RNA and the targeting sequence on the target nucleic acid molecule may contain 5 or 6 mismatches.

[0644] The length of the targeting sequence can depend on the CRISPR-Cas system and components used. For example, different Cas9 proteins in different bacterial species have different optimal targeting sequence lengths. Therefore, the targeting sequence can include a length 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 nucleotides. In some embodiments, the length of the targeting sequence may include 18-24 nucleotides. In some embodiments, the length of the targeting sequence may include 19-21 nucleotides. In some embodiments, the length of the targeting sequence may include 20 nucleotides.

[0645] 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 guide the Cas protein to a target sequence on a target nucleic acid molecule (such as a genomic DNA molecule), and the Cas protein cuts the target nucleic acid at this sequence. 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 type 2 V-type CRISPR system. In some embodiments, the endonuclease is Mad7.

[0646] iii. Cas nuclease

[0647] In some embodiments, the present disclosure provides compositions and systems (e.g., engineered CRISPR / Cas systems) comprising site-directed nucleases, wherein the site-directed nuclease is a Cas nuclease. The Cas nuclease may comprise at least one domain that interacts with a guide RNA (gRNA). In addition, the Cas nuclease is directed to the target sequence via a guide RNA. The guide RNA interacts with the Cas nuclease and the target sequence, and once directed to the target sequence, the Cas nuclease can cleave the target sequence. In some embodiments, the guide RNA provides specificity for the cleavage of the target sequence, and the Cas nuclease is universal and can be paired with different guide RNAs to cleave different target sequences.

[0648] In some embodiments, the CRISPR / Cas system includes components derived from type I, type II, or type III systems. The latest classification scheme for CRISPR / Cas loci defines type 1 and type 2 CRISPR / Cas systems, with types I to V or VI (Makarova et al., (2015) Nat Rev Microbiol, 13(11):722-36; Shmakov et al., (2015) Mol Cell, 60:385-397). The second type of CRISPR / Cas system has a single protein effector. Type II, V, and VI Cas proteins are RNA-guided single-protein nucleases, referred to herein as "type 2 Cas nucleases." Type 2 Cas nucleases include Cas9, Cpf1, C2c1, C2c2, and C2c3 proteins, among others. Cpf1 nuclease (Zetsche et al., (2015) Cell 163:1-13) is homologous to Cas9 and contains a RuvC-like nuclease domain.

[0649] In some embodiments, the Cas nuclease is from a type II CRISPR / Cas system (e.g., a Cas9 protein from a CRISPR / Cas9 system). In some embodiments, the Cas nuclease is from a class 2 CRISPR / Cas system (a single-protein Cas nuclease, such as a Cas9 protein or a Cpf1 protein). The Cas9 and Cpf1 protein families are enzymes with DNA endonuclease activity that can be directed to cleave a desired nucleic acid target by designing appropriate guide RNAs, as described herein.

[0650] Type II CRISPR / Cas system components are derived from Type IIA, Type IIB, or Type IIC systems. They include Cas9 and its orthologs. Non-limiting examples of species from which the Cas9 nuclease or other components may be derived include Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp.), Staphylococcus aureus, Listeria innocua, Lactobacillus gasseri, Francisella novicida, Wolinella succinogenes, Sutterella wadsworthensis, γproteobacterium, 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, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Treponema denticola, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthophagous naphthalenivorans), Polaromonas sp., Crocosphaera watsonii, Cyanothece sp.), Microcystis aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp.), Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas shaloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp.), Petrotoga mobilis, Thermosiphoafricanus, Streptococcus pasteurianus, Neisseria cinerea, Campylobacter lari, Parvibaculum lavamentivorans, Corynebacterium diphtheria, or Acaryochloris marina. In some embodiments, the Cas9 protein is from Streptococcus pyogenes (SpCas9). In some embodiments, the Cas9 protein is from Streptococcus thermophilus (StCas9). In some embodiments, the Cas9 protein is from Neisseria meningitidis (NmCas9). In some embodiments, the Cas9 protein is from Staphylococcus aureus (SaCas9). In some embodiments, the Cas9 protein is from Campylobacter jejuni (CjCas9).

[0651] In some embodiments, the Cas nuclease may comprise more than one nuclease domain. For example, the Cas9 nuclease may comprise at least one RuvC-like nuclease domain (such as Cpf1) and at least one HNH-like nuclease domain (such as Cas9). In some embodiments, the Cas9 nuclease introduces a DSB into the target sequence. In some embodiments, the Cas9 nuclease is modified to comprise only one functional nuclease domain. For example, the Cas9 nuclease is modified so that one of the nuclease domains is mutated or deleted in whole or in part 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 certain embodiments in which only one nuclease domain is functional, the Cas9 nuclease is a nickase that can introduce single-strand breaks ("nicks") into the target sequence. In some embodiments, conserved amino acids within the Cas9 nuclease domain are substituted to reduce or change nuclease activity. In some embodiments, the Cas nuclease nickase comprises an amino acid replacement in a RuvC-like nuclease domain. Exemplary amino acid replacements in a RuvC-like nuclease domain include D10A (based on Streptococcus pyogenes (S.pyogenes) Cas9 nuclease). In some embodiments, the nickase comprises an amino acid replacement in a HNH-like nuclease domain. Exemplary amino acid replacements in an HNH-like nuclease domain include E762A, H840A, N863A, H983A, and D986A (based on Streptococcus pyogenes (S.Pyogenes) Cas9 nuclease). In some embodiments, the nuclease system described herein comprises a nickase and a pair of guide RNAs complementary to the sense and antisense strands of the target sequence, respectively. The guide RNA guides the nickase to lock the target and introduces DSB by creating a nick (i.e., a double nick) on the opposite strand of the target sequence. When using a chimeric Cas9 nuclease, a domain or region of the protein can be replaced by a portion of a different protein. For example, the Cas9 nuclease domain is replaced with a domain of a different nuclease, such as Fok1. Cas9 nuclease is a modified nuclease.

[0652] In some embodiments, the Cas nuclease is from a type I CRISPR / Cas system. In some embodiments, the Cas nuclease is a component of the Cascade complex of a type I CRISPR / Cas system. For example, the Cas nuclease is a Cas3 nuclease. In some embodiments, the Cas nuclease is from a type III CRISPR / Cas system. In some embodiments, the Cas nuclease is from a type IV CRISPR / Cas system. In some embodiments, the Cas nuclease is from a type V CRISPR / Cas system. In some embodiments, the Cas nuclease is from a type VI CRISPR / Cas system.

[0653] In some embodiments, the Cas nuclease is a Mad endonuclease. The CRISPR / Mad system is closely related to type V (Cpf1-like) of the Cas enzyme family 2. In some embodiments, the CRISPR-Mad system utilizes the Eubacterium rectale Mad7 endonuclease or a variant thereof. The Mad7-crRNA complex cleaves target DNA by recognizing PAM5'-YTTN.

[0654] b. Engineered nucleases

[0655] In some embodiments, the cells described herein are genetically engineered to have a site-directed nuclease, wherein the site-directed nuclease is an engineered nuclease. Typical engineered nucleases include meganucleases (such as homing endonucleases), ZFNs, TALENs, and megaTALs.

[0656] Naturally occurring meganucleases can recognize and cut double-stranded DNA sequences of about 12 to 40 base pairs and are generally divided into five families. In some embodiments, the meganucleases are 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 meganucleases is engineered to recognize and bind to sequences other than their cognate target sequences. In some embodiments, the DNA binding domain of the meganucleases is fused to heterologous nuclease domains. In some embodiments, meganucleases, such as homing endonucleases, are fused to TAL modules to form hybrid proteins, such as "megaTAL" proteins. By recognizing the target sequences of the meganucleases DNA binding domains and the TAL modules, the megaTAL proteins increase the specificity of DNA targeting.

[0657] ZFN is a fusion protein consisting of a zinc finger DNA binding domain ("zinc finger" or "ZF") and a nuclease domain. Each naturally occurring ZF can bind to three consecutive base pairs (DNA triplets), and the ZF repeat sequence combination can recognize the DNA target sequence and provide sufficient affinity. Therefore, the engineered ZF repeat sequence combination can recognize longer DNA sequences, such as 9-, 12-, 15- or 18-bp, etc. In some embodiments, ZFN comprises a ZF fused to the nuclease domain of a restriction endonuclease. For example, the restriction endonuclease is FokI. In some embodiments, the nuclease domain comprises a dimerization domain, for example, when nuclease dimerization is active, a pair of ZFNs comprising a ZF repeat sequence and a nuclease domain are designed to target a target sequence, the target sequence comprising two half target sequences recognized by each ZF repeat sequence on the opposite chain of the DNA molecule, with an interconnected sequence in the middle (sometimes referred to as a spacer in the literature). For example, the interconnected sequence is 5 to 7bp in length. When two paired ZFNs bind, the nuclease domains can dimerize and introduce a DSB in the interconnected sequence. In some embodiments, the dimerization domain of the nuclease domain comprises a knob-into-hole motif that promotes dimerization. For example, the ZFN comprises a knob-into-hole motif in the FokI dimerization domain.

[0658] The DNA binding domain of TALEN is typically composed of a varying number of 34 or 35 amino acid repeats ("modules" or "TAL modules"), each module binding to a single DNA base pair of A, T, G or C. The adjacent residues at positions 12 and 13 of each module ("repeat variable diresidues" or RVDs) specify the single DNA base pair that the module binds to. Although the module used to recognize G may also have an affinity for A, the advantage of TALEN is simple base recognition, with one module for each of the four bases, which greatly simplifies the customization of DNA binding domains that recognize specific target sequences. In some embodiments, TALEN may include a nuclease domain of a restriction endonuclease. For example, the restriction endonuclease is FokI. In some embodiments, the nuclease domain can dimerize to be active, and a pair of TALENS is designed to target a target sequence, which includes two half-target sequences recognized by each DNA binding domain on opposite chains of the DNA molecule, with an interconnected sequence in the middle. For example, the length of each half target sequence ranges from 10 to 20 bp, and the length of the interconnected sequence ranges from 12 to 19 bp. When the two paired TALENs bind, the nuclease domain can dimerize and introduce a DSB in the interconnected sequence. In some embodiments, the dimerization domain of the nuclease domain can include a knob-into-hole motif that promotes dimerization. For example, TALEN can include a knob-into-hole motif in the dimerization domain of FokI.

[0659] c. Target site

[0660] In some embodiments, the site-directed nucleases described herein can direct and cleave (e.g., introduce DSBs) target nucleic acid molecules (e.g., endogenous genes). In some embodiments, the target nucleic acid molecule is a housekeeping gene. In some embodiments, the housekeeping gene is eukaryotic translation elongation factor 1α (EEF1A), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ubiquitin C (UBC), or actin beta (ACTB). In some embodiments, the target nucleic acid molecule is a blood lineage gene. In some embodiments, the blood lineage gene is protein tyrosine phosphatase receptor type C (PTPRC), IL2RG, or IL2RB. In some embodiments, the target nucleic acid is a gene associated with rapamycin response. In some embodiments, the target nucleic acid is FKBP12. In some embodiments, the target nucleic acid is B2M, TRAC, or SIRPA.

[0661] The target nucleic acid molecule is any DNA molecule endogenous or exogenous to the cell. As used herein, the term "endogenous sequence" refers to the native sequence in the cell. In some embodiments, the target nucleic acid molecule is a genomic DNA (gDNA) molecule or chromosome from a cell or a cell. In some embodiments, the target sequence of the target nucleic acid molecule is a genomic sequence from a cell or a cell. In some embodiments, the target sequence can be located in the coding sequence of a gene, the intron sequence of a gene, the transcription control sequence of a gene, the translation control sequence of a gene, or the non-coding sequence between genes. In some embodiments, the gene can be a protein coding gene. In other embodiments, the gene can be a non-coding RNA gene. In some embodiments, the target sequence can include all or part of a disease-related gene.

[0662] In some embodiments, the target sequence can be located at a non-gene functional site in the genome that controls chromatin organization, such as a scaffold site or a locus control region. In some embodiments, the target sequence can be a gene safe harbor site, i.e., a site that is conducive to safe genetic modification.

[0663] In some embodiments, the target sequence can be adjacent to a pre-spacer adjacent motif (PAM), which is a short sequence recognized by the CRISPR / Cas complex. In some embodiments, the PAM can be adjacent to 1, 2, 3, or 4 nucleotides at the 3' end of the target sequence or within 1, 2, 3, or 4 nucleotides. In some embodiments, the target sequence can include a PAM. The length and sequence of the PAM can depend on the Cas protein used. For example, the PAM can be selected from a common or specific PAM sequence of a specific Cas nuclease or Cas straight homolog, including those disclosed in Figure 1 of Ran et al., (2015) Nature, 520: 186-191 (2015), which are incorporated herein by reference. In some embodiments, the length of the PAM can include 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.Non-limiting example PAM sequences include NGG (SpCas9WT, SpCas9nickase, dimeric dCas9-Fok1, SpCas9-HF1, SpCas9K855A, eSpCas9 (1.0), eSpCas9 (1.1)), NGAN or NGNG (SpCas9VQR variant), NGAG (SpCas9EQR variant), NGCG (SpCas9VRER variant), NAAG (SpCas9QQR1 variant), NNGRRT or NNGRRN (SaCas9), NNNRRT (KKHSaCas9), NNNNRYAC (CjCas9), NNAGAAW (St1Cas9), NAAAC (TdCas9), NGGNG (St3Cas9), NG (FnCas9), NAAAAN (TdCas9), NNAAAAW (StCas9), NNNNACA (CjCas9), GNNNCNNA (PmCas9), and NNNNGATT (NmCas9) (see, e.g., Cong et al., (2013) Science 339:819-823; Kleinstiver et al. (2015) Nat Biotechnol 33:1293-1298; Kleinstiver et al. (2015) Nature 523:481-485; Kleinstiver et al. (2016) Nature 529:490-495; Tsai et al. (2014) Nat Biotechnol 32:569-576; Slaymaker et al. (2016) Science 351:84-88; Anders et al. (2016) Mol Cell 61:895-902; Kim et al. (2017) Nat Comm 8:14500; Fonfara et al. (2013) Nucleic Acids Res 42:2577-2590; Garneau et al., (2010) Nature 468:67-71; Magadan et al., (2012) PLoS ONE 7:e40913; Esvelt et al., (2013) Nat Methods 10(11):1116-1121 (wherein N is defined as any nucleotide, W is defined as A or T, R is defined as a purine (A) or (G), and Y is defined as a pyrimidine (C) or (T)). In some embodiments, the PAM sequence is NGG. In some embodiments, the PAM sequence is NGAN. In some embodiments, the PAM sequence is NGNG. In some embodiments, the PAM is NNGRRT. In some embodiments, the PAM sequence is NGGNG. In some embodiments, the PAM sequence can be NNAAAAW.

[0664] In some embodiments, the PAM s...

Claims

1. An engineered stem cell 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 intracellular domain of interleukin-2 receptor subunit gamma (IL-2RG), and A synthetic β-chain polypeptide comprises a second dimerization domain, a second transmembrane domain and an intracellular domain, wherein the intracellular domain is selected from the intracellular domain of interleukin-2 receptor subunit β (IL-2RB), the intracellular domain of interleukin-7 receptor subunit β (IL-7RB) and / or the intracellular domain of interleukin-21 receptor subunit β (IL-21RB). 2 . The engineered stem cell according to claim 1 , wherein the first dimerization domain and the second dimerization domain are extracellular domains.

3. The engineered stem cell according to claim 1 or 2, wherein the synthetic γ chain polypeptide comprises, in order from N-terminus to C-terminus, a first dimerization domain, a first transmembrane domain and an intracellular domain of interleukin-2 receptor subunit γ (IL-2RG), and The synthetic β chain polypeptide comprises a second dimerization domain, a second transmembrane domain and an intracellular domain in order from N-terminus to C-terminus.

4. The engineered stem cell 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 a polypeptide sequence as shown in SEQ ID NO:

1.

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

6. The engineered stem cell of claim 5, wherein the IL-2RG intracellular domain comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO: 8 or 31, or a polypeptide sequence as shown in SEQ ID NO: 8 or 31.

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

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

2.

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

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

3.

11. The engineered stem cell according to any one of claims 1-6, wherein the β chain intracellular domain comprises an IL-21RB intracellular domain. 12 . The engineered stem cell of claim 11 , wherein the IL-21RB intracellular domain comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO: 4, or a polypeptide sequence as shown in SEQ ID NO:

4.

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

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

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

2.

16. The engineered stem cell according to any one of claims 1 to 15, wherein the first dimerization domain and the second dimerization domain are heterologous dimerization domains selected from the group consisting of a 12 kD sized FK506-binding protein (FKBP) and a FKBP12-rapamycin binding (FRB) domain; and / or The non-physiological ligand is rapamycin or a rapamycin analog.

17. The engineered stem cell of 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 cell according to claim 16, wherein the FRB domain comprises a polypeptide sequence as shown in SEQ ID NO: 6 or SEQ ID NO:

7.

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

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

30.

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

30.

22. The engineered stem cell according to any one of claims 1-8 and 13-21, wherein the synthetic γ chain polypeptide has the amino acid sequence shown 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 β chain polypeptide has the amino acid sequence shown 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 1-8 and 13-22, wherein the synthetic γ chain polypeptide has the amino acid sequence shown in SEQ ID NO:28, and the synthetic β chain polypeptide has the amino acid sequence shown in SEQ ID NO:

33.

24. The engineered stem cell according to any one of claims 1 to 15, wherein the first dimerization domain and the second dimerization domain are homologous dimerization domains selected from the following: i) FK506 binding protein (FKBP) with a size of 12 kD; ii) Cyclophilin A (CypA); or iii) gyrase B (CyrB); And the non-physiological ligands are: i) FK1012, AP1510, AP1903 or AP20187 or their analogs; ii) cyclosporine-A (CsA) or its analogs; or iii) Coumarin or its analogs.

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

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

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

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

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

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

7.

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

7.

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

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

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

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

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

37. The engineered stem cell of claim 36, wherein the insertion reduces expression of an endogenous gene in the locus.

38. The engineered stem cell of claim 36 or 37, wherein the insertion knocks out an endogenous gene in a locus.

39. The engineered stem cell of any one of claims 36-38, wherein the insertion is performed by homology directed repair.

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

41. The engineered stem cell of claim 40, wherein 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).

42. The engineered stem cell of claim 40, wherein 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.

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

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

45. The engineered stem cell of any one of claims 1-43, wherein the stem cell comprises a B2M knockout.

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

47. The engineered stem cell of claim 46, wherein the disruption is a FKBP12 knockout that inactivates a gene encoding FKBP12.

48. The engineered stem cell of any one of claims 1-44, wherein the stem cell comprises a B2M knockout and a FKBP12 knockout.

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

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

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

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

53. A method of genetically engineering stem cells to express synthetic cytokine receptors, comprising: contacting a population of stem cells with (i) a guide RNA (gRNA) that targets a target site in an endogenous gene, (ii) an RNA-guided endonuclease, and (iii) a recombinant vector comprising a nucleotide sequence encoding a synthetic cytokine receptor for a non-physiological ligand, Thereby inserting the nucleotide sequence into the endogenous gene; Wherein the cytokine receptors include: 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 β chain polypeptide comprising a second dimerization domain, a second transmembrane domain and an intracellular domain, wherein the intracellular domain is selected from the intracellular domain of interleukin-2 receptor subunit β (IL-2RB), the intracellular domain of interleukin-7 receptor subunit β (IL-7RB) and / or the intracellular domain of interleukin-21 receptor subunit β (IL-21RB).

54. The method of claim 53, wherein the nucleotide sequence is inserted by homology directed repair (HDR).

55. The method of claim 54, wherein the vector comprises a nucleic acid comprising, 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.

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

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

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

59. The method of any one of claims 53-57, 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 the group consisting of B2M, TRAC and SIRPA.

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

62. The method of any one of claims 53-61, wherein the gRNA comprises the sequence shown in SEQ ID NO:

18.

63. The method of any one of claims 55-62, wherein 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 with the nucleic acid sequence shown 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 with the nucleic acid sequence shown in SEQ ID NO:

23.

64. The method of any one of claims 53-63, wherein the nucleotide sequence encoding the synthetic cytokine receptor comprises a first nucleic acid sequence encoding a γ chain and a second nucleic acid sequence encoding a β chain, wherein the first nucleic acid sequence has 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 shown in SEQ ID NO:37; and the second nucleic acid sequence has 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 shown in SEQ ID NO:

38.

65. The method of 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 of claim 65, wherein the cleavable linker is a protein quantitation reporter linker (PQR), optionally as shown in SEQ ID NO:

42.

67. The method of any one of claims 53-66, wherein the nucleotide sequence encoding the synthetic cytokine receptor for a non-physiological ligand is under the operable control of a heterologous promoter.

68. The method of claim 67, wherein the heterologous promoter is the EF1α promoter or the MND promoter.

69. The method of any one of claims 53-68, wherein the nucleotide sequence encoding the synthetic cytokine receptor comprises a polyadenylation sequence.

70. The method according to any one of claims 53-69, wherein the recombinant vector comprises the sequence shown 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 shown in SEQ ID NO:

40.

71. The method of any one of claims 53-70, further comprising engineering the stem cell population to be resistant to rapamycin-mediated inhibition of mTOR.

72. The method of claim 71, wherein engineering the stem cell population to be resistant to rapamycin comprises knocking out the FKBP12 gene.

73. The method of claim 72, wherein the method comprises further contacting the stem cell population with the RNA-guided endonuclease and a guide RNA (gRNA) targeted to a target site in the FKBP12 gene.

74. The method of claim 73, wherein the RNA-guided endonuclease is selected from the group consisting of a Cas endonuclease, a Mad endonuclease, and a Cpf1 endonuclease.

75. The method of claim 73 or claim 74, wherein the RNA-guided endonuclease is Cas9.

76. The method of claim 73 or claim 74, wherein the RNA-guided endonuclease is Mad7.

77. The method of any one of claims 73-76, wherein the further contacting is performed simultaneously with contacting with (i) a guide RNA (gRNA) targeting a target site in an endogenous gene, optionally in combination with the same RNA-guided endonuclease.

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

21.

79. The method of claim 78, wherein the one or more gRNAs is a gRNA pool comprising 2 or 3 gRNAs.

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

81. The engineered stem cell of claim 80, wherein the CAR is an anti-FITC CAR.

82. The method of any one of claims 53-81, wherein the stem cell is a pluripotent stem cell.

83. The method of any one of claims 53-82, wherein the stem cell is an iPSC.

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

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

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

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

88. The method of claim 87, wherein the culturing comprises: 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 the plurality of cells, wherein the start of culturing in b) is day 0; c) culturing the cells produced in b) under conditions that cause the cells to differentiate into a hematopoietic progenitor (HP) population; as well as d) culturing the cells produced in c) under conditions for producing iCIL cells, wherein at least a portion of one or more of steps a) to d) is performed in the presence of a non-physiological ligand for the synthetic cytokine receptor.

89. A method of generating cytotoxic innate lymphoid (iCIL) cells, the method comprising: a) culturing a cell population comprising the engineered stem cell according to any one of claims 1 to 51 under conditions where aggregates are formed; b) culturing the cells produced in a) under conditions that induce mesoderm formation in the plurality of cells, wherein the start of culturing in b) is day 0; c) culturing the cells produced in b) under conditions that cause the cells to differentiate into a hematopoietic progenitor (HP) population; as well as d) culturing the cells produced in c) under conditions for producing iCIL cells, At least a portion of one or more of steps a) to d) is performed in the presence of a non-physiological ligand for a synthetic cytokine receptor.

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

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

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

93. The method of claim 92, wherein the non-adherent culture container is Aggrewell TM plate.

94. The method of any one of claims 86 and 88-93, wherein the aggregates in a) are embryoid bodies (EBs).

95. The method of any one of claims 86-89, wherein the culturing is performed in suspension.

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

97. The method of claim 94 or claim 95, wherein step a) comprises: (i) performing a first incubation, comprising culturing a cell population of the engineered stem cells under conditions that form a first aggregate; (ii) contacting the aggregates with a dissociation agent to form a dissociated cell population; as well as (iii) performing a second incubation comprising culturing the dissociated cell population under conditions where a second aggregate is formed.

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

99. The method according to any one of claims 86-98, wherein the culturing in b) is carried out in a culture medium comprising BMP4, FGF2, VEGF and Y27632.

100. The method according to any one of claims 86-98, wherein the culturing in b) is carried out in a culture medium comprising BMP4, FGF2 and VEGF.

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

102. The method according to any one of claims 86-96 and 101, wherein the culturing in b) is performed in a culture medium comprising a non-physiological ligand without any additional growth factors.

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

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

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

106. The method according to any one of claims 86-103, wherein the culturing in c) is carried out in a culture medium comprising BMP4 and FGF2.

107. The method of claim 106, wherein the culturing with BMP4 and FGF2 in c) is performed for 3 to 15 days.

108. The method of any one of claims 86-106, wherein the culturing in c) comprises a PI3K inhibitor.

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

110. The method of any one of claims 108 and 109, wherein the PI3K inhibitor is added during a portion of the culturing in c).

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

112. The method according to any one of claims 86-109, wherein the culturing in c) is carried out in a culture medium without SCF and TPO.

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

114. The method of any one of claims 86-102 and 112, wherein the culturing in c) is performed in a culture medium comprising a non-physiological ligand without any additional growth factors, cytokines, or both.

115. The method according to any one of claims 86-112, wherein the culturing in c) is performed on days 3 to 15.

116. The method of any one of claims 86-115, wherein during at least a portion of the culturing in c), the culture medium comprises an aryl hydrocarbon receptor (AHR) antagonist, e.g., a pyrimidine-[4,5-b]-indole derivative, e.g., or both.

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

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

119. The method according to any one of claims 116-118, wherein the SR1 and the UM729 are added to the culture in c) starting from one of day 6 to day 9.

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

121. The method according to any one of claims 88-119, wherein the culturing in d) is carried out in a culture medium comprising 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 88-121, wherein the culturing in d) is carried out in a culture medium comprising a non-physiological ligand.

123. The method of any one of claims 88-120 and 122, wherein the culturing in d) is performed in a culture medium comprising a non-physiological ligand without any additional growth factors, cytokines, or both.

124. The method according to any one of claims 88-123, wherein the culturing in d) is performed at a time between day 15 and day 40.

125. The method according to any one of claims 88-123, wherein the culturing in d) is performed from day 15 to day 30 or from day 15 to day 30.

126. A method of generating cytotoxic innate lymphoid (iCIL) cells, the method comprising: a) culturing a cell population comprising the engineered stem cells according to any one of claims 1 to 48 under conditions where aggregates are formed; 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 the plurality of cells, wherein the start of culturing 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 hematopoietic progenitor cell (HP) population, wherein the start of culturing in c) is day 3; and d) culturing the cells produced in d) in a medium comprising SCF and IL-15 to generate the iCIL cells, wherein the start of the culturing in b) is day 15, At least a portion of one or more of steps a) to d) is performed in the presence of a non-physiological ligand for a synthetic cytokine receptor.

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

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

129. A method of producing cytotoxic innate lymphoid (iCIL) cells, comprising contacting a cell population comprising the engineered stem cells of any one of claims 1-48 with a non-physiological ligand for a first period of time sufficient to produce CLPs, and contacting the CLPs with a differentiation medium for a second period of time sufficient to produce iCILs.

130. The method of 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 of claim 130, wherein the differentiation medium comprises a non-physiological ligand.

132. The method of claims 129-131, wherein the first time period is 1-15 days and the second time period is 1-15 days.

133. The method of any one of claims 129-132, 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 of time sufficient to generate mature iCIL.

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

135. The kit of claim 133 or 134, wherein the third time period is 1-10 days.

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

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

138. The method of claim 137, wherein the analog of rapamycin is a rapamycin analog (rapalog).

139. The method of any one of claims 86-138, wherein the non-physiological ligand is added to the culture medium at a concentration of 5 nM-200 nM, 5 nM-150 nM, 5 nM-100 nM, 5 nM-50 nM, 5 nM-20 nM, 5 nM-10 nM, 10 nM-200 nM, 10 nM-150 nM, 10 nM-1 00nM, 10nM-50nM, 10nM-20nM, 20nM-200nM, 20nM-150nM, 20nM-100nM, 20nM-50nM, 50nM-200nM, 50nM-150nM, 50nM-100nM, 100nM-200nM, 100nM-150nM and 150nM-200nM.

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

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

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

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

144. A hematopoietic progenitor (HP) cell produced by the method of any one of claims 86 and 90-119.

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

146. The HP cells of 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 times as compared to CD34+ umbilical cord blood cells.

147. The HP cells of claim 145 or 146, wherein the CD34+ cord blood cells comprise hematopoietic stem cells (HSCs).

148. A cytotoxic innate lymphoid (iCIL) cell produced by the method of any one of claims 87-136.

149. A hematopoietic progenitor (HP) cell that has been differentiated from the engineered stem cell of any one of claims 1-51, wherein the HP comprises a synthetic cytokine receptor.

150. A cytotoxic innate lymphoid cell (iCIL) that has been differentiated from the engineered stem cell of any one of claims 1-51, wherein the iCIL comprises a synthetic cytokine receptor.

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

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

153. The population of claim 153, 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-fold as compared to a population of CD34+ cord blood cells.

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

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

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

157. An iCIL according to claim 148 or claim 150 or a population of iCILs according to claim 155 or claim 156, wherein the iCIL comprises a B2M knockout.

158. An iCIL according to claim 148 or claim 150 or a population of iCILs according to claim 155 or claim 156, wherein the iCIL comprises a B2M knockout and a FKBP12 knockout.

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

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

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

162. The method of claim 160 or 161, performed in vitro or ex vivo.

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

164. The method of claim 163, wherein the analog of rapamycin is a rapamycin analog (rapalog).

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

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

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

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

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

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

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

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

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

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

175. The method of any one of claims 160-174, wherein the iCIL expresses a CAR that targets a cancer cell of the subject.

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

177. The method of claim 176, wherein the FITC ligand is FITC-folate or FITC-folate.

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

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

180. The method of claim 179, wherein the analog of rapamycin is a rapamycin analog (rapalog).

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

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

183. The method of claim 182, wherein the plurality of doses are administered intermittently or periodically, optionally for a predetermined period of time, following administration of the population of iCILs or composition thereof to the subject.

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

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

186. The method of any one of claims 172-185, wherein the iCIL population or composition thereof is at or about 1 x 10 8 iCIL cells are about 100x10 9 iCIL cells were administered at a dose of 100 μg / ml.

187. The method of any one of claims 172-186, wherein the iCIL population or composition thereof is present in an amount greater than or about 5×10 9 5×10 iCIL cells, optionally wherein the dose is or is about 5×10 9 iCIL cells are about 100×10 9 iCIL cells.

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

189. The method of claim 188, wherein the multiple doses of iCIL cells are administered intermittently or periodically, optionally for a predetermined period of time.

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

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

192. A kit comprising the engineered stem cells of any one of claims 1-51 and 84, and instructions for differentiating the cell population into cytotoxic innate lymphoid cells.

193. A kit comprising an iCIL or a population of iCILs according to any one of claims 148-158 or a pharmaceutical composition according to claim 159, and instructions for administration to a subject in need thereof.

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

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

196. A population of induced cytotoxic innate lymphoid (iCIL) cells, wherein the iCILs are mature iCILs expressing CD56 and LFA1, and wherein: At least 25% of iCILs expressed cytotoxic receptors; No more than 75% of iCILs expressed dysfunctional receptors; and / or At least 25% of the iCILs were proliferative.

197. The population of claim 196, wherein at least 25% of the iCILs express a cytotoxic receptor.

198. A population according to claim 196 or claim 197, wherein the cytotoxic receptor is one or more of NKp30, NKp46 and NKG2D.

199. The population of any one of claims 196-198, 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 the iCILs express NKp30+.

200. The population of any one of claims 196-199, wherein at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80% of the iCILs express NKp46.

201. The population of any one of claims 196-200, 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 the iCILs express NKG2D.

202. The population of any one of claims 196-201, wherein no more than 75% of the iCILs express a dysfunctional receptor.

203. A population according to any one of claims 196-202, wherein the dysfunctional receptor is one or more of KLRG1, CD73 and CD38.

204. The population of any one of claims 196-203, wherein no more than 45%, no more than 40%, no more than 35%, no more than 30%, no more than 25%, no more than 20%, no more than 15%, no more than 10% or no more than 5% of the iCILs express KLRG1.

205. A population according to any one of claims 196-204, wherein no more than 10%, no more than 9%, no more than 8%, no more than 7%, no more than 6%, no more than 5%, no more than 4%, no more than 3%, no more than 2% or no more than 1% of the iCILs express CD73.

206. A population according to any one of claims 196-205, wherein no more than 75%, no more than 65%, no more than 55%, no more than 45%, no more than 35%, no more than 25%, no more than 15% or no more than 5% of the iCILs express CD38.

207. The population of any one of claims 196-206, 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. A population according to any one of claims 196-207, wherein the proliferating iCIL is CD56 bright CD57-.

209. The population of any one of claims 196-208, wherein the iCIL further comprises a synthetic cytokine receptor for a non-physiological ligand, Wherein the cytokine receptors include: 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 β chain polypeptide comprising a second dimerization domain, a second transmembrane domain and an intracellular domain, wherein the intracellular domain is selected from the intracellular domain of interleukin-2 receptor subunit β (IL-2RB), the intracellular domain of interleukin-7 receptor subunit β (IL-7RB) and / or the intracellular domain of interleukin-21 receptor subunit β (IL-21RB).

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

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

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

1.

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

214. The population of claim 213, wherein the IL-2RG intracellular domain comprises a polypeptide sequence at least 95% identical to SEQ ID NO: 8 or 31, or a polypeptide sequence as shown in SEQ ID NO: 8 or 31.

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

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

2.

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

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

3.

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

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

4.

221. A population according to any one of claims 209-220, wherein the second transmembrane domain comprises a transmembrane domain from the same β chain intracellular domain.

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

223. A population according to any one of claims 209-216, 221 and 222, wherein: The synthetic γ chain polypeptide comprises an IL-2RG TM domain, wherein the IL-2RG TM domain comprises the sequence shown in SEQ ID NO: 8 or 31; and an IL-2RG intracellular domain, wherein the IL-2RG intracellular domain comprises the sequence shown in SEQ ID NO: 1; and The synthetic β chain polypeptide comprises an IL-2RB TM domain, wherein the IL-2RB TM domain comprises the sequence shown in SEQ ID NO: 35 or 36; and an IL-2RB intracellular domain, wherein the IL-2RB intracellular domain comprises the sequence shown in SEQ ID NO:

2.

224. A population according to any one of claims 209-223, wherein the first dimerization domain and the second dimerization domain are heterologous dimerization domains selected from the 12kD size FK506-binding protein (FKBP) and FKBP12-rapamycin binding (FRB) domain; and / or The non-physiological ligand is rapamycin or a rapamycin analog.

225. A population according to claim 224, wherein the FRB domain comprises a polypeptide sequence at least 95% identical to SEQ ID NO:6 or SEQ ID NO:

7.

226. A population according to claim 224, wherein the FRB domain comprises a polypeptide sequence as shown in SEQ ID NO:6 or SEQ ID NO:

7.

227. A population according to any one of claims 209-223, wherein the first dimerization domain and the second dimerization domain are heterologous dimerization domains selected from FK506-binding protein (FKBP) of size 12kD and a calcineurin domain; and / or The non-physiological ligand is FK506 or an analog thereof.

228. A population according to any one of claims 209 to 224, wherein the FKBP domain comprises a polypeptide sequence at least 95% identical to SEQ ID NO:5 or SEQ ID NO:

30.

229. A population according to any one of claims 209 to 224, wherein the FKBP domain comprises a polypeptide sequence as shown in SEQ ID NO:5 or SEQ ID NO:

30.

230. A population according to any one of claims 209-216 and 221-229, wherein the synthetic γ chain polypeptide has the amino acid sequence shown 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 β chain polypeptide has the amino acid sequence shown 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. A population according to any one of claims 209-216 and 221-230, wherein the synthetic γ chain polypeptide has the amino acid sequence shown in SEQ ID NO:28, and the synthetic β chain polypeptide has the amino acid sequence shown in SEQ ID NO:

33.

232. A population according to any one of claims 209-223, wherein the first dimerization domain and the second dimerization domain are homologous dimerization domains selected from the following: i) FK506 binding protein (FKBP) with a size of 12 kD; ii) Cyclophilin A (CypA); or iii) gyrase B (CyrB); And the non-physiological ligands are: i) FK1012, AP1510, AP1903 or AP20187 or their analogs; ii) cyclosporine-A (CsA) or its analogs; or iii) Coumarin or its analogs.

233. A population according to any one of claims 196-232, wherein the population comprises about 1x10 6 About 1x10 12 iCIL, about 1x10 6 About 1x10 10 iCIL, about 1x10 6 About 1x10 8 iCIL, about 1x10 8 About 1x10 12 iCIL, about 1x10 8 About 1x10 10 iCIL or about 1x10 10 About 1x10 12 iCIL.

234. A population according to any one of claims 196-233, wherein 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.

235. A pharmaceutical composition comprising a population of iCILs according to any one of claims 196-234.

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

237. A cryopreserved composition comprising a population of iCILs according to any one of claims 196-234.

238. The composition of any one of claims 235-237, wherein the composition comprises about 1 x 10 6 About 1x10 12 iCIL, about 1x10 6 About 1x10 10 iCIL, about 1x10 6 About 1x10 8 iCIL, about 1x10 8 About 1x10 12 iCIL, about 1x10 8 About 1x10 10 iCIL or about 1x10 10 About 1x10 12 iCIL.

239. The composition of any one of claims 235-238, wherein the volume of the composition 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.

240. A method of killing a target cell or inhibiting the proliferation of a target cell, comprising contacting the target cell with a population of iCILs according to any one of claims 196-234 or a composition according to any one of claims 235-239.

241. The method of claim 240, wherein the target cell is a cancer cell.

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

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

244. The method of claim 242 or claim 243, wherein the non-physiological ligand is rapamycin or an analog of rapamycin.

245. The method of claim 244, wherein the analog of rapamycin is a rapamycin analog (rapalog).

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

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

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

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

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

251. The method of any one of claims 240-242 and 244-248, wherein the method is performed in a subject and the population of iCILs or a composition thereof is administered to the subject.

252. The method of claim 251, wherein 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.

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 196-234 or a composition according to any one of claims 235-239.

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 196-234 or a composition according to any one of claims 235-239.

255. The method of any one of claims 251-254, wherein the subject has not been administered lymphodepletion therapy prior to administering the population of iCILs or composition thereof.

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

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

258. The method of claim 257, wherein the FITC ligand is FITC-folate or FITC-folate.

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

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

261. The method of claim 260, wherein the analog of rapamycin is a rapamycin analog (rapalog).

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

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

264. The method of claim 263, wherein the plurality of doses are administered intermittently or periodically, optionally for a predetermined period of time, following administration of the population of iCILs or composition thereof to the subject.

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

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

267. The method of any one of claims 251-266, wherein the iCIL population or composition thereof is at or about 1 x 10 8 iCIL cells are about 100x10 9 iCIL cells were administered at a dose of 100 μg / ml.

268. The method of any one of claims 251-267, wherein the iCIL population or composition thereof is present in an amount greater than or about 5×10 9 5×10 iCIL cells, optionally wherein the dose is or is about 5×10 9 iCIL cells are about 100×10 9 iCIL cells.

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

270. The method of claim 269, wherein the multiple doses of iCIL cells are administered intermittently or periodically, optionally for a predetermined period of time.

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

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

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

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

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

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