Cytotoxic innate lymphoid cells and uses thereof

Abstract

Compositions and methods are provided for cell populations comprising engineered cytotoxic innate lymphoid cells engineered for controlled expansion and / or activity, the engineered cytotoxic innate lymphoid cells comprising a synthetic cytokine receptor for a non-physiological ligand. The cytokine receptor of the present application may comprise a synthetic gamma chain polypeptide as a first dimerization domain, a first transmembrane domain and an interleukin-2 receptor subunit gamma (IL-2RG) intracellular domain, and a synthetic beta chain polypeptide as an intracellular domain selected from a second dimerization domain, a second transmembrane domain and an interleukin-2 receptor subunit beta (IL-2RB) intracellular domain, an interleukin-7 receptor subunit beta (IL-7RB) intracellular domain and / or an interleukin-21 receptor subunit beta (IL-21RB) intracellular domain. The non-physiological ligand of the present application activates the synthetic cytokine receptor in the cytotoxic innate lymphoid cells to induce expansion and / or activation of the engineered cytotoxic innate lymphoid cells. TIFF2025500894000055.tif52159

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. Provisional Application No. 63 / 291,235, filed December 17, 2021, U.S. Provisional Application No. 63 / 291,237, filed December 17, 2021, U.S. Provisional Application No. 63 / 392,862, filed July 27, 2022, U.S. Provisional Application No. 63 / 392,864, filed July 27, 2022, U.S. Provisional Application No. 63 / 392,865, filed September 28, 2022, This application claims priority to U.S. Provisional Application No. 63 / 411,056, filed September 28, 2022, U.S. Provisional Application No. 63 / 411,063, filed November 4, 2022, and U.S. Provisional Application No. 63 / 422,848, filed November 4, 2022, and U.S. Provisional Application No. 63 / 422,830, filed November 4, 2022, the entire contents of which are incorporated herein by reference.

[0002] INCORPORATION BY REFERENCE TO SEQUENCE LISTING This application has been filed with an electronic sequence listing, which is provided as a file entitled 260132000140SeqList.xml, created on December 16, 2022, and is 85,737 bytes in size. The information in the electronic format of the sequence listing is incorporated by reference in its entirety.

[0003] Technical Field The present invention relates generally to engineered lymphocytes, such as cytotoxic innate lymphoid cells (CILs), methods for making such cells, and methods for their use in immunotherapy. [Background technology]

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

[0005] CIL cells (e.g., NK cells) for use in immunotherapy can be obtained from primary sources such as peripheral blood or umbilical cord blood. Artificial sources of CIL cells include pluripotent stem cells, including induced pluripotent stem cells (iPSCs), which are cells derived from somatic cells (commonly fibroblasts or peripheral blood mononuclear cells [PBMCs]) that have been induced to allow unlimited proliferation and differentiation into other cell types when subjected to appropriate differentiation conditions, and human embryonic stem cells (hESCs). CIL cells can be derived from iPSCs by sequentially differentiating iPSCs into hematopoietic progenitor cells (HPCs), also called hematopoietic stem cells (HSCs), HPCs into common lymphoid progenitors (CLPs), and then CLPs into CIL cells, called iPSC-derived cytotoxic innate lymphoid cells (iPSC-CILs). In general, iPSC-CIL cells, like NK cells, express CD56 and have cytotoxic activity, although iPSC-CIL cells may differ from NK cells in phenotype and other respects.

[0006] After primary CIL cells or iPSC-CIL cells are obtained, they can be expanded ex vivo before being administered to a patient. Methods for expanding CIL cells ex vivo include contacting the cells with the cytokines interleukin 2 (IL-2) or interleukin 15 (IL-15). Known expansion protocols generally combine IL-2 or IL-15 with exogenous soluble factors, bead-bound factors or feeder cells.

[0007] It is known that such factors or feeder cells are required in effective methods for CIL cell expansion for manufacturing purposes. Such methods may further provide CIL cells with other factors to promote expansion. For example, antigen-presenting cells (APCs) can be genetically engineered to create artificial APCs (aAPCs). Expansion is generally further enhanced by engineering aAPCs to secrete interleukin 15 (IL-15) or interleukin 21 (IL-21), or to present recombinant membrane-bound IL-15 (mbIL-15) or membrane-bound IL-21 (mbIL-21). In summary, known methods for effective CIL cell expansion generally rely on a variety of exogenous soluble factors and feeder cell-based factors.

[0008] Methods are known for differentiating iPSCs into CD34+ HPCs using either embryoid bodies (EBs) or culturing single-cell iPSCs with feeder cells. CD34+ HPCs may then be differentiated into CLPs using a combination of stem cell factor (SCF), bone morphogenetic protein 4 (BMP4) and vascular endothelial growth factor (VEGF) in a conventional manner. Known methods for differentiating CLPs into CILs generally involve contacting CLPs with SCF, interleukin 17 (IL-7), interleukin 15 (IL-15), FMS-like tyrosine kinase 3 ligand (FLT3L) and optionally interleukin (IL-3). The resulting iPSC-CIL cells may then be expanded using methods previously described.

[0009] There remains a need in the art for compositions and methods relating to engineered cytotoxic innate lymphoid cells, methods for making such cells, and methods for their immunotherapy use. Summary of the Invention

[0010] The present disclosure is based in part on the surprising discovery by the inventors of unexpected properties of lymphocytes, such as cytotoxic innate lymphoid (CIL) cells (e.g., NK cells), that have been engineered to express a synthetic cytokine receptor. In some aspects, lymphocytes, such as CIL cells (e.g., NK cells), that have been engineered to express a synthetic cytokine receptor can expand in response to the receptor's cognate non-physiological ligand and have other desirable characteristics, such as functional activity, over and above unengineered lymphocytes, such as CIL cells from other sources.

[0011] According to the methods described herein, lymphocytes such as CIL cells can be generated in large quantities with desired functional characteristics from primary cells or iPSC-derived lymphocytes such as cytotoxic innate lymphoid (iPSC-CIL) cells. Non-limiting advantages of certain embodiments may include the ability of engineered lymphocytes such as CIL cells (e.g., NK cells) described herein to be expanded without the use of exogenous factors, for example, without IL-2, IL-7, IL-15 and / or IL-21. Since it is well known in the relevant art that the expansion of CIL cells (e.g., NK cells) generally depends on having various exogenous stimuli for the CIL cells, the expansion of engineered CIL cells that omits one or more of these stimuli as described herein is surprising and unexpected. In certain embodiments, the methods disclosed herein may further enhance the expansion by providing other expansion factors to engineered lymphocytes such as engineered CIL cells. Engineered lymphocytes, such as the engineered CIL cells described herein, and related compositions, may be used for immunotherapy using ex vivo expansion.

[0012] Further non-limiting advantages described herein may include increased proliferation capacity, resistance to senescence, and cytotoxic activity compared to lymphocytes that do not express synthetic cytokine receptors (e.g., CIL cells such as NK cells). In some cases, advantages may also include improved expression of surface markers characteristic of NK cells.

[0013] Among the embodiments provided are engineered lymphocytes or progenitor cells that contain a synthetic cytokine receptor for a non-physiological ligand, the cytokine receptor comprising (i) a synthetic gamma chain polypeptide comprising a first dimerization domain, a first transmembrane domain, and an interleukin-2 receptor subunit gamma (IL-2RG) intracellular domain, and (ii) a synthetic alpha chain polypeptide or a synthetic beta chain polypeptide comprising a second dimerization domain, a second transmembrane domain, and an intracellular domain selected from an interleukin-2 receptor subunit beta (IL-2RB) intracellular domain, an interleukin-7 receptor subunit beta (IL-7RB) intracellular domain, an interleukin-21 receptor subunit beta (IL-21RB) intracellular domain, or an interleukin-9 receptor subunit alpha (IL-9RA) intracellular domain. In some embodiments, engineering the synthetic cytokine receptor in the engineered lymphocytes or progenitor cells results in controlled differentiation and / or expansion of the engineered cells. In some embodiments, binding of a non-physiological ligand to the synthetic cytokine receptor activates the synthetic cytokine receptor in the engineered lymphocytes or progenitor cells, inducing differentiation and / or expansion of the engineered lymphocytes and / or progenitor cells within the cell population.

[0014] In some embodiments, the engineered cells are engineered progenitor cells that are hematopoietic progenitor cells (HPCs; also called hematopoietic stem cells (HSCs)) or common lymphoid progenitor cells (CLPs). In some embodiments, the engineered cells are cytotoxic innate lymphoid cells (CILs), NK cells, or T cells. In some embodiments, the T cells are γδ T cells or αβ T cells. In certain embodiments, the engineered cells are CILs, such as NK cells.

[0015] In some embodiments, provided herein are engineered cytotoxic innate lymphoid (CIL) cells comprising a synthetic cytokine receptor for a non-physiological ligand, the cytokine receptor comprising a synthetic gamma chain polypeptide comprising a first dimerization domain, a first transmembrane domain, and an interleukin-2 receptor subunit gamma (IL-2RG) intracellular domain, and a synthetic alpha chain polypeptide or a synthetic beta chain polypeptide comprising a second dimerization domain, a second transmembrane domain, and an intracellular domain selected from an interleukin-2 receptor subunit beta (IL-2RB) intracellular domain, an interleukin-7 receptor subunit beta (IL-7RB) intracellular domain, an interleukin-21 receptor subunit beta (IL-21RB) intracellular domain, or an interleukin-9 receptor subunit alpha (IL-9RA) intracellular domain. In some embodiments, the synthetic cytokine receptor for the non-physiological ligand is engineered to enter the engineered CIL cells for controlled expansion and / or activity of the engineered CIL. In some embodiments, binding of a non-physiological ligand to the synthetic cytokine receptor activates the synthetic cytokine receptor in the engineered CIL cells, inducing expansion and / or activation of the engineered CIL cells within the cell population.

[0016] Also provided herein is a cell population containing engineered lymphocytes or progenitor cells according to any of the embodiments provided. Also provided herein is a cell population containing engineered CIL cells according to any of the embodiments provided. Also provided herein is a method of genetically engineering cells to express a synthetic cytokine receptor by introducing a nucleic acid or vector encoding the synthetic cytokine receptor into the cells. In some embodiments, the synthetic cytokine receptor is inserted into an endogenous gene in the cells. In such embodiments, the method comprises contacting a population of lymphocytes or progenitor cells, e.g., CIL cells, with a recombinant vector comprising a guide RNA (gRNA) targeting a target site in the endogenous gene, (ii) an RNA-guided endonuclease, and (iii) a nucleotide sequence encoding a synthetic cytokine receptor for a non-physiological ligand, thereby inserting the nucleotide sequence into the endogenous gene. Any method for gene editing and insertion of a synthetic cytokine gene can be used, including any described herein. In some embodiments, the insertion method is by homology-directed repair (HDR). Also provided is a cell population produced by any of the methods provided.

[0017] Also provided are methods of expanding engineered lymphocytes, such as engineered CIL cells, by contacting any of the engineered cells provided with a non-physiological ligand for the synthetic cytokine receptor. Also provided are methods of expanding engineered lymphocytes, such as engineered CIL cells, by contacting any of the engineered cell populations provided with a non-physiological ligand for the synthetic cytokine receptor. In some embodiments, the non-physiological ligand is rapamycin or a rapamycin analog.

[0018] Also provided herein are pharmaceutical compositions containing any of the engineered cells, e.g., engineered CILs, provided. In some embodiments, the pharmaceutical composition contains a pharma- ceutically acceptable carrier.

[0019] In some embodiments, methods of treatment and use of any of the engineered cells or engineered cell populations provided are also provided herein. Methods of treatment and use of any of the pharmaceutical compositions provided are also provided herein. Use of any of the engineered cells, engineered cell populations, or pharmaceutical compositions provided in the preparation of medicaments for carrying out such methods and treatments, as well as in the preparation of medicaments for carrying out such methods of treatment, are provided herein. In some embodiments, the methods are carried out by administering the engineered cells or compositions comprising the same to a subject having, having, or suspected of having a disease or condition or disorder. In some embodiments, the methods thereby treat the disease or condition or disorder in the subject. In some embodiments, the methods can be used to treat any disease or condition, and immunotherapy by adoptive cell therapy can be used to treat the disease or condition. In some embodiments, the engineered cells express a recombinant receptor, such as a chimeric antigen receptor (CAR), that targets an antigen on cells associated with the disease or condition. In some embodiments, the disease or condition is cancer. Exemplary methods and uses are described herein.

[0020] In certain aspects, provided herein are therapeutic methods, kits and pharmaceutical compositions for use in immunotherapy with ex vivo generated lymphocytes (e.g., CIL cells such as NK cells). In certain aspects, the engineered lymphocytes described herein, e.g., CIL cells, are generated, optionally expanded ex vivo, and then administered to a subject in need of immunotherapy.

[0021] In a preferred embodiment, the synthetic cytokine receptor is the rapamycin-activated cytokine receptor (RACR), which can be activated by either rapamycin or its derivatives (rapalogs).

[0022] Lymphocytes, such as CIL cells (e.g., NK cells), can be derived from iPSCs, common lymphoid progenitors (CLPs), or other stem or progenitor cells. Further provided herein are CLPs engineered to express synthetic cytokine receptors, and methods for differentiating the engineered CLPs into CIL cells by contacting the CLPs with the cognate non-physiological ligand for the cytokine receptor.

[0023] The present disclosure also provides other compositions and methods, including, without limitation, pharmaceutical compositions, methods of treatment, kits, etc. The foregoing advantages may be present in some, but not all, of the embodiments described herein, and other advantages of the compositions and methods described herein will become apparent from the detailed description that follows. [Brief description of the drawings]

[0024] [Figure 1A] FIG. 1A is a diagram of an embodiment showing the expansion of engineered cytotoxic innate lymphoid cells (eg, natural killer (NK) cells). [Figure 1B] FIG. 1B is a diagram of an embodiment showing differentiation of engineered stem or progenitor cells. [Figure 2A] Figure 2A is a panel of graphs showing RACR expansion of CD19 CAR blood-derived NK (bdNK) cells. The top panel shows the timeline of the experiment. The percent CAR expression over time for each group is shown in the left panel, and the total CAR+NK cells over time are shown in the right panel. [Figure 2B] Figure 2B is a panel of graphs showing CAR expression analysis by flow cytometry. CAR expression is shown in the top panel for bdNK cells expanded in the presence of either IL-2 (1000U / ml) or AP21967 (20nM). CD56 and CD16 expression of CAR+ cells is shown in the bottom panel. [Figure 2C]FIG. 2C is a panel of graphs showing the ability of RACR-expanded blood-derived NK cells to recognize and target tumor cells. The top panel shows the timeline of the experiment. Flow cytometry plots showing CAR expression 31 days after transduction are shown in the top right panel. 1×105 NK cells were incubated with 1×105 K562 cells, Nalm6 cells, or Nalm6 CD19 knockout cells in the presence of brefeldin A, monensin, and anti-CD107 in 100 μl of RPMI medium in a 96-well plate for 5 hours. CD107a expression levels (left panel) and IFNγ expression levels (middle panel) in AP21967-expanded CAR+ cells (AP-20) or non-transduced controls are shown. % death (right panel) was calculated by comparing the total number of viable cell trace violet+ cells in each well with non-effector control wells. NK effector cells were added at various effector cell:target cell (E:T) ratios. [Figure 3A] FIG. 3A is a diagram of the iPSC-derived IL cell (eg, NK cell) generation process. [Figure 3B] FIG. 3B is a graph showing the % CD34+ cells before and after cell selection and analysis on day 12 of the differentiation process. [Figure 3C] FIG. 3C is a panel of graphs showing CD34+ selection by flow cytometry analysis on day 12 of the differentiation process. [Figure 3D] Figure 3D is a graph showing the percentage of CD45+ cells among white blood cells and the percentage of white blood cells among progenitor and CIL cells (NK cells). The percentage of CD45+ cells is plotted as % white blood cells, CD7+ cells are plotted as progenitor cells, and CD56+ cells are plotted as CIL cells. [Figure 3E] FIG. 3E is a panel of graphs showing flow cytometry analysis of differentiated leukocytes (CD45+), precursor (CD45+ / CD5- / CD7+) and CIL cells (CD45+ / CD5- / CD7+ / CD56+). [Figure 3F]FIG. 3F is a panel of graphs showing the % of leukocytes or CIL cells at day 40 (left panel) and flow cytometry analysis of differentiated cells at day 40 (right panel). [Figure 3G] FIG. 3G is a panel of graphs showing the percentage of iCIL cells that were harvested and immunophenotyped by flow cytometry for detection of markers: CD16, IL7R, KIR, NKp30 and NKp46 (left panel) and cytotoxicity as measured by % 4H lysis of K562 cells for various effector:target cell (E:T) ratios. [Figure 4A] FIG. 4A shows the experimental timeline. [Figure 4B] FIG. 4B is a panel of plots showing representative flow cytometry analysis of CD56+ cells and TagCAR (anti-fluorescein isothiocyanate (FITC) chimeric antigen receptor (CAR)) enrichment over the course of the experiment. The x-axis is TagCAR detection with FITC-AF647 and the y-axis is side scatter. Cells analyzed after "mock" transduction, 32 days, 35 days, 38 days and 42 days post-transduction. [Figure 4C] Figure 4C is a panel of graphs showing RACR enrichment of TagCAR cells over time (left panel), total cell number (middle panel), and percentage of cells expressing CD45 and CD56 over time (right panel) for the BXS cell line. [Figure 4D] FIG. 4D is a panel of graphs showing RACR enrichment of TagCAR cells over time (left panel), total cell number (middle panel), and percentage of cells expressing CD45 and CD56 over time (right panel) for NHS cell lines. [Diagram 5]Figure 5A is a panel of histograms showing different activation markers of cytokine-differentiated mock-CIL cells (BXS line) and RACR-iCIL cells (BXS and NH5 lines). Cells were gated for CD45+CD7+CD5- and then plotted for activation markers CD56, CD16, NKp30, NKp40 and NKG2D. Figure 5B is a graph of a cytotoxicity assay showing the killing of MDA-mCherry tumor cell lines by cytokine-differentiated mock-CIL cells (BXS cell line) and RACR-iCIL cells (BXS and NH5 cell lines). [Figure 6A] FIG. 6A shows the experimental timeline. [Figure 6B] FIG. 6B is a graph showing % RACR iCIL cells over time and total number of RACR iCIL cells over time for cells treated with Expansion Medium containing IL-2, IL-15, IL-21, IL-18, IL-7 (cytokine mix) or cells treated with A / C Heterodimerizer AP21967 (AP 100 nM). [Figure 6C] FIG. 6C is a panel of flow cytometry plots showing cells stained for CD56 clone HCD56 positive cells and RACR-FRB positive cells detected by mCherry. [Figure 6D] FIG. 6D is a panel of graphs showing % FITC-CAR iCIL cells over time (top panel) and total number of FLCAR iCIL cells over time (bottom panel) for cells transduced with viral constructs containing TagCAR-RACR-FRB(206) and FRB-RACR-TagCAR(205). [Figure 6E] FIG. 6E is a panel of flow cytometry plots showing cells stained for CD56 clone HCD56 positive cells and FLCAR positive cells. [Figure 7A] Figure 7A is a panel of flow cytometry plots showing cells stained for CD56 and Tag-CAR. [Figure 7B]FIG. 7B is a panel of histograms showing various markers of activation and cytotoxicity, including NKp30, NKp46, NKG2D, NKG2A and CD57, in TagCAR+RACR expanded iCIL cells, mock IL2 expanded CIL cells, and unstained cells. [Figure 7C] FIG. 7C is a panel of histograms showing CD107a secretion in response to antigen in mock IL2-expanded CIL cells and TagCAR RACR-expanded iCIL cells. [Figure 8] FIG. 8 is a diagram of an exemplary CAR of the disclosure, where the fusion protein is encoded by a lentiviral expression vector, "SP" is the signal peptide, the CAR is an anti-FITC CAR, the CD8α hinge is present, the transmembrane domain is present ("TM"), the costimulatory domain is 4-1BB, and the activation signaling domain is CD3ζ. [Figure 9]9 shows the differentiation factors involved in the transition from iPSC to CIL cells along with a timeline. The differentiation factors involved in each stage of differentiation are shown in open boxes. In bold are differentiation factors that exemplify embodiments of the present disclosure. In some embodiments, starting at week 4, cells are differentiated in medium containing IL7, IL15, SCF, FLT3L and UM729, optionally with or without a rapalog. Starting at week 6, these cells are expanded in medium containing IL7, IL15, SCF, FLT3L, UM729 and CD2 / NKp46 beads, optionally with or without a rapalog. In some embodiments, starting at week 4, cells are differentiated in medium containing SCF, FLT3L and UM729, optionally with or without a rapalog. Starting at week 6, the cells are expanded in a medium containing SCF, FLT3L, UM729 and CD2 / NKp46 beads, optionally with or without a rapalog. In some embodiments, starting at week 4, the cells are differentiated in a medium containing IL7, IL15 and UM729, optionally with or without a rapalog. Starting at week 6, the cells are expanded in a medium containing IL7, IL15, UM729 and CD2 / NKp46 beads, optionally with or without a rapalog. In some embodiments, starting at week 4, the cells are differentiated in a medium containing UM729, optionally with or without a rapalog. Starting at week 6, the cells are expanded in a medium containing UM729 and CD2 / NKp46 beads, optionally with or without a rapalog. [Figure 10]Figure 10 is a graph showing the % of RACR CD19-CAR positive cells as a function of days in culture. Blood-derived NK cells were transduced with CD19-CAR-RACR (IL-2RG / IL-2RB)-, CD19-CAR-RACR (IL-2RG / IL-7RB)- or CD19-CAR-RACR (IL-2RG / IL-21RB)-containing viruses. Cells were then expanded in 100 IU / mL human IL-2 or 100 nM AP219667 in complete medium supplemented with membrane-bound IL-21 (mbIL-21) 41BBL and K562 feeder cells weekly. RACR(IL-2RG / IL-2RB) "RACR2" supported the highest expansion of AP219667, while RACR(IL-2RG / IL-7RB) "RACR7" and RACR(IL-2RG / IL-21RB) "RACR21" both supported RACR expansion to a lesser extent. [Figure 11] Figure 11 is a panel of graphs showing RACR enrichment of CAR-expressing cells over time (left panel), fold change of progenitor cells (0-4 weeks) and fold change of RACR-iCIL cells (4-9 weeks) (middle panel), and cytotoxicity as measured by % killing of K562 cells by iCIL and RACR-iCIL cells for various effector:target cell (E:T) ratios (right panel). The top of the figure shows a diagram of hematopoietic lineage differentiation and the corresponding cell culture. [Figure 12] Figure 12A is a graph showing 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. Figure 12B is a graph showing the % of CAR+ cells containing the markers CD45+CD5-, CD45+CD5-CD7+, or CD45+CD5-CD7+CD56+ for cells thawed on day 26 and mock-transduced or analyzed on day 29 post-viral vector transduction. [Figure 13]Figure 13A is a graph showing the % of CAR+iCIL cells after analysis of cells plated in media containing IL7, IL15, SCF, FLT3L and UM729 (Full Mix), IL7, IL15 and UM729 (IL7 / IL15), or UM729 (none). Each media condition was treated with or without a rapalog. Figure 13B is a graph showing the fold change (FC) of the % of CAR+iCIL cells after analysis of cells plated in media containing IL7, IL15, SCF, FLT3L and UM729 (Full Mix), IL7, IL15 and UM729 (IL7 / IL15), or UM729 (none). Each media condition was treated with or without a rapalog. Figure 13C is a graph showing the total number of CAR+iCIL cells after analysis of cells plated in medium containing IL7, IL15, SCF, FLT3L and UM729 (Full Mix), IL7, IL15 and UM729 (IL7 / IL15), or UM729 (none). Each medium condition was treated with or without a rapalog. Figure 13D is a graph showing the fold change (FC) of the total number of CAR+iCIL cells after analysis of cells plated in medium containing IL7, IL15, SCF, FLT3L and UM729 (Full Mix), IL7, IL15 and UM729 (IL7 / IL15), or UM729 (none). Each medium condition was treated with or without a rapalog. [Figure 14] FIG. 14A is a panel of histograms showing various markers including CD45, CD7 and CD56 on TagCAR+RACR-iCIL cells plated in media containing IL7, IL15, SCF, FLT3L and UM729 (Full 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 rapalog. FIG. 14B is a histogram showing the CD56 marker on progenitor cells at day 26 (CD56-) and cells plated in media containing IL7, IL15, SCF, FLT3L and UM729 (Full Mix) analyzed at day 40 (CD56+). [Figure 15A]Figure 15A shows a graph showing tumor cell proliferation 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 shown in thick black line. Tumor cells were reintroduced 50 hours later (i.e., tumor cell reloading). [Figure 15B] Figure 15B shows a graph showing tumor cell proliferation after incubation of bladder cancer cells with unstimulated NK cells, cytokine-stimulated NK cells, or RACR-stimulated NK cells. Bladder cancer cells that were not incubated with NK cells are shown in thick black lines. Tumor cells were reintroduced at 50 and 100 hours (i.e., tumor cell reloading). [Figure 15C] Figure 15C shows a graph depicting tumor cell proliferation after incubation of breast adenocarcinoma cells with unstimulated NK cells, cytokine-stimulated NK cells, or RACR-stimulated NK cells. Breast adenocarcinoma cells not incubated with NK cells are shown in thick black line. Tumor cells were reintroduced after 40 hours (aka tumor cell reloading). [Figure 15D] FIG. 15D shows a graph depicting RACR-NK cell proliferation after incubation without rapamycin or cytokines (unstimulated NK), with rapamycin (RACR-stimulated NK), or with cytokines (cytokine-stimulated). [Figure 16A] Figure 16A shows a graph showing tumor cell killing in tumor cells incubated without RACR-engineered iCIL (tumor only or T), with RACR-engineered iCIL and no CAR antigen expression (R+NCA), with RACR-engineered iCIL and moderate CAR antigen expression (R+MCA), or with RACR-engineered iCIL and high CAR antigen expression (R+HCA). The CAR antigen is specific for FITC conjugated to folate, which binds to the folate receptor on the tumor cells. [Figure 16B] Figure 16B shows a graph depicting 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. [Figure 16C] FIG. 16C shows a graph depicting RACR-CAR-NK cell proliferation after incubation without rapamycin or cytokines (unstimulated NK), with rapamycin (RACR-stimulated NK), or with cytokines (cytokine-stimulated). [Figure 17A] Figure 17A shows a timeline for an in vivo mouse model of breast cancer. 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 subcutaneously injected with FITC-Folate for two weeks. On day 1 of the experiment, mice were intraperitoneally injected with RACR-TagCAR-NK-92 cells, followed by treatment with rapamycin three times a week or IL2 / IL15 three times a week. [Figure 17B] FIG. 17B shows a graph depicting quantification of luminescence correlating with tumor growth in mice receiving various treatments over a 3 week period. [Figure 17C] FIG. 17C shows raw luminescence images of tumor growth in mice receiving various treatments over a 3-week period. [Figure 17D] FIG. 17D shows RACR-NK in the blood of the mice in FIG. 22C. [Figure 17E] FIG. 17E shows RACR-NK detection in tissues of the mice in FIG. 22C. [Figure 18] Figure 18 shows in vitro tumor cell killing in co-cultures of RACR iCIL with MDA-MB-231 breast cancer cells. Breast cancer cells were cultured alone and left untreated or treated with cytokines or rapalogs (left legend) or co-cultured with RACR iCIL and left untreated or treated with cytokines or rapalogs (right legend). [Figure 19A]Figure 19A shows a timeline for an in vivo mouse model of breast cancer. Five days before the start of the experiment, mice were injected with MDA-231 mCherry / luciferase cells. Mice were imaged one day before the start of the experiment and bled once a week for the duration of the experiment. On day 1 (D0) of the experiment, mice were injected intraperitoneally with RACR-iCIL cells and subsequently treated with rapamycin three times a week or IL2 / IL15 three times a week. [Figure 19B] FIG. 19B shows raw luminescence images of tumor growth in mice receiving various treatments over a 3-week period. [Figure 19C] FIG. 19C shows a timeline for an in vivo mouse model of breast cancer. 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 once a week for the duration of the experiment. On day 1 (D0) of the experiment, 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 of the experiment, mice were injected again with RACR-iCIL cells. [Figure 19D] FIG. 19D shows raw luminescence images of tumor growth in mice receiving various treatments over a 6-week period. [Figure 19E] FIG. 19E shows a survival plot of the mice described in FIG. 19D. [Figure 20] Figures 20A-D show a panel of graphs depicting RACR-dependent expansion of Vd2 cells. The percentage of RACR+Vd2+ expression over time for each treatment group (e.g., IL2, RAPA, AC) is shown in Figure 20A. The total number of RACR+Vd2+ cells over time is shown in Figure 20B. The fold changes of Figures 20A and 20B are shown in Figures 20C and 20D, respectively. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] Detailed Description of the Invention Induced pluripotent stem cells (iPSCs) are a renewable, modifiable and scalable source of material for cell therapy manufacturing. However, significant challenges exist with current iPSC-based approaches to cell therapy. Specifically, current approaches for differentiating iPSCs into therapeutic immune cell types require the presence of exogenous growth factors and, in some cases, feeder cells.

[0026] iPSCs can be created by reprogramming adult cells to a cellular state similar to embryonic stem cells. iPSCs can differentiate into any cell type found in the human body and are believed to have unlimited expansion proliferation capacity, meaning that they can replicate and grow indefinitely, providing a nearly infinite supply of starting material. In addition, iPSCs are amenable to precise multiplex genome editing, allowing for the safe introduction of multiple genetic modifications. Because of these properties, iPSCs provide a consistent starting material derived from a single cell (clone), allowing for consistent genomic integrity in process intermediates and final cell products.

[0027] Current approaches to cell therapy manufacturing include either autologous or allogeneic cell starting materials from which disease-targeting therapeutic cell products are engineered. In some aspects, allogeneic or "off-the-shelf" cell therapies have the potential to transform cell therapy from personalized medicine to routine treatment. However, current "off-the-shelf" cell therapies struggle to demonstrate the same cell engraftment and persistence in vivo that is achieved by approved autologous cell therapy products. In some aspects, this is due to the foreignness of the allogeneic or even engineered elements of the autologous cells, which can be recognized and rejected by the host immune system. To date, cell therapy achieves cell engraftment by treating patients with a highly toxic chemotherapy regimen called lymphodepletion (LD), which is given prior to administration of the cell therapy product. LD essentially eliminates the host immune system, providing many benefits to cell therapy products, including initially providing free “homeostatic cytokines” for ex vivo cell therapy products, as well as reducing the anti-graft response to the foreign graft by the host immune system. However, LD is a transient solution, and the host immune system rapidly reconstitutes. Thus, multiple LDs and cell infusions are required to sustain allogeneic cell exposure. Additionally, exogenous cytokines such as IL-2 are administered, but these cytokine treatments have short exposure times and high toxicity associated with their use. Finding even better ways to enhance cell persistence is key to achieving sustained tumor remission and has proven to be a challenge in the allogeneic cell therapy field.

[0028] Allogeneic cells can be further classified as donor-derived cells or iPSC-derived cells. Donor-derived cells are generally sourced from circulating blood or umbilical cord blood of healthy donors, and the therapeutic cell type (e.g., natural killer cells or NK cells) is selected in a complex cell culture process that generally includes multiple cytokines, growth factors, genetic manipulations and feeder cells, and is subsequently harvested and expanded to yield many doses. Alternatively, for iPSC-derived cells, which also require multiple complex cell culture conditions, these conditions must be implemented stepwise to drive the cells through the precursor stages required to ultimately obtain the intended final cell product (e.g., immune effector cells). For example, an effective method for CIL cell expansion for clinical-scale purposes typically involves exogenous cytokines including IL-2, IL-15 and / or IL-7, as well as antigenic molecules, costimulatory molecules and / or cell adhesion molecules. CIL cells are cytotoxic lymphocytes characterized by their ability to distinguish self from non-self by monitoring the expression of MHC class I molecules, release of cytokines, and directly kill non-self cells or infected target cells. It is known in the art that CIL cells are not a homogenous population. Rather, there are many distinct subsets of CIL cells. In many studies, these exogenous factors are replenished during the ex vivo expansion of CIL cells and / or after injecting CIL cells into subjects. Effective expansion of CIL cells that rely on large amounts of various exogenous factors using currently known methods often requires complex and expensive manufacturing processes. A further challenge in the art involves regulating the activity of endogenous CIL cells in vivo, especially considering that cancer patients exhibit significantly reduced NK cell activity compared to healthy patients.

[0029] Moreover, the gold standard of cell therapy is autologous derived chimeric antigen receptor (CAR) T cell therapy. Decades of CAR T cell therapy attempts, starting with the "first generation" CAR T cell therapy in the 1990s and culminating with the first CAR T cell therapy approved by the FDA in 2017, have successfully treated B cell malignancies, achieving long-term remission in 30-40% of certain patient populations. Importantly, the efficacy of CAR T cells requires lymphodepleting chemotherapy to eliminate nidus for survival factors such as IL-15. Although CAR T cell therapy has revolutionized the treatment of malignancies (e.g., hematological malignancies), significant limitations have hindered its widespread application. Although the allogeneic CAR T therapy field has shown promising early clinical results, sustained response profiles have generally been poor compared to autologous CAR T cell therapy, despite the use of LD regimens of ever-increasing intensity. This is likely due to limitations in the formulation cell type, manufacturing process, and anti-allograft responses to the therapeutic cells. Thus, despite the promising clinical efficacy of CAR T cells in hematological malignancies, significant challenges remain, including patient access, complex manufacturing, and high cost. The engineered CAR T cells and associated methods provided offer an "off-the-shelf" cancer therapy to overcome these challenges.

[0030] Also, iPSCs can be modified via CRISPR to express CARs, for example, to overcome the challenges associated with targeting the heterogeneous solid tumor microenvironment.

[0031] Overall, iPSC-based cell therapies are generally inefficient in generating the necessary intermediate progenitor cells, resulting in low initial yields of therapeutic cell types (e.g., cytotoxic innate lymphoid (CIL) cells) followed by the need for feeder cell-facilitated expansion, which can dramatically reduce the proliferation capacity of the final cell therapy product. Thus, high cell numbers (approximately 1 billion cells) and repeated administrations are required in addition to repeated cycles of lymphodepleting chemotherapy to achieve the engraftment required to have any therapeutic effect.

[0032] Provided herein is a platform for expanding immune effector cells in the absence of exogenous cytokines and feeder cells by genetically modifying lymphocytes, such as CILs (e.g., NK cells), to express synthetic cytokine receptors. In some aspects, lymphocytes can be primary cells, or lymphoid progenitor cells, or cells derived or differentiated from lymphoid progenitor cells. In some embodiments, provided herein is a platform for expanding immune effector cells in the absence of exogenous cytokines and feeder cells by genetically modifying iPSC-derived progenitor cells (e.g., common lymphoid cells) to express synthetic cytokine receptors for the expansion of lymphocyte differentiation, such as CILs. Synthetic small molecule ligands (e.g., rapamycin) activate the receptor to promote the expansion of immune effector cells. In some embodiments, the synthetic cytokine receptor is the rapamycin-activated cytokine receptor (RACR), which can be associated with rapamycin or an analog, e.g., a rapalog. In some embodiments, RACR-induced lymphocytes are lymphocyte types that are promoted by RACR during either differentiation and / or expansion. Examples of such "RACR-induced" cells include cytotoxic innate lymphoid cells (RACR-iCIL), NK (RACR-iNK), or T cells (RACR-iT), including γδ T cells or αβ T cells. In certain embodiments, RACR-induced cells, such as RACR-iCIL, are the resultant cells that have used RACR for differentiation or expansion instead of the normal cytokine pathway, for example, instead of the normal cytokine pathway from a precursor cell (e.g., a common lymphoid progenitor cell or a pre-NK cell).

[0033] The compositions and methods provided herein include engineered lymphocytes, such as CIL cells (e.g., NK cells), to express synthetic cytokine receptors.Non-limiting advantages of engineered lymphocytes, such as CIL cells (e.g., NK cells), include superior and controllable expansion when administered to a subject, similar cytotoxic activity compared to natural lymphocytes (e.g., CIL cells), improved iPSC-derived cell production, and enhanced anti-tumor activity.

[0034] Improved cell manufacturing. In provided aspects, the RACR engineering platform provided herein improves cell therapy manufacturing by controlling cell production. In some aspects, the RACR engineering platform provided herein improves iPSC-derived cell manufacturing by controlling cell production. The RACR engineering platform also reduces manufacturing costs, since RACR activation eliminates the need to add expensive growth factors, cytokines and other raw materials. In certain embodiments, the methods disclosed herein can further enhance expansion via the ability of the engineered lymphocytes described herein, e.g., CIL cells, to expand without exogenous factors or with reduced exogenous factors, e.g., without IL-2, IL-15 and / or IL-7. The RACR engineering platform increases the yield of high purity intermediate and final cell products. The RACR engineering platform enhances the patient compatibility of cells, since the manufacturing process is completely free of feeder cells and xenogeneic cells. The RACR engineering platform is also compatible with cells in suspension, facilitating the scalability of cell production.

[0035] Enhancement of antitumor activity. In a provided aspect, the RACR engineering platform provided herein improves the antitumor activity of engineered lymphocytes, for example, CILs comprising iPSC-derived cells, by increasing the engraftment, persistence and effector function of cells. The RACR engineering platform provided herein also improves the antitumor activity of engineered lymphocytes, for example, CILs comprising iPSC-derived cells, by inhibiting host immune response via rapamycin administration, thereby further enabling the engraftment of cells (for example, CILs). The RACR engineering platform provided herein also improves the antitumor activity of engineered lymphocytes, for example, CILs comprising iPSC-derived cells, by removing the need for toxic lymphodepletion (LD) by activating the RACR system to selectively support the expansion and survival of RACR cells.

[0036] In some aspects, the advantages of the RACR system over lymphocytes, such as CILs (e.g., NK cells), including engineered synthetic cytokine receptors and their activation by rapamycin or rapalogs, include the ability to engineer unlimited starting materials that are efficient in generating immediate precursors and feature minimal expansion requirements for final cell types; the ability to efficiently edit cells; no need for feeder cells, thereby minimizing complex raw materials; no need for lymphodepletion in subjects administered RACR-engineered cells; reduced or no cytokine release syndrome (CRS) or immune effector cell-associated neurotoxicity syndrome (ICAN); and promotion of engraftment, expansion and persistence by administration of rapamycin or rapalogs. In some embodiments, engineered lymphocytes such as RACR-iCILs provide a cell source for allogeneic cell therapy that can be achieved in some aspects with minimal or no immune manipulation requirements.

[0037] Provided herein is a synthetic cytokine receptor, e.g., RACR, that can be applied to support the induction of cytotoxic innate lymphoid (CIL) cells, e.g., NK cells. CIL cells can be derived from stem cells or progenitor cells, and such cells are referred to herein as "induced cytotoxic innate lymphoid" (iCIL) cells. iCIL cells share characteristic cell surface markers and functional attributes as described herein. As used herein, the term "induced cytotoxic innate lymphoid cells" or "iCIL" refers to CILs made by inducing the differentiation of progenitor cells. As disclosed herein, iCILs can be made and / or expanded by expressing synthetic cytokine receptors in stem cells or progenitor cells and acting on the synthetic cytokine receptors with non-physiological ligands. Such a process can involve the differentiation of progenitor cells (e.g., lymphoid progenitor cells) engineered to express synthetic cytokine receptors by activating the synthetic cytokine receptors. The process may also, or alternatively, involve the expansion of progenitor cells or CILs through activation of a synthetic cytokine receptor.

[0038] In some embodiments, synthetic cytokine receptors, such as RACR, are provided herein that can be induced to support the induction or expansion of NK cells. NK cells derived from endogenous NK cells are herein referred to as primary NK cells. NK cells can also be derived from stem cells or progenitor cells, and such cells are herein referred to as iPSC-derived natural killer (iNK) cells. Primary NK cells and iNK cells share characteristic cell surface markers and functional attributes, but are not necessarily identical.

[0039] In a preferred embodiment, the present disclosure provides CIL cells, such as NK cells, engineered to express the rapamycin-activated cytokine receptor (RACR), a synthetic cytokine receptor that is activated by the small molecule rapamycin or a rapalog. CIL cells that contain RACR and are activated by rapamycin or a rapalog are referred to herein as "RACR-iCIL" cells, e.g., RACR-iNK cells. RACR has been demonstrated to support the differentiation and / or expansion of RACR-iCIL cells in a feeder-free manufacturing process. RACR-iCIL cells express multiple natural tumor targeting receptors and, when engineered to express a chimeric antigen receptor (CAR), can exert CAR-induced cytolytic activity. Thus, RACR-iCIL cells provide an "off-the-shelf" allogeneic cell therapy.

[0040] This disclosure relates in part to the surprising discovery by the inventors that lymphocytes, such as cytotoxic innate lymphoid (CIL) cells (e.g., NK cells), engineered to express a synthetic cytokine receptor can be expanded in response to the receptor's cognate non-physiological ligand. The engineered CIL cells can be produced in large quantities with functional activity equal to or greater than CIL cells from other sources.

[0041] The present disclosure relates in part to the surprising discovery by the present inventors that common lymphoid progenitor (CLP) cells engineered to express synthetic cytokine receptors can differentiate into CIL cells in response to the receptor's cognate non-physiological ligand without exogenous factors, and the resulting CIL cells exhibit cytotoxic phenotypes. It is well known in the relevant art that the expansion of CIL cells generally depends on having various exogenous stimuli. The engineered CIL cells described herein have the ability to be expanded without exogenous factors or with reduced exogenous factors, for example, without IL-2, IL-15 and / or IL-7.

[0042] The engineered CIL cells described herein, and related compositions, may be used for immunotherapy using ex vivo expansion.

[0043] As shown in the diagram of FIG. 1A, isolated lymphocytes, such as CIL cells (e.g., NK cells), including such primary cells isolated from peripheral blood, may be transduced with a vector comprising at least one polynucleotide encoding a synthetic cytokine receptor, such as RACR. When the transduced lymphocyte cells, e.g., CIL cells, are contacted with a non-physiological ligand, the extracellular domains of the cytokine receptors dimerize upon mutual binding of the non-physiological ligand. This dimerization generates an expanded proliferation signal within the cells, e.g., CIL cells, which produces a population of phenotypically enriched and functionally active engineered cells, e.g., engineered CIL cells.

[0044] As shown in the diagram in Figure 1B, stem or progenitor cells may be transduced with a vector containing at least one polynucleotide encoding a synthetic cytokine receptor, such as RACR. When the transduced cells are contacted with a non-physiological ligand, the extracellular domains of the cytokine receptor dimerize. Dimerization generates a differentiation signal within the stem or progenitor cells, which in turn induces differentiation into CIL cells, such as NK cells.

[0045] CIL cells can be derived from iPSCs, common lymphoid progenitors (CLPs), or other stem or progenitor cells.Further provided herein are stem or progenitor cells engineered to 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-physiological ligand for the cytokine receptor.

[0046] In certain embodiments, ex vivo generated lymphocytes are provided herein. In some embodiments, lymphocytes or progenitor cells (common lymphoid progenitor cells or pre-NK cells) are engineered with a provided synthetic cytokine receptor (e.g., RACR). In such aspects, non-physiological ligands of the synthetic cytokine receptors can be used to expand and / or differentiate lymphocytes or progenitor cells ex vivo to produce a population of ex vivo generated lymphocytes. Lymphocytes include innate lymphoid cells (ILCs), including T cells, cytotoxic innate lymphoid cells (CILs), such as natural killer (NK) cells. In some embodiments, the T cells are αβ T cells. In some embodiments, the T cells are γδ T cells.

[0047] In certain embodiments, ex vivo generated CIL cells are provided herein. In some embodiments, CIL cells or common lymphoid progenitor cells are engineered with synthetic cytokine receptors (e.g., RACR) provided. In such aspects, non-physiological ligands of synthetic cytokine receptors can be used to expand or differentiate cells to produce ex vivo generated CIL cells.

[0048] The engineered cells described herein and related compositions can be used for immunotherapy using ligand-controlled ex vivo expansion.Further provided herein is a method for expanding engineered lymphocytes, such as CIL cells, with synthetic cytokine receptors (e.g., RACR) by contacting cells with the cognate non-physiological ligand for synthetic cytokine receptors.Furthermore, the engineered lymphocytes, such as engineered CIL cells, disclosed herein can be further engineered to express chimeric antigen receptors (CARs), allowing engineered lymphocytes, such as engineered CIL cells, to target cells that express the antigen recognized by the CAR or are marked by the antigen recognized by the CAR.

[0049] In some embodiments, engineered lymphocytes, e.g., engineered CIL cells, and methods are provided for improved immunotherapy compared to existing strategies. Chimeric antigen receptor (CAR) T cell therapy has revolutionized the treatment of hematological malignancies, but significant limitations prevent their widespread application.

[0050] In some embodiments, engagement of a synthetic cytokine receptor (e.g., RACR) can increase cell proliferation and promote expansion through engagement of a synthetic cytokine receptor on the provided engineered lymphocytes, e.g., CIL cells. In some aspects, the provided engineered cells, including iCIL cells, and related methods can be used to increase cell proliferation and expansion in vivo of the engineered cell therapy by administering a non-physiological ligand of the synthetic cytokine receptor to the patient after the cell therapy product (e.g., administering rapamycin to the patient). In some embodiments, the non-physiological ligand (e.g., rapamycin) simultaneously expands and protects the cells. In some embodiments, expansion is achieved by JAK / STAT signal activation, and protection is achieved by rapamycin suppression of the host anti-graft response. In some embodiments, lymphodepletion and the need for exogenous cytokine administration are not required. In some embodiments, the provided methods of administration and treatment with engineered lymphocytes, e.g., engineered iCIL cells, can be performed without lymphodepletion (e.g., without the need to administer lymphodepleting therapy such as cyclophosphamide and / or fludarabine). In some embodiments, the provided methods of administration and treatment with engineered lymphocytes, e.g., engineered iCIL cells, can be performed without exogenous cytokine administration (e.g., without the need to administer IL-2 and / or IL-15).

[0051] Thus, the embodiments provided use synthetic cytokine receptor systems such as rapamycin or analogs, e.g., rapalogs, such as the rapamycin-activated cytokine receptor (RACR), to protect and expand cells in a single technique. In addition, the further inclusion of genetic disruptions, e.g., knockouts of specific immune genes such as beta-2-microglobulin (B2M), can produce "stealth" cells, which have additional advantages for allogeneic cell therapy.

[0052] All publications referenced in this application, including patent documents, scientific papers, and databases, are incorporated by reference in their entirety for all purposes as if each individual publication were individually incorporated by reference. To the extent that the definitions set forth herein conflict or are otherwise inconsistent with the definitions set forth in the patents, patent applications, published patent applications, and other publications incorporated herein by reference, the definitions set forth herein shall take precedence over the definitions incorporated herein by reference.

[0053] 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 several embodiments are possible within the scope and spirit of the present disclosure. The following description is illustrative of the present disclosure and, of course, should not be construed as limiting the scope of the invention described herein in any way.

[0054] I. Definition All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0055] Unless the context indicates 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 a combination.

[0056] As used herein, the singular forms "a," "an," and "the" include the plural forms unless the context indicates otherwise. The use of "and / or" refers to any and all possible combinations of one or more of the listed items.

[0057] As used herein, a "subject" refers to a recipient of engineered CIL cells or other agents. The term includes mammals, such as primates, mice, rats, dogs, cats, cows, horses, goats, camels, sheep or pigs, preferably humans.

[0058] As used herein, "treat," "treating," or "treatment" refers to any type of action or administration that benefits a subject with a disease or disorder, including amelioration of the patient's condition (i.e., improvement, reduction, or amelioration of one or more symptoms, and partial or complete response to treatment).

[0059] The term "effective amount" refers to an amount effective to bring about a desired biochemical, cellular, or physiological response. The term "therapeutically effective amount" refers to an amount, dosage, or administration regimen of a treatment effective to elicit a desired therapeutic effect.

[0060] As used herein, a "polynucleotide" refers to a biopolymer composed of two or more nucleotide monomers covalently linked via an ester bond between the phosphoryl group of one nucleotide and the hydroxyl group of the sugar moiety of the next nucleotide in the chain. DNA and RNA are non-limiting examples of polynucleotides.

[0061] As used herein, "polypeptide" refers to a polymer of amino acid residues linked together by peptide bonds that forms part (or the whole) of a protein.

[0062] It will be understood by those skilled in the art that many different polynucleotides and nucleic acids can encode the same polypeptide as a result of the degeneracy of the genetic code. It will further be understood that those skilled in the art may use routine techniques to make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides described herein to reflect the codon usage of any particular host organism in which the polypeptide is expressed.

[0063] Nucleic acids may include DNA or RNA. They may be single-stranded or double-stranded. They may also be polynucleotides that contain synthetic or modified nucleotides within them. Several different types of modifications to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, the addition of acridine or polylysine chains at the 3' and / or 5' ends of the molecule. It should be understood that for the uses described herein, polynucleotides may be modified by any method available in the art. Such modifications may be made to enhance the in vivo activity or life span of the polynucleotide of interest.

[0064] The term "variant" refers to a polynucleotide or polypeptide that has at least one substitution, insertion or deletion in its sequence compared to a reference polynucleotide or polypeptide. A "functional variant" is a variant that retains one or functions of the reference polynucleotide or polypeptide.

[0065] As used herein, the term "sequence identity" or "identity" in relation to a polynucleotide or polypeptide sequence refers to the degree to which two optimally aligned polynucleotide or polypeptide sequences match at each position in the alignment over the entire length of the reference sequence. "Percent identity" is the number of matched positions in the optimal alignment divided by the sum of the length of the reference sequence plus the length of any gaps in the reference sequence in the alignment. The optimal alignment is the alignment that results in the highest percent identity. Alignment of sequences to determine percent identity can be achieved by several well-known methods, including, for example, using mathematical algorithms such as those in the BLAST suite or Clustal Omega sequence analysis program. Unless otherwise stated, the term "sequence identity" in the claims refers to the sequence identity calculated by BLAST version 2.12.0 using default parameters. Also, unless otherwise stated, the alignment is the alignment of all or part of the polynucleotide or polypeptide sequence of interest over the entire length of the reference sequence.

[0066] As used herein, "small molecule" refers to an organic compound with a low molecular weight (<1000 Daltons). Small molecules can bind to specific biological macromolecules and can have a variety of biological functions or uses, including, but not limited to, cell signaling molecules, drugs, secondary metabolites, or a variety of other modes of action.

[0067] The term "analog" in reference to a small molecule refers to a compound that has a similar structure and / or function to another compound, but differs from it with respect to certain components. Analogs may differ in that one or more atoms, functional groups or substructures are replaced by other atoms, groups or substructures. Analogs may have different physical, chemical, physicochemical, biochemical or pharmacological properties despite high structural and / or functional similarity.

[0068] The term "rapalog" refers to an art-recognized group of analogues of rapamycin that share structural and functional similarities with rapamycin.Certain rapalogs are known to share some, but not all, of the functional attributes of rapamycin.For example, some rapalogs promote dimerization but have substantially no immunosuppressive activity, making them suitable for use as non-physiological ligands (e.g., AP21967, AP23102, or iRAP).

[0069] An exemplary rapalog of the present disclosure is AP21967 The file is TIFF2025500894000002.tif45128.

[0070] An exemplary rapalog of the present disclosure is AP23102. The file is TIFF2025500894000003.tif45128.

[0071] An exemplary rapalog of the present disclosure is iRAP The file is TIFF2025500894000004.tif45128.

[0072] The term "cell population" refers to a mixture of cells suspended in a solution, attached to a substrate, or stored in a container. The characteristics of the entire cell population can be examined using a bulk measurement of a sample volume having a plurality of cells. To reduce the background fluorescence problems encountered in bulk cell population measurements, flow cytometry methods can be used.

[0073] As used herein, the term "cytotoxic innate lymphoid cells" or "CIL cells" is used to refer to a class of cytotoxic lymphocytes that constitute a major component of the innate immune system. In humans, cytotoxic innate lymphoid cells usually express surface markers CD16 (FCyRIII), CD56, and may also express CD127. They may express one or more of CD45, CD94, CD122, KIR, NKG2A, NKG2D, NKp30, NKp44, NKP46, NKp80. Cytotoxic innate lymphoid cells generally do not express CD3 or express lower levels of CD3 than CD3+ T cells. CIL cells are cytotoxic and contain small granules in the cytoplasm that contain special proteins, such as perforin, and proteases known as granzymes. CIL cells provide a rapid response to virus-infected cells and respond to transformed cells. When perforin is released in the vicinity of the cell to be killed, it creates pores in the plasma membrane of the target cell, through which granzymes and related molecules can enter, inducing apoptosis. CIL cells can act as effectors of lymphoid cell populations 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 clarity, the term "CIL cells" excludes adaptive immune cells and their precursors, and excludes common lymphoid progenitors (CLPs). However, CILs can be derived from CLPs.

[0074] As used herein, the term "engineered" refers to cells that have been stably transduced with a heterologous polynucleotide or that have been subjected to gene editing to introduce, delete or modify a polynucleotide within the cell, or cells that have been transiently transduced with a polynucleotide to cause a stable phenotypic change in the cell.

[0075] As used herein, the term "stem cell" is used to refer to a cell with an undifferentiated phenotype that can differentiate into, for example, common lymphoid progenitors, cytotoxic innate lymphoid cells and / or NK cells.

[0076] As used herein, the term "pluripotent" means that stem cell can form substantially all of the differentiated cell types of an organism, at least during culture.For example, embryonic stem cell is a kind of pluripotent stem cell that can form cells from each of three germ layers, namely ectoderm, mesoderm and endoderm.

[0077] As used herein, the term "induced pluripotent stem cell" and "iPSC" are used to refer to the cell derived from somatic cell that is reprogrammed into pluripotent state and can proliferate, selectively differentiate and mature.iPSC is the stem cell that is produced from differentiated adult, neonatal or fetal cells that are induced or changed into cells that can differentiate into all three germ layers or layers, i.e., mesoderm, endoderm and ectoderm tissues, i.e., reprogrammed.Produced iPSC does not refer to the cell that is found in nature.

[0078] As used herein, the term "hematopoietic stem cell" refers to a stem cell that can give rise to both mature myeloid and lymphoid cell types, including natural killer cells, T cells, and B cells. Hematopoietic stem cells are typically characterized as CD34+.

[0079] The term "progenitor" refers to a cell that is partially differentiated into a desired cell type. Progenitor cells retain some degree of pluripotency and can differentiate into multiple cell types.

[0080] As used herein, the term "hematopoietic progenitor cell" refers to the intermediate cell type that can differentiate into blood lineage, and hematopoietic progenitor cell can differentiate into either common myeloid progenitor cell or common lymphoid progenitor cell.Hematopoietic progenitor cell is typically characterized by CD34+ and CD45+.CD38 is also considered as a marker for hematopoietic progenitor cell.CD45 is considered as a hematopoietic lineage marker.

[0081] As used herein, the term "lymphoid progenitor cell" or "lymphoblast" or "common lymphoid precursor" refers to a cell that is a precursor of lymphoid cells, such as CIL cells and NK cells. Lymphoid progenitor cells are the first stage of differentiation of hematopoietic stem cells that follow the lymphoid lineage of differentiation. As used herein, the term "lymphoid precursor" refers to a cell that is capable of hematopoietic transition to hematopoietic cell types. Lymphoid progenitor cells may be characterized as CD45+CD7+CD5+ / lo CD3-CD56-. Lymphoid progenitor cells may be characterized as CD45+CD5+ / lo CD7+.

[0082] As used herein, "differentiate" or "differentiated" is used to refer to the process and conditions in which an undifferentiated or immature (e.g., unspecialized) cell acquires the characteristics of becoming a mature (specialized) cell, thereby acquiring a specific morphology and function. Stem cells (unspecialized) are often exposed to various conditions (e.g., growth factors and morphogenetic factors) to induce a specific lineage commitment or differentiation of the stem cell.

[0083] As used herein, "expanding" or "expansion" refers to an increase in the number and / or purity of a cell type within a cell population by mitosis of cells with limited proliferative capacity, e.g., CIL cells.

[0084] As used herein, "activity," "activate," or "activation" refers to the stimulation of activating receptors on cytotoxic innate lymphoid cells, resulting in cell division, cytokine secretion (e.g., IFNγ and / or TNFα), and / or release of cytolytic granules to regulate or support the immune response.

[0085] II. Source Cells and Synthetic Cytokine Receptor (e.g., RACR) Engineered Cells In some aspects, provided herein are engineered cells that contain synthetic cytokine receptors for non-physiological ligands, such as synthetic small molecule ligands (e.g., rapamycin), which can activate the synthetic cytokine receptors to promote differentiation and / or expansion of the engineered cells. In some embodiments, the engineered cells are immune effector cells. In some embodiments, the engineered cells are lymphocytes. In some embodiments, the lymphocytes include innate lymphoid cells (ILCs), including cytotoxic innate lymphoid cells (CILs), such as T cells, natural killer (NK) cells, etc. In some embodiments, the T cells are αβ T cells. In some embodiments, the T cells are γδ T cells. In some embodiments, the engineered cells are precursor cells, such as common lymphoid precursor cells, that can differentiate into lymphocytes, e.g., CILs or NK cells. Also provided are resulting populations of cells induced to differentiate or expand from such engineered lymphocytes or precursor cells. In some embodiments, the cell population is iCILs.

[0086] In some embodiments, the synthetic cytokine receptor is comprised of a synthetic gamma chain polypeptide comprising a first dimerization domain, a first transmembrane domain, and an interleukin-2 receptor subunit gamma (IL-2RG) intracellular domain, and a synthetic alpha chain polypeptide or a synthetic beta chain polypeptide comprising a second dimerization domain, a second transmembrane domain, and an intracellular domain selected from an interleukin-2 receptor subunit beta (IL-2RB) intracellular domain, an interleukin-7 receptor subunit beta (IL-7RB) intracellular domain, an interleukin-21 receptor subunit beta (IL-21RB) intracellular domain, and / or an interleukin-9 receptor subunit alpha (IL-9RA) intracellular domain. In some embodiments, the synthetic cytokine receptor is a RACR synthetic cytokine receptor. Exemplary synthetic receptors are described in Section V. Methods of generating and / or expanding a population of engineered cells are also provided herein.

[0087] In some embodiments, the population of engineered cells is obtained by introducing a polynucleotide or vector containing a polynucleotide encoding a synthetic cytokine receptor into source cells. The source cells can be obtained from a primary source, including, but not limited to, peripheral blood mononuclear cells (PBMC) or umbilical cord blood (UCB). Sources of cells that can be engineered can also include cells differentiated from stem cells, such as pluripotent stem cells (iPSCs), which are cells derived from somatic cells (generally fibroblasts or peripheral blood mononuclear cells [PBMCs]), and human embryonic stem cells (hESCs). In some embodiments, the iPSCs are human iPSCs. In some embodiments, the stem cells can be differentiated into other cell types when subjected to appropriate differentiation conditions and then engineered with synthetic cytokine receptors. In some embodiments, the differentiation conditions include appropriate differentiation factors for differentiating the cells into lymphoid progenitor cells or lymphocytes, such as ILCs, including CILs.

[0088] In some embodiments, the engineered cells are ILCs. ILCs are a family of innate immune cells that have been discovered only recently, and originate from common lymphoid precursors (CLPs). ILCs can be cytotoxic innate lymphoid (CIL) cells or non-cytotoxic. Among CILs are natural killer (NK) cells. In some aspects, the characteristics that distinguish ILCs from other immune cells include their regular lymphoid morphology, the absence of rearranged antigen receptors found on T cells and B cells, and phenotypic markers that are usually present on myeloid or dendritic cells (Spits and Cupedo, Annual Review of Immunology. (2012) 20:647-75).

[0089] In some aspects, provided herein is a pharmaceutical composition comprising any of the engineered cells or cell populations (e.g., lymphocytes, e.g., ILCs, e.g., CILs) provided herein. In yet further aspects, provided herein is a method for expanding engineered cells, a method for treating a subject with engineered cells, and a method for killing or inhibiting cancer cells with engineered cells. In some aspects, provided herein is a kit comprising any of the engineered cells provided herein.

[0090] In some embodiments, the cells are engineered from peripheral blood cells. As used herein, the term "peripheral blood cells" refers to cells derived from circulating blood and including hematopoietic stem cells capable of proliferation, selective differentiation, and maturation. Thus, peripheral blood NK cells may alternatively be referred to as differentiated blood-derived NK cells (bdNK).

[0091] In some embodiments, lymphocytes, such as those used to generate engineered CIL cells, may be obtained from a donor or subject (for autologous therapy) by various means well known in the art. For example, lymphocytes may be obtained by collecting peripheral blood, including peripheral blood mononuclear cells (PBMCs), from a patient, subjecting the blood to Ficoll density gradient centrifugation and / or leukapheresis, and then isolating a population of lymphocytes from the peripheral blood using an isolation kit. In an exemplary embodiment, the population of lymphocytes need not be pure of the selected cell type, but may contain other cell types, such as T cells, monocytes, macrophages, natural killer cells, and B cells. In some embodiments, the collected cell population may comprise at least about 90% of the selected cell type, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% of the selected cell type.

[0092] In some embodiments, NK cells can be generated from NK cell lines. In some embodiments, NK cell lines can be engineered as described herein. In some embodiments, engineered NK cells include engineered NK cell lines. In some aspects, engineered cell lines allow for the production of larger amounts of cells without the need to expand a small number of NK cells from a subject. Engineered cell lines also have the advantage of being well characterized.

[0093] In some embodiments, the cell line is a clonal cell line.In some embodiments, the cell line is derived from a patient with NK cell leukemia or NK cell lymphoma.In some embodiments, the NK cell line is NK-92, NK-YS, KHYG-1, NKL, NKG, SNK-6 or IMC-1.

[0094] The NK-92 cell line is an NK-like cell line that was originally isolated from the blood of a subject suffering from large granular lymphoma and subsequently expanded in cell culture. The NK-92 cell line has been previously described (Gong et al., 1994; Klingemann, 2002). NK-92 cells have a CD3- / CD56+ phenotype characteristic of NK cells. They express all known NK cell activating receptors except CD16, but lack all known NK cell inhibitory receptors except NKG2A / CD94 and ILT2 / LIR1, which are expressed at low levels. Furthermore, NK-92 is a clonal cell line that expresses these receptors in a consistent manner, both in terms of type and cell surface concentration, unlike polyclonal NK cells isolated from blood. Similarly, NK-92 cells are not immunogenic and do not induce immune rejection when administered therapeutically to human subjects. Indeed, NK-92 cells are well tolerated in humans without any known deleterious effects on normal tissues.

[0095] Vectors for engineered cells into viral and non-viral vectors. For example, virus-free methods, adenovirus, plasmid, minicircle vector, episomal vector, Sendai virus, synthetic mRNA, self-replicating RNA, retrovirus, lentivirus, PhiC31 integrase, excisable transposon, episomal vector, CRISPR-based gene editing, or methods known in the art. In some embodiments, the source cells for engineering include hematopoietic stem cells (HSCs) characterized as CD34+ and / or CD45+; common lymphoid progenitor cells (CLPs) characterized as CD45+CD7+CD56-; CIL progenitor cells characterized as CD45+CD5-CD7+; and / or CIL cells characterized as CD45+CD56+CD3-, optionally CD5- and / or CD7+.

[0096] In some embodiments, source cells can be human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs) that can differentiate into lymphocytes or lymphoid progenitor cells, including any of the above.The lymphocytes or lymphoid progenitor cells can then be engineered with synthetic cytokine receptors (e.g., RACRs) to promote expansion and / or differentiation instead of the normal cytokine pathway from progenitor cells (common lymphoid progenitor cells or pre-NK cells).In immunotherapy, source cells are allogeneic or autologous, which means that they are derived from a donor or subject, respectively.

[0097] In some embodiments, CIL cells can be generated from induced pluripotent stem cells (iPSCs). CIL cells can be derived from iPSCs by sequentially differentiating iPSCs into hematopoietic progenitor cells (HPCs), also called hematopoietic stem cells (HSCs), HPCs into common lymphoid progenitors (CLPs), and then CLPs into CIL cells, called iPSC-derived cytotoxic innate lymphoid cells (iPSC-CILs, or iCILs).

[0098] iPSCs are a type of pluripotent stem cell derived from adult somatic cells (e.g., fibroblasts or PBMCs) that have been genetically reprogrammed into an embryonic stem cell-like state by forced expression of genes and factors important for maintaining the distinct properties of embryonic stem cells. iPSCs can be generated from tissues that have somatic cells, including, but not limited to, skin, dental tissue, peripheral blood and urine. To generate iPSCs, somatic cells can be reprogrammed by methods including, but not limited to, transient expression of reprogramming factors, virus-free methods, adenoviruses, plasmids, minicircle vectors, episomal vectors, Sendai virus, synthetic mRNA, self-replicating RNA, retroviruses, lentiviruses, PhiC31 integrase, excisable transposons, CRISPR-based gene editing, or recombinant proteins.

[0099] While the cell therapy industry has demonstrated the transformative potential of using genetically engineered patient-derived cells to treat specific disease indications, many challenges remain with the use of patient-derived material, including limited expansion and scalability, manufacturing complexity, high cost, patient-to-patient variability, and patient access. In contrast, iPSCs are pluripotent stem cells, a type of cell that is theoretically capable of differentiating into any other cell type, including cytotoxic innate lymphoid (CIL) cells applicable to the treatment of cancer. It is possible to use iPSCs to provide scalable and simplified manufacturing of targeted cell-fighting (e.g., cancer-fighting) cell therapies, such as CIL cells, thereby reducing costs and improving patient access to cell therapies. iPSCs have infinite expansion and proliferation capacity, meaning that they can replicate and grow indefinitely, potentially resulting in a nearly infinite supply of differentiated immune cells for therapies such as cancer treatment. iPSCs are also amenable to precise multiplex genome editing, allowing for the introduction of multiple genetic modifications to enhance their disease-targeting capabilities and the safety of the immune cells they ultimately become. iPSCs can similarly be engineered to protect them from allogeneic rejection by the patient's own immune system and improve their initial expansion and duration of engraftment. Furthermore, whereas either patient-derived or donor-derived blood material is variable, iPSCs provide a consistent starting material derived from a single cell clone, which may allow for genomic consistency and integrity in the final cell product.

[0100] Various differentiation protocols of CIL cells are known in the art. Examples of methods for differentiating stem cells (e.g., iPSCs) into pluripotent hematopoietic progenitor cells are provided in U.S. Patent No. 9,624,470, U.S. Patent Application No. 2020 / 0080059 and Mesquitta et al., Sci.Rep.9:6622 (2019), the entire disclosures of which are incorporated herein by reference. Examples of methods for differentiating cells into NK cells are provided in International Patent Application Nos. WO 2013 / 163171 and WO 2020 / 124256, U.S. Patent No. 9,260,696 and U.S. Patent No. 10,626,372, as well as Zhu and Kaufman, Methods Mol. Biol. 2048:107-119 (2019) and Matsubara et al., Biochem. Biophys. Res. Commun. 515(1):1-8 (2019), the entire disclosures of which are incorporated herein by reference.

[0101] Generally, techniques for differentiating cells involve modulating specific cellular pathways directly or indirectly using polynucleotide, polypeptide and / or small molecule-based approaches. The developmental potency of cells can be modulated, for example, by contacting the cells with one or more modulators. In some embodiments, cells are cultured in the presence of one or more agents (e.g., small molecules, proteins, peptides, etc.) to induce cell differentiation. In some embodiments, one or more differentiation agents are introduced into the cells during in vitro culture. The cells can be maintained in a culture medium containing one or more agents for a period of time sufficient for the cells to achieve the desired differentiation phenotype.

[0102] In some embodiments, stem cells are adapted to feeder-free culture.As used herein, "feeder-free" (FF) environment refers to the environment, such as culture conditions, cell culture or culture medium, that does not essentially contain feeder cells or stromal cells and / or is not preconditioned by culturing feeder cells."Preconditioned" medium refers to the medium that is harvested after feeder cells are cultured in medium for a certain period of time, for example, for at least one day.Preconditioned medium contains many mediator substances, including growth factors and cytokines secreted by feeder cells cultured in medium.

[0103] In some embodiments, the culture platform comprises one or more of nutrients, extracts, growth factors, hormones, cytokines, and media additives. Exemplary nutrients and extracts may include, for example, DMEM / F-12 (Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12), a basal medium widely used to support the growth of many different mammalian cells; KOSR (Knockout Serum Replacement); L-glut; NEAA (Non-Essential Amino Acids). Media additives may include, but are not limited to, MTG, ITS, (ME, antioxidants (e.g., ascorbic acid). In some embodiments, the culture media of the present invention may further comprise 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), neurotrophic factor (N ... Growth factors (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 through 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).

[0104] These cytokines may be commercially available and may be either natural or recombinant. In some other embodiments, the culture medium of the present disclosure comprises one or more of bone morphogenetic protein (BMP4), insulin-like growth factor-1 (IGF-1), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), hematopoietic growth factors (e.g., SCF, GMCSF, GCSF, EPO, IL3, TPO, EPO), Fms-related tyrosine kinase 3 ligand (FLT3L), and one or more cytokines from leukemia inhibitory factor (LIF), IL3, IL6, IL7, IL11, IL15. In some embodiments, the growth factors, mitogens and cytokines are stage and / or cell type specific concentrations determined empirically or guided by established cytokine technology. Examples of exogenous cell culture media additives and supplements and cell selection kit components are provided in WO 2020 / 124256, the entire disclosure of which is incorporated herein by reference.

[0105] In some other aspects, the culture medium of the present disclosure comprises Roswell Park Memorial Institute (RPMI) medium, cRPMI1640 medium, fetal bovine serum (FBS), Glutamax, penicillin, streptomycin, Rosuvastatin, BX795, protamine sulfate, brefeldin A, monensin, UM729, IL-2, IL-15, IL-21, IL-18, IL-7, or any combination thereof.

[0106] In some aspects, the method of producing CIL cells of the present disclosure comprises forming embryoid bodies (EBs) comprising stem cell aggregates; 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.

[0107] In some embodiments, the differentiation medium contains supplements such as serum, extracts, growth factors, hormones, cytokines, etc.

[0108] Differentiation of cells to generate CIL cells requires changes in the culture system, such as stimulants in the culture medium or changing the physical state of the cells. Conventional strategies utilize the formation of embryoid bodies as a common key intermediate to initiate lineage-specific differentiation. Embryoid bodies are aggregates of stem cells that are induced to differentiate by changes in environmental stimuli (e.g., exposure and / or removal of specific molecules / chemical factors; and / or exposure to / interaction with three-dimensional structures). The formation of embryoid bodies induces cells to differentiate into mesoderm specification.

[0109] The pluripotent stem cell aggregates are transferred to a differentiation medium that provides induction cues for a selected lineage (e.g., lymphoid lineage). In some embodiments, once the EBs are formed, they are dissociated and then cultured in medium to induce mesodermal specification of the cells.

[0110] In some aspects of the present invention, the induction of the above-mentioned embryoid bodies and differentiation into hematopoietic progenitor cell platforms can be performed under serum-free conditions. Examples of commercially available serum-free media suitable for cell attachment and / or induction include mTeSR™1 or TeSR™2 from Stem Cell Technologies (Vancouver, Canada), Primate ES / iPS cell medium from ReproCELL (Boston, Mass.), StemPro™-34 from Invitrogen (Carlsbad, Calif.), StemPro™ hESC SFM from Invitrogen, and X-VIVO™ from Lonza (Basel, Switzerland).

[0111] In some embodiments, the medium for the mesoderm-specific induction step comprises one or more of bone morphogenetic protein 4 (BMP4), vascular endothelial growth factor (VEGF), stem cell factor (SCF), thrombopoietin (TPO), Fms-related tyrosine kinase 3 ligand (FLT3L), basic fibroblast growth factor (bFGF, also known as FGF2). In some embodiments, the medium for the mesoderm-specific induction step comprises one or more of bone morphogenetic protein 4 (BMP4), vascular endothelial growth factor (VEGF), stem cell factor (SCF), thrombopoietin (TPO), Fms-related tyrosine kinase 3 ligand (FLT3L), basic fibroblast growth factor (bFGF, also known as FGF2) and / or ROCK inhibitor.

[0112] In some embodiments, the methods include differentiating the mesoderm specified cells into hematopoietic stem cells in a first differentiation medium.

[0113] In some embodiments, the hematopoietic stem cells in the first differentiation medium comprise one or more of bone morphogenetic protein 4 (BMP4), fibroblast growth factor 2 (FGF2), vascular endothelial growth factor (VEGF), stem cell factor (SCF), Fms-related tyrosine kinase 3 ligand (FLT3L), thrombopoietin (TPO), interleukin-6 (IL-6), interleukin-3 (IL-3), a TGF-β inhibitor, a PI3K inhibitor, or any combination thereof.

[0114] In some embodiments, the TGF-β inhibitor is GW788388. In some embodiments, the PI3K inhibitor is LY294002.

[0115] In some embodiments, the stem cells are differentiated into a mesodermal lineage in a differentiation medium comprising BMP4, FGF2 and VEGF.

[0116] In some embodiments, the mesodermal cells are differentiated into hematopoietic stem cells in a differentiation medium comprising BMP4, FGF2, VEGF and SCF.

[0117] In some embodiments, the hematopoietic stem cells are differentiated into lymphoid progenitor cells in a differentiation medium comprising BMP4, FGF2, VEGF and SCF.

[0118] In some embodiments, lymphoid progenitor cells are differentiated into cytotoxic innate lymphoid cells in a differentiation medium that includes non-physiological ligands, SCF, FLT3L and UM729.In some embodiments, the non-physiological ligand is a rapalog.In some embodiments, the non-physiological ligand is rapamycin.

[0119] In some embodiments, differentiating the mesoderm-specified cells into hematopoietic stem cells in the first differentiation medium is carried out for a period of time sufficient for the mesoderm-specified cells to become CD34+ hematopoietic stem cells.

[0120] In some embodiments, the method comprises differentiating the hematopoietic stem cells into lymphoid progenitor cells in a second differentiation medium.

[0121] In some embodiments, the lymphoid progenitor cell differentiation medium comprises one or more of bone morphogenetic protein 4 (BMP4), fibroblast growth factor 2 (FGF2), vascular endothelial growth factor (VEGF), stem cell factor (SCF), Fms-related tyrosine kinase 3 ligand (FLT3L), thrombopoietin (TPO), interleukin-6 (IL-6), interleukin-3 (IL-3), a TGF-β inhibitor, a PI3K inhibitor, or any combination thereof.

[0122] In some embodiments, differentiating the hematopoietic stem cells into lymphoid progenitor cells in the second differentiation medium is performed for a period of time sufficient for the hematopoietic stem cells to become Lin-CD34+CD38- / lo CD45RA+CD90- lymphoid progenitor cells.

[0123] 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.

[0124] 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).

[0125] 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 pyrimido-indole derivative, or any combination thereof. The pyrimido-indole derivative is UM 729 (a pyrimido-[4,5-b]-indole derivative).

[0126] In some embodiments, differentiating lymphoid progenitor cells into CIL cells in differentiation medium is performed for a period of time sufficient for lymphoid progenitor cells to become CD3-CD56+CD45+CD94+CD122+ / IL-2Rβ+CD127 / IL-7Rα-FcγRIII / CD16+KIR+NKG2A+NKG2D+NKp30+NKp44+NKP46+NKp80+. In some embodiments, differentiating lymphoid progenitor cells into CIL cells in differentiation medium is performed for a period of time sufficient for lymphoid progenitor cells to become CD45+CD56+. In some embodiments, differentiating lymphoid progenitor cells into CIL cells in differentiation medium is performed for a period of time sufficient for lymphoid progenitor cells to become CD45+CD5-CD7+CD56+.

[0127] In some embodiments, differentiation of lymphoid progenitor cells to CIL cells in differentiation medium is performed for about 8 to about 18 days. In some embodiments, differentiation of lymphoid progenitor cells to CIL cells in differentiation medium is performed for about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 days.

[0128] 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.

[0129] In some embodiments, after differentiation, the CIL cells are expanded in medium comprising a non-physiological ligand and a CD2 / NKp46 stimulus. In some embodiments, the CIL cell expansion medium comprises activation beads comprising conjugated anti-CD2 and anti-NKp46 antibodies that stimulate and activate the CIL cells.

[0130] 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 initiating feeder cells.

[0131] Induced cytotoxic innate lymphoid cells (iCIL) In some embodiments, CIL cells may be derived from iPSCs by sequentially differentiating iPSCs into hematopoietic progenitor cells (HPCs), HPCs into common lymphoid progenitors (CLPs), and then the CLPs into CIL cells, referred to as "iCIL" cells. In a variation, CIL cells may be derived from HPCs by sequentially differentiating HPCs into CLPs, and then the CLPs into iCIL cells. In a further variation, CIL cells may be derived by differentiating CLPs into iCIL cells. Engineering cells to express synthetic cytokine receptors may be performed at the HPC, CLP, or iCIL cell stage of the differentiation process.

[0132] In some embodiments, the iCIL cells are characterized as being 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 being CD3-CD56+CD45+ cells. In some embodiments, the iCIL cells are characterized as being 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 any combination thereof. iCIL cells can be CD45+; CD45+CD5-; or CD45+CD5-CD56+.

[0133] In some embodiments, the iCIL cells are CD45+CD7+CD56 + / lo It is characterized in that:

[0134] In some embodiments, after transduction of the iCIL cells with a CAR, the cells are cultured under conditions that promote activation of the cells.

[0135] Culture conditions may be such that the cells can be administered to a patient without consideration of reactivity to components of the culture medium. For example, culture conditions may omit bovine serum products such as bovine serum albumin. In an exemplary aspect, activation may be achieved by introducing a known activator into the culture medium. In one aspect, a population of iCIL cells may be cultured for about 1 to about 4 days under conditions that promote activation. In one embodiment, the appropriate activation level may be determined by cell size, proliferation rate, or activation markers determined by flow cytometry. In some embodiments, any of the culture methods disclosed herein may be used to promote activation of iCIL cells.

[0136] In some embodiments, provided herein are methods for generating and / or expanding a population of engineered cells that contain a synthetic cytokine receptor for a non-physiological ligand. In some embodiments, the methods include: a. optionally providing an engineered cell containing a synthetic cytokine receptor by introducing a polynucleotide encoding the synthetic cytokine receptor into a source cell; and b. Incubating the engineered cells in a medium containing a non-physiological ligand. Includes.

[0137] In this embodiment, the cytokine receptor comprises a synthetic gamma chain polypeptide comprising a first dimerization domain, a first transmembrane domain, and an interleukin-2 receptor subunit gamma (IL-2RG) intracellular domain, and a synthetic alpha chain polypeptide or a synthetic beta chain polypeptide comprising a second dimerization domain, a second transmembrane domain, and an intracellular domain selected from an interleukin-2 receptor subunit beta (IL-2RB) intracellular domain, an interleukin-7 receptor subunit beta (IL-7RB) intracellular domain, an interleukin-21 receptor subunit beta (IL-21RB) intracellular domain, and / or an interleukin-9 receptor subunit alpha (IL-9RB), and the non-physiological ligand activates the synthetic cytokine receptor within the engineered cell to induce expansion and / or activation of the engineered cell.

[0138] In this embodiment, the cytokine receptor comprises a synthetic gamma chain polypeptide comprising a first dimerization domain, a first transmembrane domain, and an interleukin-2 receptor subunit gamma (IL-2RG) intracellular domain, and a synthetic beta chain polypeptide comprising a second dimerization domain, a second transmembrane domain, and an intracellular domain selected from an interleukin-2 receptor subunit beta (IL-2RB) intracellular domain, an interleukin-7 receptor subunit beta (IL-7RB) intracellular domain, and / or an interleukin-21 receptor subunit beta (IL-21RB) intracellular domain, and the non-physiological ligand activates the synthetic cytokine receptor within the engineered cell to induce expansion and / or activation of the engineered cell.

[0139] In some embodiments, the iPSCs are genetically edited using lentivirus.

[0140] In some embodiments, HSCs are genetically edited using lentivirus. In some embodiments, blood progenitor cells are genetically edited using lentivirus. In some embodiments, common lymphoid progenitor cells are genetically edited using lentivirus. In some embodiments, NK progenitor cells are genetically edited using lentivirus. In some embodiments, common lymphoid progenitor (CLP) cells are genetically edited using lentivirus.

[0141] III. Retroviral particles Retroviruses include lentiviruses, gamma-retroviruses, and alpha-retroviruses, each of which can be used to deliver polynucleotides to cells using methods known in the art. Lentiviruses are complex retroviruses that contain other genes with regulatory or structural functions in addition to the common retroviral genes gag, pol, and env. The increased complexity allows the virus to regulate its life cycle, such as during the process of latent infection. Some examples of lentiviruses include human immunodeficiency viruses (HIV-1 and HIV-2) and simian immunodeficiency viruses (SIV). Retroviral vectors have been generated by multiple attenuation of HIV pathogenic genes, for example, genes env, vif, vpr, vpu, and nef are deleted to make the vector biologically safe.

[0142] Exemplary lentiviral vectors include those described in Naldini et al. (1996) Science 272:263-7; Zufferey et al. (1998) J. Virol. 72:9873-9880; Dull et al. (1998) J. Virol. 72:8463-8471; U.S. Patent No. 6,013,516; and U.S. Patent No. 5,994,136, each of which is incorporated herein by reference in its entirety. Generally, these vectors are constructed to have the necessary sequences for selection of cells containing the vector, incorporation of the foreign nucleic acid into the lentiviral particle, and transfer of the nucleic acid into target cells.

[0143] The commonly used lentiviral vector system is the so-called third generation system. The third generation lentiviral vector system includes four plasmids. The "transfer plasmid" encodes the polynucleotide sequence that is delivered to the target cell by the lentiviral vector system. The transfer plasmid generally has one or more transgene sequences of interest flanked by long terminal repeat (LTR) sequences, which facilitates the integration of the transfer plasmid sequence into the host genome. For safety reasons, the transfer plasmid is generally designed to disable the resulting vector replication. For example, the transfer plasmid lacks the genetic elements required for the production of infectious particles in the host cell. In addition, the transfer plasmid can be designed with a deletion of the 3'LTR, which causes the virus to "self-inactivate" (SIN). See Dull et al. (1998) J.Virol. 72:8463-71; Miyoshi et al. (1998) J.Virol. 72:8150-57. The viral particle may also contain a 3' untranslated region (UTR) and a 5' UTR. The UTR contains retroviral regulatory elements that support packaging, reverse transcription and integration of the proviral genome into a cell following contact of the cell with the retroviral particle.

[0144] Third generation systems also typically include two "packaging plasmids" and an "envelope plasmid." The "envelope plasmid" typically encodes an Env gene operably linked to a promoter. In an exemplary third generation system, the Env gene is VSV-G and the promoter is a CMV promoter. Third generation systems use two packaging plasmids, one encoding gag and pol, and the other encoding an additional safety feature, namely rev, as an improvement over the single packaging plasmid of the so-called second generation system. Third generation systems, although safer, are more cumbersome to use and may result in lower viral titers due to the addition of the additional plasmid. Exemplary packaging plasmids include, but are not limited to, pMD2.G, pRSV-rev, pMDLG-pRRE, and pRRL-GOI.

[0145] Many retroviral vector systems rely on the use of "packaging cell lines". In general, packaging cell lines are cell lines that can produce infectious retroviral particles when the transfer plasmid, packaging plasmid and envelope plasmid are introduced into the cell. Various methods of introducing plasmids into cells can be used, including transfection or electroporation. In some cases, packaging cell lines are adapted for high-efficiency packaging of retroviral vector systems into retroviral particles.

[0146] As used herein, the term "retroviral vector" or "lentiviral vector" is intended to mean a nucleic acid that encodes the retroviral or lentiviral cis nucleic acid sequence required for genome packaging and one or more polynucleotide sequences that are delivered to target cells.Retroviral and lentiviral particles generally comprise an RNA genome (derived from a transfer plasmid), a lipid bilayer envelope in which Env protein is embedded, and other accessory proteins, including integrase, protease, and matrix protein.As used herein, the term "retroviral particle" and "lentiviral particle" refer to a viral particle that comprises an envelope, has one or more characteristics of lentivirus, and can enter target host cells. Such characteristics include, for example, infecting non-dividing host cells, transducing non-dividing host cells, infecting or transducing host immune cells, containing a retroviral or lentiviral virion that includes one or more of the gag structural polypeptides, containing a retroviral or lentiviral envelope that includes one or more of the env-encoded glycoproteins, containing a genome that includes one or more retroviral or lentiviral cis-acting sequences that function in replication, proviral integration, or transcription, containing a genome that encodes a retroviral or lentiviral protease, reverse transcriptase, or integrase, or containing a genome that encodes a regulatory activity such as Tat or Rev. The transfer plasmid may include a cPPT sequence as described in U.S. Pat. No. 8,093,042.

[0147] The efficiency of the system is an important concern in vector engineering. The efficiency of retroviral or lentiviral vector systems can be evaluated by various methods known in the art, including measuring vector copy number (VCN) or vector genome (vg), such as by quantitative polymerase chain reaction (qPCR), or viral titer in infectious units per milliliter (IU / mL). For example, titer can be evaluated using a functional assay performed on the cultured tumor cell line HT1080, as described in Humbert et al. Development of third-generation Cocal Envelope Producer Cell Lines for Robust Retroviral Gene Transfer into Hematopoietic Stem Cells and T-cells. Molecular Therapy 24:1237-1246 (2016). When evaluating titer on a continuously dividing cultured cell line, no stimulation is required, so the measured titer is not affected by surface engineering of retroviral particles. Other methods for assessing the efficiency of retroviral vector systems are provided in Gaererts et al. Comparison of retroviral vector titration methods. BMC Biotechnol. 6:34 (2006).

[0148] In some embodiments, the retroviral and / or lentiviral particles of the present disclosure comprise a polynucleotide comprising a sequence encoding a receptor that specifically binds to a gating adaptor. In some embodiments, the sequence encoding the receptor that specifically binds to the gating adaptor is operably linked to a promoter. Exemplary promoters include, but are not limited to, a cytomegalovirus (CMV) promoter, a CAG promoter, an SV40 promoter, an SV40 / CD43 promoter, and an MND promoter.

[0149] In some aspects, the retroviral particle comprises a transduction enhancer, hi some aspects, the retroviral particle comprises a tag protein.

[0150] In some embodiments, each retroviral particle comprises a polynucleotide comprising, in 5' to 3' order, (i) a 5' long terminal repeat (LTR) or untranslated region (UTR), (ii) a promoter, (iii) a sequence encoding a receptor that specifically binds a ligand, and (iv) a 3' LTR or 3' UTR.

[0151] In some embodiments, the retroviral particle contains a cell surface receptor that binds to a surface marker on a target host cell and allows host cell transduction. The viral vector can contain a heterologous viral envelope glycoprotein that results in a pseudotyped viral vector. For example, the viral envelope glycoprotein can be derived from RD114 or one of its mutants, VSV-G, Gibbon-ape leukaemia virus (GALV), or is an amphotropic envelope, measles envelope, or baboon retrovirus envelope glycoprotein. In some embodiments, the cell surface receptor is the VSV G protein from Cocal strain or a functional mutant thereof.

[0152] Various fusion glycoproteins can be used to pseudotype lentiviral vectors. The most commonly used example is the envelope glycoprotein from vesicular stomatitis virus (VSVG), but many other viral proteins have also been used to pseudotype lentiviral vectors. See Joglekar et al. Human Gene Therapy Methods 28:291-301 (2017). The present disclosure contemplates the substitution of various fusion glycoproteins. In particular, some fusion glycoproteins increase vector efficiency.

[0153] In some embodiments, pseudotyping of the fusion glycoprotein or functional variant thereof facilitates targeted transduction of specific cell types, including, but not limited to, innate lymphoid cells, cytotoxic innate lymphoid cells, or NK cells. In some embodiments, the fusion glycoprotein or functional variant thereof is selected from the group consisting of human immunodeficiency virus (HIV) gp160, murine leukemia virus (MLV) gp70, gibbon ape leukemia virus (GALV) gp70, feline leukemia virus (RD114) gp70, amphotropic retrovirus (Ampho) gp70, 10A1 MLV (10A1) gp70, narrow-skinned retrovirus (Eco) gp70, baboon ape leukemia virus (Baboon ... virus (BaEV) gp70, measles virus (MV) H and F, Nipah virus (NiV) H and F, rabies virus (RabV) G, Mokola virus (MOKV) G, Ebola Zaire virus (EboZ) G, lymphocytic choriomeningitis virus (LCMV) GP1 and GP2, baculovirus GP64, chikungunya virus (CHIKV) E1 and E2, Ross River virus (RRV) E1 and E2, Semliki Forest virus (SFV) E1 and E2, Sindbis virus (SV) E1 and E2, Venezuelan equine encephalitis virus (VEEV) E1 and E2, Western equine encephalitis virus (WEEV) E1 and E2, influenza A, B, C, or D The polypeptide may be a full-length polypeptide, a functional fragment, a homologue or a functional variant of HA, Avian plague virus (FPV) HA, Vesicular stomatitis virus VSV-G, or Chandipura virus and Pili virus CNV-G and PRV-G.

[0154] In some embodiments, the fusion glycoprotein or functional variant thereof is a full-length polypeptide, functional fragment, homolog, or functional variant of the G protein of Vesicular Stomatitis Alagoas Virus (VSAV), Carajas Vesiculovirus (CJSV), Chandipura Vesiculovirus (CHPV), Cocal Vesiculovirus (COCV), Vesicular Stomatitis Indiana Virus (VSIV), Isfahan Vesiculovirus (ISFV), Maraba Vesiculovirus (MARAV), Vesicular Stomatitis New Jersey Virus (VSNJV), Bas-Congo Virus (BASV). In some embodiments, the fusion glycoprotein or functional variant thereof is a coccoccal virus G protein.

[0155] In some embodiments, the fusion glycoprotein or functional variant thereof is a full-length polypeptide, functional fragment, homolog, or functional variant of the G protein of Vesicular Stomatitis Alagoas Virus (VSAV), Carajas Vesiculovirus (CJSV), Chandipura Vesiculovirus (CHPV), Cocal Vesiculovirus (COCV), Vesicular Stomatitis Indiana Virus (VSIV), Isfahan Vesiculovirus (ISFV), Maraba Vesiculovirus (MARAV), Vesicular Stomatitis New Jersey Virus (VSNJV), Bas-Congo Virus (BASV). In some embodiments, the fusion glycoprotein or functional variant thereof is a coccoccal virus G protein.

[0156] The present disclosure further provides various retroviral vectors, including, but not limited to, gamma-retroviral vectors, alpha-retroviral vectors, and lentiviral vectors. In some embodiments, the vector can be a viral vector, a retroviral vector, a lentiviral vector, or a gamma-retroviral vector. In some embodiments, the viral vector comprises a VSV G protein or a functional variant thereof. In some embodiments, the viral vector comprises a coccus G protein or a functional variant thereof.

[0157] IV. Nucleic Acid Vectors As used herein, the term "nucleic acid vector" is intended to mean any nucleic acid that carries, carries, or functions to express a nucleic acid of interest. Nucleic acid vectors can have specialized functions, such as, for example, expression, packaging, pseudotyping, transduction, or sequencing. Nucleic acid vectors can also have operational functions, such as, for example, cloning vectors or shuttle vectors. The structure of a vector can include any desired form that is feasible and desirable for a particular use. Such forms include, for example, circular forms, such as plasmids and phagemids, as well as linear or branched forms. Nucleic acid vectors can be composed of, for example, DNA or RNA, and can contain partially or completely nucleotide derivatives, analogs, and mimetics. Such nucleic acid vectors can be obtained from natural sources, can be recombinantly produced, or can be chemically synthesized.

[0158] Non-limiting examples of vector systems of the present disclosure include retroviruses, lentiviruses, foamy viruses and Sleeping Beauty transposons.

[0159] V. Synthetic Cytokine Receptor Complexes and Synthetic Cytokine Receptor Systems In some embodiments, synthetic cytokine receptors are provided herein for manipulating cells, including cells that can be used for immunotherapy.In some embodiments, synthetic cytokine receptors are composed of two polypeptide chains that can dimerize to initiate signal transduction in response to non-physiological ligands of synthetic cytokine receptors.In certain embodiments, synthetic cytokine receptors are rapamycin-activated cytokine receptors (RACR).

[0160] Any synthetic receptor system that provides a source of cytokine or cytokine signal (e.g., JAK / STAT) without the need to add exogenous cytokines can be used. In some embodiments, the synthetic cytokine receptor or synthetic cytokine system induces common cytokine receptor gamma chain signaling. The common cytokine receptor gamma chain is the signaling chain for the receptor complex of the cytokines IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21. Illustrative examples of cytokines that share a common gamma cytokine chain include, but are not limited to, IL-2, IL-7, IL-15, IL-21 and IL-9. A variety of synthetic cytokine receptor systems are known and can be used, including orthogonal receptors to the common gamma chain receptor (e.g., Kalbasi et al. Nature, 2022 607:360; Zhang et al. Sci. Transl. Med., 2021 13(625):eabg6986), drug-inducible systems of engineered cytokine fusions with drug-responsive domains (DRDs) for controlled or inducible expression of cytokines, e.g., as described in published U.S. Patent No. 20200172879; and synthetic receptors with dimerization domains that are responsive to chemical inducers of dimerization (CIDs).

[0161] In some embodiments, the synthetic cytokine receptor contains the dimerization domain of CID ligand.In some embodiments, the synthetic cytokine receptor is a heterodimer of common cytokine receptor gamma signaling chain and either alpha cytokine receptor signaling chain or beta cytokine receptor signaling chain, each fused to dimerization domain.In some embodiments, the dimerization domain can be derived from FKBP, cyclophilin receptor, steroid receptor, tetracycline receptor, estrogen receptor, glucocorticoid receptor, vitamin D receptor, calcineurin A, CyP-Fas, FRB domain of mTOR, GyrB, GAI, GID1, Snap-tag and / or HaloTag, or their parts or derivatives. Examples of CIDs and corresponding dimerization domains are known in the art (see, e.g., U.S. Patent Application Publication No. 2016 / 0046700; Clackson et al. (1998) Proc Natl Acad Sci US A. 95(18):10437-42; Spencer et al. (1993) Science 262(5136):1019-24; Farrar et al. (1996) Nature 383(6596):178-81; Miyamoto et al. (2012) Nature Chemical Biology 8(5):465-70; Erhart et al. (2013) Chemistry and Biology 20(4):549-57). In some embodiments, the CID is estrogen, glucocorticoid, vitamin D, steroid, tetracycline, cyclosporine, rapamycin, coumermycin, gibberellin, FK1012, FK506, FKCsA, rimiducid or HaXS, or an analog or derivative thereof. The dimerization domain can be extracellular (N-terminal to the transmembrane domain) or intracellular. In some embodiments, the CID is a non-physiological ligand or a synthetic ligand. In some embodiments, the CID is rapamycin or an analog thereof. Exemplary synthetic receptor dimerization systems are described below.

[0162] The non-physiological ligand may activate a synthetic cytokine receptor in an engineered lymphocyte, such as a cytotoxic innate lymphoid cell, to induce the expansion and / or activation of the engineered cell, such as a cytotoxic innate lymphoid cell. In a preferred embodiment, the non-physiological ligand is rapamycin or a rapalog, such a synthetic cytokine receptor referred to as the rapamycin-activated cytokine receptor (RACR).

[0163] In some embodiments, the non-physiological ligand activates a synthetic cytokine receptor in the engineered lymphocyte (e.g., CIL cells) to induce expansion of the engineered lymphocyte, e.g., CIL cells. In some embodiments, activation of the synthetic cytokine receptor results in at least about 10-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 1000-fold, at least about 1500-fold, at least about 2000-fold, at least about 2500-fold, at least about 3000-fold, at least about 3500-fold, or at least about 4000-fold greater numbers of lymphocytes, e.g., CIL cells, compared to non-induced cells.

[0164] In some embodiments, lymphocytes, e.g., CIL cells, are increased by about 10-fold to about 100-fold, about 50-fold to about 200-fold, about 100-fold to about 300-fold, about 200-fold to about 400-fold, about 300-fold to about 500-fold, about 400-fold to about 1000-fold, about 500-fold to about 1500-fold, about 1000-fold to about 2000-fold, about 1500-fold to about 2500-fold, about 2000-fold to about 3000-fold, about 2500-fold to about 3500-fold, about 3000-fold to about 4000-fold, or any value between these ranges.

[0165] A. Exemplary Synthetic Cytokine Receptors (e.g., RACR) In some aspects, a synthetic cytokine receptor of the disclosure comprises a first transmembrane receptor protein that is a first synthetic cytokine chain (e.g., a synthetic gamma chain containing a common receptor gamma signaling chain) and a second transmembrane receptor protein that is a synthetic cytokine chain (e.g., a synthetic alpha chain or a synthetic beta chain containing an intracellular signaling chain of a cytokine receptor alpha chain or beta chain, respectively), each comprising a dimerization domain. The dimerization domain can controllably dimerize in the presence of a non-physiological ligand, thereby activating signaling of the synthetic cytokine receptor.

[0166] In some embodiments, the first cytokine chain is a synthetic gamma chain polypeptide comprising a first dimerization domain, a first transmembrane domain, and an interleukin-2 receptor subunit gamma (IL-2RG) intracellular domain. In some embodiments, the second cytokine chain comprises a second dimerization domain, a second transmembrane domain, and an intracellular domain of a cytokine receptor alpha or beta chain. In some embodiments, the second cytokine chain is a synthetic alpha chain containing an intracellular domain of an interleukin-9 receptor subunit alpha (IL-9RA) intracellular domain. In some embodiments, the second cytokine chain is a synthetic beta chain containing an intracellular domain selected from an interleukin-2 receptor subunit beta (IL-2RB) intracellular domain, an interleukin-7 receptor subunit beta (IL-7RB) intracellular domain, or an interleukin-21 receptor subunit beta (IL-21RB) intracellular domain. The dimerization domain can be extracellular (N-terminal to the transmembrane domain) or intracellular (C-terminal to the transmembrane domain, and N-terminal or C-terminal to the intracellular domain.

[0167] In some embodiments, the synthetic cytokine receptor of the present disclosure comprises a synthetic gamma chain and a synthetic alpha chain, each of which comprises a dimerization domain. The dimerization domain can controllably dimerize in the presence of a non-physiological ligand, thereby activating the signal transduction of the synthetic cytokine receptor. In some embodiments, the synthetic gamma chain polypeptide comprises a first dimerization domain, a first transmembrane domain, and an interleukin-2 receptor subunit gamma (IL-2RG) intracellular domain. The dimerization domain can be extracellular (N-terminal to the transmembrane domain) or intracellular (C-terminal to the transmembrane domain, and N-terminal or C-terminal to the IL-2G intracellular domain. The synthetic alpha chain polypeptide comprises a second dimerization domain, a second transmembrane domain, and an intracellular domain selected from an interleukin-9 receptor subunit alpha (IL-2RA) intracellular domain. The dimerization domain can be extracellular (N-terminal to the transmembrane domain) or intracellular (C-terminal to the transmembrane domain, and N-terminal or C-terminal to the IL-9RA intracellular domain).

[0168] In some embodiments, the synthetic cytokine receptor of the present disclosure comprises a synthetic gamma chain and a synthetic beta chain, each of which comprises a dimerization domain. The dimerization domain can controllably dimerize in the presence of a non-physiological ligand, thereby activating the signal transduction of the synthetic cytokine receptor. In some embodiments, the synthetic gamma chain polypeptide comprises a first dimerization domain, a first transmembrane domain, and an interleukin-2 receptor subunit gamma (IL-2RG) intracellular domain. The dimerization domain can be extracellular (N-terminal to the transmembrane domain) or intracellular (C-terminal to the transmembrane domain, and N-terminal or C-terminal to the IL-2G intracellular domain. The synthetic beta chain polypeptide comprises a second dimerization domain, a second transmembrane domain, and an intracellular domain selected from an interleukin-2 receptor subunit beta (IL-2RB) intracellular domain, an interleukin-7 receptor subunit beta (IL-7RB) intracellular domain, or an interleukin-21 receptor subunit beta (IL-21RB) intracellular domain. The dimerization domain can be extracellular (N-terminal to the transmembrane domain) or intracellular (C-terminal to the transmembrane domain, and N-terminal or C-terminal to the IL-2RB intracellular domain, the IL-7RB intracellular domain, or the IL-21RB intracellular domain).

[0169] In some aspects, the synthetic gamma chain polypeptide is encoded by a nucleic acid sequence encoding a signal peptide. In some aspects, the synthetic alpha chain polypeptide is encoded by a nucleic acid sequence encoding a signal peptide. In some aspects, the synthetic beta chain polypeptide is encoded by a nucleic acid sequence encoding a signal peptide. Those skilled in the art are readily familiar with signal peptides that can provide a signal for transporting a nascent protein within a cell. Any of a variety of signal peptides can be used.

[0170] In some embodiments, the signal peptide is The CD8a signal sequence is shown as TIFF2025500894000005.tif4128.

[0171] In some embodiments, the signal peptide is The signal sequence is shown as TIFF2025500894000006.tif4128.

[0172] 1. Intracellular domain 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 to SEQ ID NO:1.

[0173] The sequence of the IL2RG common gamma chain intracellular domain is SEQ ID NO:1: Write it in TIFF2025500894000007.tif12144.

[0174] In some embodiments, the synthetic cytokine receptor comprises a first transmembrane receptor protein comprising an IL-2RG intracellular domain and a first dimerization domain, and a second transmembrane receptor protein comprising an IL-2RB intracellular domain and a second dimerization domain.

[0175] In some embodiments, the synthetic beta chain comprises an interleukin-2 receptor subunit beta (IL2RB) intracellular domain. IL2RB is also known as IL15RB or CD122. Thus, when referred to herein, IL2RB may also mean IL15RB. That is, the terms are used interchangeably in this disclosure. In some embodiments, the IL2RB intracellular domain comprises the sequence set forth in SEQ ID NO:2. In some embodiments, the IL2RB intracellular domain has at least 80% amino acid identity, at least 85% amino acid identity, at least 90% amino acid identity, at least 95% amino acid identity, or 100% amino acid identity to SEQ ID NO:2.

[0176] The sequence of the IL2RB intracellular domain is SEQ ID NO:2: Write it in TIFF2025500894000008.tif41147.

[0177] In some embodiments, the synthetic cytokine receptor comprises a first transmembrane receptor protein comprising an IL-2RG intracellular domain and a first dimerization domain, and a second transmembrane receptor protein comprising an IL-7RB intracellular domain and a second dimerization domain.

[0178] In some embodiments, the synthetic beta chain comprises an interleukin-7 receptor subunit beta (IL7RB) intracellular domain. In some embodiments, the IL7RB intracellular domain comprises the sequence set forth in SEQ ID NO:3. In some embodiments, the IL7RB intracellular domain has at least 80% amino acid identity, at least 85% amino acid identity, at least 90% amino acid identity, at least 95% amino acid identity, or 100% amino acid identity to SEQ ID NO:3.

[0179] The sequence of the IL7RB intracellular domain is SEQ ID NO:3: Write it in TIFF2025500894000009.tif26147.

[0180] In some embodiments, the synthetic cytokine receptor comprises a first transmembrane receptor protein comprising an IL-2RG intracellular domain and a first dimerization domain, and a second transmembrane receptor protein comprising an IL-21RB intracellular domain and a second dimerization domain.

[0181] In some embodiments, the synthetic beta chain comprises an 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 to SEQ ID NO:4.

[0182] The sequence of the IL21RB intracellular domain is SEQ ID NO:4: Write it in TIFF2025500894000010.tif41147.

[0183] In some embodiments, the synthetic cytokine receptor comprises a first transmembrane receptor protein comprising an IL-2RG intracellular domain and a first dimerization domain, and a second transmembrane receptor protein comprising an IL-9RA intracellular domain and a second dimerization domain.

[0184] In some embodiments, the synthetic alpha chain comprises an interleukin-9 receptor subunit alpha (IL9RA) intracellular domain. In some embodiments, the IL9RA intracellular domain comprises a sequence set forth in SEQ ID NO:51. In some embodiments, the IL9RA intracellular domain has at least 80% amino acid identity, at least 85% amino acid identity, at least 90% amino acid identity, at least 95% amino acid identity, or 100% amino acid identity to SEQ ID NO:51.

[0185] The sequence of the IL9RA intracellular domain is SEQ ID NO:51: Write it in TIFF2025500894000011.tif33147.

[0186] 2. Dimerization Domain The dimerization domain may be a heterodimerization domain including, but not limited to, FK506-binding protein (FKBP), 12 kD in size, which is known in the art to dimerize in the presence of rapamycin or a rapalog, and FKBP12-rapamycin binding (FRB) domain. In some embodiments, the FRB domain may comprise a polypeptide sequence 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 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 may comprise a polypeptide sequence 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: 48. In some embodiments, the FKBP domain may comprise a polypeptide sequence 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. In some embodiments, the FKBP domain may comprise a polypeptide sequence 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.

[0187] In some embodiments, the sequence of an exemplary FKBP domain is SEQ ID NO:5: Write it in TIFF2025500894000012.tif18144.

[0188] In some embodiments, the sequence of an exemplary FKBP domain is SEQ ID NO:48: Write it in TIFF2025500894000013.tif18144.

[0189] In some embodiments, the sequence of an exemplary FKBP domain is SEQ ID NO:30: Write it in TIFF2025500894000014.tif18144.

[0190] In some embodiments, the sequence of an exemplary FKBP domain is SEQ ID NO:49: Write it in TIFF2025500894000015.tif11140.

[0191] In some embodiments, the sequence of an exemplary FRB domain is SEQ ID NO:6: Write it in TIFF2025500894000016.tif12146.

[0192] In some embodiments, the sequence of the mutant FRB domain (FRB variant domain) is SEQ ID NO:7: Write it in TIFF2025500894000017.tif12146.

[0193] In some embodiments, the first dimerization domain is set forth in SEQ ID NO:5 and the second dimerization domain is set forth in SEQ ID NO:6.

[0194] In some embodiments, the first dimerization domain is set forth in SEQ ID NO:48 and the second dimerization domain is set forth in SEQ ID NO:6.

[0195] In some embodiments, the first dimerization domain is set forth in SEQ ID NO:30 and the second dimerization domain is set forth in SEQ ID NO:6.

[0196] In some embodiments, the first dimerization domain is set forth in SEQ ID NO:5 and the second dimerization domain is set forth in SEQ ID NO:7.

[0197] In some embodiments, the first dimerization domain is set forth in SEQ ID NO:48 and the second dimerization domain is set forth in SEQ ID NO:7.

[0198] In some embodiments, the first dimerization domain is set forth in SEQ ID NO:30 and the second dimerization domain is set forth in SEQ ID NO:7.

[0199] Alternatively, the first dimerization domain and the second dimerization domain may be FK506-binding protein (FKBP) of size 12 kD and calcineurin domains, which are known in the art to dimerize in the presence of FK506 or an analog thereof.

[0200] In some aspects, the dimerization domain comprises: (i) FK506-binding protein (FKBP) with a size of 12 kD; (ii) Cyclophilin A (CypA); or (iii) Gyrase B (CyrB) and a homodimerization domain selected from and the corresponding non-physiological ligands are (i) FK1012, AP1510, AP1903 or AP20187; (ii) Cyclosporine A (CsA); or (iii)(iii) coumermycin or its analogues It is.

[0201] In some embodiments, the first and second dimerization domains of the transmembrane receptor protein are an FKBP domain and a cyclophilin domain.

[0202] In some embodiments, the first and second dimerization domains of the transmembrane receptor protein are an FKBP domain and a bacterial dihydrofolate reductase (DHFR) domain.

[0203] In some embodiments, the first and second dimerization domains of the transmembrane receptor protein are a calcineurin domain and a cyclophilin domain.

[0204] 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).

[0205] 3. Transmembrane domain The transmembrane domain is the sequence of the synthetic cytokine receptor that spans the membrane. The transmembrane domain may include a hydrophobic alpha helix. In some aspects, the transmembrane domain is derived from a human protein.

[0206] The sequence of the transmembrane (TM) domain is Shown as TIFF2025500894000018.tif4128.

[0207] The sequence of the TM domain Shown as TIFF2025500894000019.tif4128.

[0208] The sequence of the TM domain Shown as TIFF2025500894000020.tif4128.

[0209] The sequence of the TM domain Shown as TIFF2025500894000021.tif4128.

[0210] The sequence of the TM domain Shown as TIFF2025500894000022.tif4128.

[0211] In some embodiments, the TM domain and the intracellular signaling domain are derived from the same cytokine receptor. In some embodiments, the synthetic gamma chain polypeptide contains an IL-2RG TM domain and an IL-2RG intracellular domain. In some embodiments, the synthetic beta chain polypeptide contains an IL-2RB TM domain and an IL-2RB intracellular domain. In some embodiments, the synthetic beta chain polypeptide contains an IL-7RB TM domain and an IL-7RB intracellular domain. In some embodiments, the synthetic beta chain polypeptide contains an IL-21RB TM domain and an IL-21RB intracellular domain. In some embodiments, the synthetic alpha chain polypeptide contains an IL-9RA TM domain and an IL-9RA intracellular domain.

[0212] In some embodiments, one or more additional contiguous amino acids of the ectodomain immediately adjacent to the TM domain of the cytokine receptor may also be included as part of the polypeptide sequence of the synthetic cytokine receptor chain. In some embodiments, 1 to 20 contiguous amino acids of the ectodomain adjacent to the TM domain of the cytokine receptor are included as part of the polypeptide sequence of the synthetic cytokine receptor chain. The portion of the ectodomain may 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 immediately adjacent to the TM sequence (e.g., N-terminal to the TM sequence).

[0213] In some embodiments, the synthetic cytokine receptor is composed of a synthetic gamma chain polypeptide containing an FKBP12 dimerization domain, an IL-2RG transmembrane domain, and an IL-2RG intracellular domain, and a synthetic beta chain polypeptide containing an FRB dimerization domain, an IL-2RB transmembrane domain, and an IL-2RB intracellular domain. In some embodiments, the synthetic cytokine receptor is composed of a synthetic gamma chain polypeptide containing an FKBP12 dimerization domain, an IL-2RG transmembrane domain, and an IL-2RB intracellular domain, and a synthetic beta chain polypeptide containing an FKBP12 dimerization domain, an IL-2RB transmembrane domain, and an IL-2RB intracellular domain.

[0214] In some embodiments, the synthetic gamma chain polypeptide contains an IL-2RG TM domain comprising the sequence set forth in SEQ ID NO:8 and an IL-2RG intracellular domain comprising the sequence set forth in SEQ ID NO:1. In some embodiments, the synthetic gamma chain polypeptide contains an IL-2RG TM domain comprising the sequence set forth in SEQ ID NO:31 and an IL-2RG intracellular domain comprising the sequence set forth in SEQ ID NO:1.

[0215] In some embodiments, the synthetic beta chain polypeptide contains an IL-2RB TM domain comprising the sequence set forth in SEQ ID NO:36 and an IL-2RB intracellular domain comprising the sequence set forth in SEQ ID NO:2. In some embodiments, the synthetic beta chain polypeptide contains an IL-2RB TM domain comprising the sequence set forth in SEQ ID NO:35 and an IL-2RB intracellular domain comprising the sequence set forth in SEQ ID NO:2.

[0216] In some embodiments, the synthetic cytokine receptor is composed of a synthetic gamma chain polypeptide containing an FKBP12 dimerization domain and an IL-2RG intracellular domain, and a synthetic beta chain polypeptide containing an FRB dimerization domain and an IL-2RB intracellular domain. In some embodiments, the synthetic gamma chain polypeptide has an amino acid sequence set forth in SEQ ID NO:28, or an amino acid sequence 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 an amino acid sequence set forth in SEQ ID NO:33, or an amino acid sequence 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.

[0217] In some embodiments, the synthetic cytokine receptor is composed of a synthetic gamma chain polypeptide containing an FKBP12 dimerization domain and an IL-2RG intracellular domain, and a synthetic beta chain polypeptide containing an FRB dimerization domain and an IL-2RB intracellular domain. In some embodiments, the synthetic gamma chain polypeptide has an amino acid sequence set forth in SEQ ID NO:28, or an amino acid sequence 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 an amino acid sequence set forth in SEQ ID NO:33, or an amino acid sequence 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.

[0218] In some embodiments, the synthetic cytokine receptor is composed of a synthetic gamma chain polypeptide set forth in SEQ ID NO:28 and a synthetic beta chain polypeptide set forth in SEQ ID NO:33.

[0219] In some embodiments, the synthetic cytokine receptor is composed of a synthetic gamma chain polypeptide containing an FKBP12 dimerization domain, an IL-2RG transmembrane domain, and an IL-2RG intracellular domain, and a synthetic beta chain polypeptide containing an FRB dimerization domain, an IL-7RB transmembrane domain, and an IL-7RB intracellular domain. In some embodiments, the synthetic cytokine receptor is composed of a synthetic gamma chain polypeptide containing an FRB dimerization domain, an IL-2RG transmembrane domain, and an IL-2RG intracellular domain, and a synthetic beta chain polypeptide containing an FKBP12 dimerization domain, an IL-7RB transmembrane domain, and an IL-7RB intracellular domain.

[0220] In some embodiments, the synthetic cytokine receptor is composed of a synthetic gamma chain polypeptide containing an FKBP12 dimerization domain, an IL-2RG transmembrane domain, and an IL-2RG intracellular domain, and a synthetic beta chain polypeptide containing an FRB dimerization domain, an IL-21RB transmembrane domain, and an IL-21RB intracellular domain. In some embodiments, the synthetic cytokine receptor is composed of a synthetic gamma chain polypeptide containing an FKBP12 dimerization domain, an IL-2RG transmembrane domain, and an IL-21RB intracellular domain, and a synthetic beta chain polypeptide containing an FKBP12 dimerization domain, an IL-21RB transmembrane domain, and an IL-21RB intracellular domain.

[0221] In some embodiments, the synthetic cytokine receptor is composed of a synthetic gamma chain polypeptide containing an FKBP12 dimerization domain, an IL-2RG transmembrane domain, and an IL-2RG intracellular domain, and a synthetic beta chain polypeptide containing an FRB dimerization domain, an IL-9RA transmembrane domain, and an IL-9RA intracellular domain. In some embodiments, the synthetic cytokine receptor is composed of a synthetic gamma chain polypeptide containing an FKBP12 dimerization domain, an IL-2RG transmembrane domain, and an IL-2RG intracellular domain, and a synthetic beta chain polypeptide containing an FKBP12 dimerization domain, an IL-9RA transmembrane domain, and an IL-9RA intracellular domain.

[0222] In some embodiments, the synthetic cytokine receptor can be bound by the non-physiological ligand rapamycin or a rapamycin analog. In some embodiments, the synthetic cytokine receptor is responsive to the non-physiological ligand rapamycin or a rapamycin analog, and 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 via the JAK / STAT pathway.

[0223] 4. Non-physiological ligands In various embodiments of the compositions and methods of the present disclosure, the system comprises a non-physiological ligand.Exemplary small molecules useful as ligands include, but are not limited to, rapamycin, fluorescein, fluorescein isothiocyanate (FITC), 4-[(6-methylpyrazin-2-yl)oxy]benzoic acid (aMPOB), folate, rhodamine, acetazolamide, and CA9 ligand.

[0224] In some embodiments, the synthetic cytokine receptor is activated by a ligand. In some embodiments, the ligand is a non-physiological ligand.

[0225] In some embodiments, the non-physiological ligand is a rapalog.

[0226] In some embodiments, the non-physiological ligand is rapamycin.

[0227] In some embodiments, the non-physiological ligand is AP21967.

[0228] In some embodiments, the non-physiological ligand is FK506.

[0229] 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 coumermycin.

[0230] In some embodiments, synthetic cytokine receptor complexes activated by folate, fluorescein, aMPOB, acetazolamide, CA9 ligand, tacrolimus, rapamycin, rapalogs (rapamycin analogs), CD28 ligand, poly(his)-tag, Strep-tag, FLAG-tag, VS-tag, Myc-tag, HA-tag, NE-tag, biotin, digoxigenin, dinitrophenol, or derivatives thereof.

[0231] In some embodiments, the non-physiological ligand can be an inorganic or organic compound less than 1000 daltons.

[0232] In some embodiments, the ligand can be rapamycin or a rapamycin analog (rapalog). In some embodiments, the rapalog includes variants of rapamycin that have one or more of the following modifications to rapamycin: demethylation, removal or replacement of the methoxy at C7, C42 and / or C29; removal, derivatization or replacement of the hydroxy at C13, C43 and / or C28; reduction, removal or derivatization of the ketone at C14, C24 and / or C30; replacement of the 6-membered pipecolate ring with a 5-membered prolyl ring; and replacement of the cyclohexyl ring with a substituted cyclopentyl ring.

[0233] Thus, in some embodiments, the rapalog is everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, zotarolimus, temsirolimus (CCI-779), C20-methallylrapamycin, C16-(S)-3-methylindolerapamycin, C16-(S)-3-methylindolerapamycin (C16-iRap), AP21967 (A / C Heterodimerizer, Takara Bio®), mycophenolate sodium, benidipine hydrochloride, rapamine, AP23573 (ridaforolimus), AP1903 (rimizucide), or metabolites, derivatives and / or combinations thereof.

[0234] In some embodiments, the ligand comprises FK1012 (a semi-synthetic dimer of FK506), tacrolimus (FK506), FKCsA (a complex of FK506 and cyclosporine), rapamycin, coumermycin, gibberellin, HaXS dimerizer (a chemical dimerizer of HaloTag and SNAP-tag), TMP-HTag (trimethoprim haloenzyme protein dimerizer), or ABT-737 or a functional derivative thereof.

[0235] In some embodiments, the non-physiological ligand is administered in an amount between 0 nM and 1000 nM, e.g., 0.05 nM, 0.1 nM, 0.5. nM, 1.0 nM, 5.0 nM, 10.0 nM, 15.0 nM, 20.0 nM, 25.0 nM, 30.0 nM, 35.0 nM, 40.0 nM, 45.0 nM, 50.0 nM, 55.0 nM, 60.0 nM, 65 0.0nM, 70.0nM, 75.0nM, 80.0nM, 90.0nM, 95.0nM, 100nM, 200nM, 300nM, 400nM, 500nM, 600nM, 700nM, 800nM, 900nM or 1000nM, or an amount within a range defined by any two of the above amounts.

[0236] 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.

[0237] 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.

[0238] In some embodiments, the non-physiological ligand is a rapalog and is present or provided at 1 nM. In some embodiments, the non-physiological ligand is a rapalog and is present or provided at 10 nM. In some embodiments, the non-physiological ligand is a rapalog and is present or provided at 20 nM. In some embodiments, the non-physiological ligand is a rapalog and is present or provided at 50 nM. In some embodiments, the non-physiological ligand is a rapalog and is present or provided at 100 nM.

[0239] In some embodiments, the non-physiological ligand is present or provided at 1 nM.

[0240] In some embodiments, the non-physiological ligand is present or provided at 10 nM.

[0241] In some embodiments, the non-physiological ligand is present or provided at 100 nM.

[0242] In some embodiments, the non-physiological ligand is present or provided at 1000 nM.

[0243] B. Cytosolic FRB In some embodiments, engineered cells, including iCIL cells, such as lymphocytes or their precursors, can be contacted with free cytosolic FRB (i.e., soluble FRB). As described in more detail elsewhere herein, rapamycin normally binds to FKBP12, and then the FKBP12-rapamycin complex binds to the FRB subunit of mTOR and blocks mTOR signaling. Thus, contacting cells with rapamycin can inhibit or reduce cell proliferation and expansion in some cases. In contrast to NK cell expansion, certain drugs are known to have an inhibitory effect on NK cells. The small molecule rapamycin has been reported by Wai et al.Transplantation.85(1):145-149(2008) to suppress NK cell function. In some embodiments, cells can be made "rapamycin resistant" by providing the cells with free cytosolic FRB to form a complex with rapamycin, thereby eliminating or reducing rapamycin-mediated growth inhibition of source cells or engineered cells, e.g., engineered lymphocytes, e.g., iCILs.

[0244] In some embodiments, soluble FRB can be microinjected into cells engineered with synthetic cytokine receptors, such as described cells, including lymphocytes, including CILs (e.g., NK cells), to eliminate or reduce rapamycin-mediated growth inhibition. In some embodiments, cells such as CILs 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.

[0245] In embodiments where soluble FRB is microinjected into stem cells or NK cells, the soluble FRB is injected at a concentration of 4 mg / mL, 4.5 mg / mL, 5 mg / mL, 5.5 mg / mL, or 6 mg / mL. In embodiments where soluble FRB is microinjected into cells such as CIL or NK cells, the soluble FRB is injected at a concentration of 1 μM.

[0246] FRB domain is a domain of about 100 amino acids derived from mTOR protein kinase. FRB domain can be expressed in cytosol as a freely diffusible soluble protein. Advantageously, FRB domain reduces the inhibitory effect of rapamycin on mTOR in transduced cells, promotes the constant activation of transduced cells, and gives cells a growth advantage over natural cells.

[0247] In some embodiments, the synthetic cytokine receptor complex comprises a cytosolic polypeptide that binds to a ligand, or a complex that includes the ligand.

[0248] In some embodiments, the cytosolic polypeptide comprises an FRB domain. In some embodiments, the cytosolic polypeptide comprises an FRB domain and the ligand is rapamycin. The cytosolic FRB domain may comprise a polypeptide sequence 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 be a naked FRB domain consisting essentially of a polypeptide having a polypeptide sequence 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. Advantageously, the cytosolic FRB confers resistance to the immunosuppressive effects of non-physiological ligands (e.g., rapamycin or rapalogs). In some embodiments, the cytosolic FRB domain can comprise a polypeptide sequence 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:50. In some embodiments, the FRB domain can be a naked FRB domain consisting essentially of a polypeptide having a polypeptide sequence 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:50.

[0249] In some embodiments, the cells are contacted with an FRB domain protein having the sequence set forth in SEQ ID NO:6.

[0250] In some embodiments, the cells are contacted with an FRB domain protein having the sequence set forth in SEQ ID NO:7.

[0251] In some embodiments, the cells are contacted with an FRB domain protein having the sequence set forth in SEQ ID NO:50.

[0252] VI. Gene Editing In some embodiments, the cells described herein (e.g., iPSCs or CILs) can be modified by gene editing. In some embodiments, the gene-edited iPSCs described can be used as source cells for differentiation into CILs.

[0253] Genome editing generally refers to the process of editing or changing the nucleotide sequence of genome, preferably in a precise, desired, and / or predetermined manner.The example of genome editing composition, system and method described herein uses site-specific nuclease to cut DNA at precise target position in genome, thereby creating double-strand break (DSB) in DNA.Such break can be repaired by endogenous DNA repair pathways, such as homology-directed repair (HDR) and / or non-homologous end joining (NHEJ) repair (see, for example, Cox et al., (2015) Nature Medicine 21(2):121-31).

[0254] In some embodiments, the cells (e.g., stem cells, CILs) described herein are genetically modified. In some embodiments, the modification involves knocking out one or more endogenous genes using DNA targeting proteins and nucleases or RNA-guided nucleases, and / or knocking in one or more exogenous genes of interest. In some embodiments, the gene of interest is knocked in to a specific locus of interest. In some embodiments, the gene of interest is a synthetic cytokine receptor complex. In some embodiments, the synthetic cytokine receptor complex is activated by rapamycin. In some embodiments, the synthetic cytokine receptor complex is a rapamycin-activated cytokine receptor (RACR). In some embodiments, the RACR is knocked in to a locus of interest. In some embodiments, the gene of interest is a chimeric antigen receptor.

[0255] In some embodiments, the modification comprises contacting the cell with a DNA targeting protein and nuclease or an RNA-guided nuclease. In some embodiments, the DNA targeting protein and nuclease or an RNA-guided nuclease comprises a zinc finger protein (ZFP), a clustered regularly interspaced short palindromic nucleic acid (CRISPR), or a TAL effector nuclease (TALEN). In some embodiments, CRISPR-Cas9 is used. In some embodiments, CRISPR-Mad7 is used.

[0256] Rejection of cellular therapeutics (e.g., CAR T cells) is at least due to human leukocyte antigen (HLA) mismatch between donor and recipient. One solution identified in recent years is to disrupt the expression of genes involved in this rejection, such as T cell receptor alpha constant (TRAC), beta-2-microglobulin (B2M) and signal regulatory protein alpha (SIRPA). Thus, in some embodiments, the cells described herein (e.g., iPSC, CIL) are genetically engineered to knock out the B2M locus, the TRAC locus and / or the 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.

[0257] 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 a pathway essential for cell growth and expansion. Thus, contacting cells with rapamycin can inhibit or reduce cell growth and expansion in some cases. In some embodiments, to eliminate or reduce rapamycin-mediated growth inhibition of source cells or CILs using the provided methods, endogenous genes involved in rapamycin function are disrupted in the provided cells, thereby making such cells "rapamycin-resistant". Reference to "rapamycin-resistant" cells is understood to refer to the ability of the cell's endogenous mTOR pathway to be unaffected by the presence of rapamycin or rapamycin analogs. However, it is further understood that "rapamycin-resistant" cells may nevertheless be responsive to rapamycin through a pathway that does not involve mTOR, for example, due to the activation of synthetic RACR described herein.

[0258] In some embodiments, the cells are genetically engineered to disrupt genes related to rapamycin recognition. In some embodiments, the cells are genetically engineered to disrupt mTOR gene. In some embodiments, the mTOR gene is FKBP-12 (also known as FKBP-1A, FKBP1, FKBP12, PKC12, PKCI2, PPIASE). FKBP12 is an essential binder of rapamycin and is required for its function. In some embodiments, the cells are genetically engineered to disrupt FKBP12 gene. In some embodiments, the cells are genetically engineered to knock out FKB12 ​​gene to induce rapamycin resistance. In some embodiments, the disruption of the endogenous FKBP12 gene of source stem cells (e.g., iPSCs) is by genetic knockout by CRISPR-Cas system. In normal cells without genetic disruption of FKBP12, FKBP12 is the main binder of rapamycin, and then the FKBP12-rapamycin complex binds to the FRB subunit of mTOR and blocks mTOR signaling. By disrupting the expression of the FKBP12 gene, such as by knocking out FKBP12, the results herein demonstrate successful rapamycin suppression activity, because rapamycin does not first complex with FKBP1A and has no function. Thus, genetic disruption of FKBP12, such as by gene knockout, renders stem cells (e.g., iPSCs) highly resistant to rapamycin-mediated mTOR inhibition, allowing for robust proliferation of stem cells (e.g., iPSCs) even in the presence of high doses of rapamycin. In some embodiments, the ability to render cells resistant to rapamycin proliferation inhibition allows for the engagement of RACR by rapamycin during cell differentiation without adverse effects. Furthermore, knocking out FKBP12 avoids the competition between FKBP12 and RACR for binding to rapamycin. Thus, in some embodiments, the ability to render cells resistant to rapamycin proliferation by FKBP12 knockout also allows for activation of RACR-containing cells in vivo, suppressing potential allogeneic anti-graft responses via mTOR suppression of the host immune system.

[0259] In some aspects, the cells described herein are genetically engineered to include a nucleotide sequence encoding a synthetic cytokine receptor in an endogenous gene. In some aspects, the synthetic cytokine receptor is engineered into a gene such that expression of the endogenous gene is not disrupted. In some aspects, the synthetic cytokine receptor is engineered into a safe harbor locus.

[0260] In some embodiments, the cell described herein is genetically engineered to include a nucleotide sequence encoding a synthetic cytokine receptor in a housekeeping gene. In some embodiments, the housekeeping gene is eukaryotic translation elongation factor 1 alpha (EEF1A), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ubiquitin C (UBC), or actin beta (ACTB).

[0261] In some embodiments, the gene of interest inserted into the endogenous locus is a synthetic cytokine receptor complex.In some embodiments, the endogenous promoter of a particular locus is used.In some embodiments, additional promoters can be included so that two or more promoters drive the expression of the exogenous gene of interest.

[0262] In some embodiments, the cells described herein are genetically engineered to contain a nucleotide sequence encoding a synthetic cytokine receptor complex in a disrupted gene. For example, in some embodiments, the cells contain a disrupted B2M gene and a nucleotide sequence encoding a synthetic cytokine receptor within the disrupted B2M gene.

[0263] In some aspects, the cells (e.g., iPSCs, CILs) described herein comprise (i) a disrupted B2M locus and (ii) a nucleotide sequence encoding a synthetic cytokine receptor complex (e.g., RACR) under the control of the endogenous B2M promoter and the EEF1A promoter.

[0264] In some aspects, the cells (e.g., iPSCs, CILs) described herein comprise (i) a disrupted B2M locus and (ii) a nucleotide sequence encoding a synthetic cytokine receptor complex (e.g., RACR) inserted into the endogenous B2M gene and under the control of the endogenous B2M promoter and the EEF1A promoter.

[0265] In some embodiments, a cell comprising (i) a disrupted B2M locus and (ii) a nucleotide sequence encoding a synthetic cytokine receptor complex (e.g., RACR) is produced by any of the methods described below.

[0266] A. Systems for Genome Editing In some embodiments, the system for editing cells described herein comprises a site-specific nuclease, such as a CRISPR / Cas system, and optionally a gRNA. In some embodiments, the system comprises an engineered nuclease. In some embodiments, the system comprises a site-specific nuclease. In some embodiments, the site-specific 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).

[0267] 1. CRISPR / Cas nuclease system The naturally occurring CRISPR / Cas system is a genetic defense system that provides a form of adaptive immunity in prokaryotes. CRISPR is a gene that encodes clustered regularly interspaced short palindromic repeats (CRISPRs). C Lustered R egularly I Interspaced S hort P alindromic RCRISPR / Cas is an abbreviation for CRISPR / Cas systems, a family of DNA sequences found in bacterial and archaeal genomes that contain fragments of DNA (spacer DNA) similar to foreign DNA previously exposed to the cell, for example, by a virus that has infected or attacked the prokaryote. These fragments of DNA are used by the prokaryote to detect and destroy similar foreign DNA upon reintroduction from a similar virus during a subsequent attack, for example. Transcription of the CRISPR locus results in the formation of an RNA molecule containing a spacer sequence that associates with and targets Cas (CRISPR-associated) proteins that can recognize and cleave the foreign exogenous DNA. Numerous types and classes of CRISPR / Cas systems have been described (see, for example, Koonin et al., (2017) Curr Opin Microbiol 37:67-78).

[0268] Engineered versions of the CRISPR / Cas system have been developed in many formats to mutate or edit the genomic DNA of cells from other species. A common approach using the CRISPR / Cas system involves heterologously expressing or introducing a site-specific nuclease (e.g., Cas nuclease) in combination with a guide RNA (gRNA) into a cell, resulting in a DNA cleavage event (e.g., forming single-strand or double-strand breaks (SSB or DSB)) in the backbone of the cell's genomic DNA at a precise targetable location. The manner in which the DNA cleavage event is repaired by the cell provides an opportunity to edit the genome by adding, removing, or modifying (substituting) DNA nucleotides or sequences (e.g., genes).

[0269] In some embodiments, the system for editing cells described herein comprises a nuclease capable of inducing DNA breaks in endogenous target genes in cells. In some embodiments, the DNA breaks comprise double-strand breaks (DSBs) induced by nucleases capable of inducing DSBs by cleaving both strands of double-stranded DNA at the break site. In some embodiments, the DNA breaks comprise single-strand breaks (SSBs) at the break site of the sense strand or antisense strand of the endogenous target gene. In some embodiments, the DNA breaks comprise SSBs at the break site of the sense strand and SSBs at the break site of the antisense strand, thereby resulting in a DSB. In some embodiments, the DSBs are induced by a pair of recombinant nucleases, e.g., nickases, each capable of inducing single-strand breaks (SSBs) of opposing DNA strands at different break sites, e.g., a break site upstream of a gene variant on one strand of a target gene and a break site downstream of a gene variant on the other strand. In some embodiments, the first of the pair of nickases is complexed with a first guide RNA, e.g., a first sgRNA, to target cleavage to one strand, e.g., the sense strand, and the second of the pair of nickases is complexed with a second guide RNA, e.g., a second sgRNA, to target cleavage to the other strand, e.g., the antisense strand. In some embodiments, the DSB is induced through an SSB on the opposite strand, i.e., the sense strand and the antisense strand, of an endogenous target gene in a cell.

[0270] Generally, gene is located in double-stranded DNA, which comprises mutually complementary sense and antisense strands.Sense strand is also called coding strand, because its sequence is the DNA version of transcribed RNA sequence.Antisense strand is also called template strand, because its sequence is complementary to transcribed RNA sequence.

[0271] i. Guide RNA (gRNA) An engineered CRISPR / Cas system includes at least two components: 1) a guide RNA (gRNA) molecule and 2) a Cas nuclease that interacts to form a gRNA / Cas nuclease complex. The gRNA includes at least a user-defined targeting domain, called a "spacer," that includes a nucleotide sequence and a CRISPR repeat sequence. In an engineered CRISPR / Cas system, the gRNA / Cas nuclease complex targets a specific target sequence of interest within a target nucleic acid (e.g., a genomic DNA molecule) by generating a gRNA that includes a spacer having a nucleotide sequence that can bind to the specific target sequence in a complementary manner (see Jinek et al., Science, 337, 816-821 (2012) and Deltcheva et al., Nature, 471, 602-607 (2011)). Thus, the spacer provides the targeting function of the gRNA / Cas nuclease complex.

[0272] In naturally occurring type II CRISPR / Cas systems, "gRNA" is composed of two RNA strands: 1) CRISPR RNA (crRNA) containing a spacer and CRISPR repeats, and 2) transactivating CRISPR RNA (tracrRNA). In type II CRISPR / Cas systems, a portion of the crRNA containing the CRISPR repeats and a portion of the tracrRNA hybridize to form a crRNA:tracrRNA duplex that interacts with Cas nuclease (e.g., Cas9). As used herein, the term "split gRNA" or "modular gRNA" refers to a gRNA molecule that contains two RNA strands, where a first RNA strand incorporates crRNA function and / or structure and a second RNA strand incorporates tracrRNA function and / or structure, and the first and second RNA strands are partially hybridized.

[0273] Thus, in some embodiments, the gRNA comprises two RNA molecules. In some embodiments, the gRNA comprises a CRISPR RNA (crRNA) and a transactivating CRISPR RNA (tracrRNA). In some embodiments, the gRNA is a split gRNA. In some embodiments, the gRNA is a modular gRNA. In some embodiments, the split gRNA comprises, from 5' to 3', a first strand comprising a spacer and a first complementary region, and, from 5' to 3', a second strand comprising a second complementary region, and optionally a tail domain.

[0274] In some embodiments, the crRNA comprises a spacer that comprises a nucleotide sequence that is complementary to and hybridizes with a sequence that is complementary to a target sequence on a target nucleic acid (e.g., a genomic DNA molecule). In some embodiments, the crRNA comprises a region that is complementary to and hybridizes with a portion of the tracrRNA.

[0275] In some embodiments, the target nucleic acid (e.g., an endogenous gene) is B2M. In some embodiments, the crRNA comprises a nucleotide sequence set forth in SEQ ID NO:18, or a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the nucleotide sequence set forth in SEQ ID NO:18. In some embodiments, the crRNA comprises a nucleotide sequence set forth in SEQ ID NO:18.

[0276] In some embodiments, the target nucleic acid (e.g., endogenous gene) is FKBP12. In some embodiments, the crRNA comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 19, 20, and 21, or a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the nucleotide sequence set forth in any one of SEQ ID NOs: 19, 20, and 21. In some embodiments, the crRNA comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 19, 20, and 21. In some embodiments, the crRNA comprises a nucleotide sequence set forth in SEQ ID NO: 19, or a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 19. In some embodiments, the crRNA comprises a nucleotide sequence set forth in SEQ ID NO:19. In some embodiments, the crRNA comprises a nucleotide sequence set forth in SEQ ID NO:20, or a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the nucleotide sequence set forth in SEQ ID NO:20. In some embodiments, the crRNA comprises a nucleotide sequence set forth in SEQ ID NO:20. In some embodiments, the crRNA comprises a nucleotide sequence set forth in SEQ ID NO:21, or a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the nucleotide sequence set forth in SEQ ID NO:21. In some embodiments, the crRNA comprises a nucleotide sequence set forth in SEQ ID NO:21.

[0277] In some embodiments, the tracrRNA may comprise all or part of the wild-type tracrRNA sequence from a naturally occurring CRISPR / Cas system. In some embodiments, the tracrRNA may comprise a truncated or modified mutant of the wild-type tracr RNA. The length of the tracr RNA may depend on the CRISPR / Cas system used. In some embodiments, the 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 length. In certain embodiments, the tracrRNA is at least 26 nucleotides in length. In additional embodiments, the tracrRNA is at least 40 nucleotides in length. In some embodiments, the tracrRNA may comprise a specific secondary structure, such as, for example, one or more hairpin or stem-loop structures, or one or more bulge structures.

[0278] a. Single guide RNA (sgRNA) Engineered CRISPR / Cas nuclease systems often combine crRNA and tracrRNA into a single RNA molecule, referred to herein as "single guide RNA" (sgRNA), by adding a linker between these components. Without being bound by theory, the sgRNA, like the double-stranded crRNA and tracrRNA, forms a complex with a Cas nuclease (e.g., Cas9), guides the Cas nuclease to the target sequence, and activates the Cas nuclease to cleave the target nucleic acid (e.g., genomic DNA). Thus, in some embodiments, the gRNA may comprise a operably linked crRNA and tracrRNA. In some embodiments, the sgRNA may comprise a crRNA covalently linked to the tracrRNA. In some embodiments, the crRNA and tracrRNA are covalently linked via a linker. In some embodiments, the sgRNA may comprise a stem-loop structure via base pairing between the crRNA and tracrRNA. In some embodiments, the sgRNA comprises, from 5' to 3', a spacer, a first region of complementarity, a linking domain, a second region of complementarity, and optionally a tail domain.

[0279] The sgRNA can be unmodified or modified. For example, a modified sgRNA can include one or more 2'-O-methyl phosphorothioate nucleotides.

[0280] By way of example, guide RNAs used in CRISPR / Cas systems, or other even smaller RNAs, can be easily synthesized by chemical means, as exemplified herein and described in the art. Although chemical synthesis procedures are constantly expanding, purification of such RNAs by procedures such as high performance liquid chromatography (HPLC, which avoids the use of gels such as PAGE) tends to become more difficult as the length of polynucleotides increases significantly beyond 100 nucleotides or so. One approach used to generate even longer RNAs is to generate two or more molecules that are ligated together. Much longer RNAs, such as those that code for Cas9 endonuclease, are more easily generated enzymatically. During or after chemical synthesis and / or enzymatic generation of RNA, various types of RNA modifications can be introduced, such as modifications that improve stability, reduce the likelihood or extent of innate immune response, and / or enhance other attributes, as described in the art.

[0281] b. Spacer In some embodiments, the gRNA comprises a spacer sequence. The spacer sequence is a sequence that defines a target site of the target nucleic acid (e.g., DNA). The target nucleic acid is a double-stranded molecule, one strand comprises a target sequence adjacent to a PAM sequence, called the "PAM strand", and the second strand is called the "non-PAM strand" and is complementary to the PAM strand and the target sequence. Both the gRNA spacer and the target sequence are complementary to the non-PAM strand of the target nucleic acid. In some embodiments, the spacer sequence corresponding to the target sequence adjacent to the PAM sequence is complementary to the non-PAM strand of the target nucleic acid. Thus, in some embodiments, the spacer sequence corresponding to the target sequence adjacent to the PAM sequence is identical to the PAM strand. The gRNA spacer sequence hybridizes to the complementary strand (e.g., the non-PAM strand of the target nucleic acid / target site). In some embodiments, the spacer is sufficiently complementary to the complementary strand of the target sequence (e.g., the non-PAM strand) to target the Cas nuclease to the target nucleic acid. In some embodiments, the spacer is at least 80%, at least 85%, at least 90%, or at least 95% complementary to the non-PAM strand of the target nucleic acid. In some embodiments, the spacer is 100% complementary to the non-PAM strand of the target nucleic acid. In some embodiments, the spacer comprises 1, 2, 3, 4, 5, 6, or more nucleotides that are not complementary to the non-PAM strand of the target nucleic acid. In some embodiments, the spacer comprises one nucleotide that is not complementary to the non-PAM strand of the target nucleic acid. In some embodiments, the spacer comprises two nucleotides that are not complementary to the non-PAM strand of the target nucleic acid.

[0282] 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 spacer is about 15-50, about 20-45, about 25-40, or about 30-35 nucleotides in length. In some embodiments, the spacer is about 19-22 nucleotides in length. In some embodiments, the spacer is about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30 nucleotides in length. In some embodiments, the spacer is 19 nucleotides in length. In some embodiments, the spacer is 20 nucleotides in length, and in some embodiments, the spacer is 21 nucleotides in length.

[0283] In some embodiments, the nucleotide sequence of spacer is designed or selected using a computer program.The computer program can use variables such as predicted melting temperature, secondary structure formation, predicted annealing temperature, sequence identity, genomic context, chromatin accessibility, %GC, genomic frequency (e.g., of identical or similar sequences that differ in one or more spots as a result of mismatch, insertion or deletion), methylation status, and / or the presence of SNPs.

[0284] In some embodiments, the spacer comprises at least one or more modified nucleotides, such as those described herein. The present disclosure provides gRNA molecules comprising a spacer that may comprise the nucleobase uracil (U), and any DNA encoding a gRNA comprising a spacer comprising the nucleobase uracil (U) comprises the nucleobase thymine (T) at the corresponding position.

[0285] ii. How to prepare gRNA Methods for making gRNA are known to those skilled in the art, including, but not limited to, in vitro transcription (IVT), synthesis and / or chemical synthesis methods, or combinations thereof. Enzyme (IVT) synthesis methods, solid-phase synthesis methods, liquid-phase synthesis methods, combined synthesis methods, small-area synthesis and ligation methods are utilized. In one embodiment, gRNA is made using IVT enzymatic synthesis 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, used to in vitro transcribe gRNA described herein.

[0286] In some embodiments, non-natural modified nucleobases are introduced into polynucleotides, such as gRNAs, during or after synthesis. In certain embodiments, the modifications are on internucleoside linkages, purine or pyrimidine bases, or sugars. In some embodiments, the modifications are introduced into the ends of polynucleotides by chemical synthesis or using polymerase enzymes. Examples of modified nucleic acids and their synthesis are disclosed in PCT Application No. PCT / US2012 / 058519. The synthesis of modified polynucleotides is also described in Verma and Eckstein, Annual Review of Biochemistry, vol.76, 99-134 (1998).

[0287] In some embodiments, enzymatic or chemical ligation methods are used to conjugate polynucleotides or regions thereof with various functional moieties, such as targeting or delivery agents, fluorescent labels, liquids, nanoparticles, etc. Conjugates of polynucleotides and modified polynucleotides are reviewed in Goodchild, Bioconjugate Chemistry, vol.1(3), 165-187 (1990).

[0288] In some embodiments, the disclosure provides a nucleic acid, e.g., a vector, encoding a gRNA described herein. In some embodiments, the nucleic acid is a DNA molecule. In other embodiments, the nucleic acid is an RNA molecule. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding a crRNA. In some embodiments, the nucleotide sequence encoding the crRNA comprises a spacer flanking all or a portion of a repeat sequence from a naturally occurring CRISPR / Cas system. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding a tracrRNA. In some embodiments, the crRNA and the tracrRNA are encoded by two separate nucleic acids. In other embodiments, the crRNA and the tracrRNA are encoded by a single nucleic acid. In some embodiments, the crRNA and the tracrRNA are encoded by opposing strands of a single nucleic acid. In other embodiments, the crRNA and the tracrRNA are encoded by the same strand of a single nucleic acid.

[0289] In some embodiments, the gRNA provided by the present disclosure is chemically synthesized by any means described in the art (see, for example, International Publication No. WO 2005 / 01248). Although chemical synthesis procedures are constantly expanding, purification of such RNA by procedures such as high performance liquid chromatography (HPLC, which avoids the use of gels such as PAGE) tends to become more difficult as the length of polynucleotides increases significantly beyond about 100 nucleotides. One approach used to generate even longer RNA is to generate two or more molecules that are ligated together.

[0290] In some embodiments, multiple guide RNAs can be used with CRISPR / Cas nuclease system. Each guide RNA can contain different targeting sequences, so that CRISPR / Cas system cuts multiple target nucleic acids. In some embodiments, one or more guide RNAs can have the same or different properties, such as activity or stability in Cas9 RNP complex. When multiple guide RNAs are used, each guide RNA can be coded on the same or different vectors. The promoters used to promote the expression of multiple guide RNAs can be the same or different.

[0291] The guide RNA may target any sequence of interest via the targeting sequence (e.g., spacer sequence) of the crRNA. In some embodiments, the degree of complementarity between the targeting sequence of the guide RNA and the target sequence on the target nucleic acid molecule is about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99% or about 100%. In some embodiments, the targeting sequence of the guide RNA and the target sequence on the target nucleic acid molecule are 100% complementary. In other embodiments, the targeting sequence of the guide RNA and the target sequence on the target nucleic acid molecule may contain at least one mismatch. For example, the targeting sequence of the guide RNA and the target 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 target sequence on the target nucleic acid molecule may contain 1 to 6 mismatches. In some embodiments, the targeting sequence of the guide RNA and the target sequence on the target nucleic acid molecule may contain 5 or 6 mismatches.

[0292] The length of the targeting sequence may depend on the CRISPR-Cas system and components used. For example, different Cas9 proteins from different bacterial species have various optimal targeting sequence lengths. Thus, the targeting sequence may comprise 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 length. In some embodiments, the targeting sequence may comprise 18-24 nucleotides in length. In some embodiments, the targeting sequence may comprise 19-21 nucleotides in length. In some embodiments, the targeting sequence may comprise 20 nucleotides in length.

[0293] In some embodiments of the present disclosure, the CRISPR / Cas nuclease system comprises at least one guide RNA. In some embodiments, the guide RNA and the Cas protein can form a ribonucleoprotein (RNP), e.g., a CRISPR / Cas complex. The guide RNA can guide the Cas protein to a target sequence on a target nucleic acid molecule (e.g., a genomic DNA molecule), and the Cas protein cleaves the target nucleic acid. In some embodiments, the CRISPR / Cas complex is a Cpf1 / guide RNA complex. In some embodiments, the CRISPR complex is a type II CRISPR / Cas9 complex. In some embodiments, the Cas protein is a Cas9 protein. In some embodiments, the CRISPR / Cas9 complex is a Cas9 / guide RNA complex. In some embodiments, the CRISPR / Cas complex is an engineered class 2 type V CRISPR system. In some embodiments, the endonuclease is Mad7.

[0294] iii. Cas nuclease In some embodiments, the present disclosure provides compositions and systems (e.g., engineered CRISPR / Cas systems) comprising a site-specific nuclease, wherein the site-specific nuclease is a Cas nuclease. The Cas nuclease may comprise at least one domain that interacts with a guide RNA (gRNA). Furthermore, the Cas nuclease is guided to a target sequence by the guide RNA. The guide RNA interacts with the Cas nuclease and the target sequence such that the Cas nuclease can cleave the target sequence after being guided to the target sequence. In some embodiments, the guide RNA provides specificity for cleavage of the target sequence, and the Cas nuclease is universal and pairs with various guide RNAs to cleave various target sequences.

[0295] In some embodiments, the CRISPR / Cas system comprises components from type I, type II or type III systems. Updated classification schemes for CRISPR / Cas loci define class 1 and class 2 CRISPR / Cas systems with type I-V or type VI (Makarova et al., (2015) Nat Rev Microbiol, 13(11):722-36; Shmakov et al., (2015) Mol Cell, 60:385-397). Class 2 CRISPR / Cas systems have a single protein effector. Type II, type V and type VI Cas proteins are single-protein RNA-guided endonucleases, referred to herein as "class 2 Cas nucleases". Class 2 Cas nucleases include, for example, Cas9 protein, Cpf1 protein, C2c1 protein, C2c2 protein and C2c3 protein. The Cpf1 nuclease (Zetsche et al., (2015) Cell 163:1-13) is homologous to Cas9 and contains a RuvC-like nuclease domain.

[0296] In some embodiments, the Cas nuclease is derived from a type II CRISPR / Cas system (e.g., a Cas9 protein from a CRISPR / Cas9 system). In some embodiments, the Cas nuclease is derived from a class 2 CRISPR / Cas system (a single protein Cas nuclease such as a Cas9 protein or a Cpf1 protein). The Cas9 and Cpf1 family of proteins are enzymes with DNA endonuclease activity and can be directed to cleave a desired nucleic acid target by designing an appropriate guide RNA, as further described herein.

[0297] Type II CRISPR / Cas system components are derived from Type IIA, Type IIB, or Type IIC systems, including Cas9 and its orthologues. Non-limiting exemplary species from which the Cas9 nuclease or other components may be derived include Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus species, Staphylococcus aureus, Listeria innocua, Lactobacillus gasseri, Francisella novicida, Wolinella succinogenes, Sutterella wadsworthensis, Gammaproteobacteria, Neisseria meningitidis, Campylobacter jejuni, and / or other strains of the bacteria. jejuni, Pasteurella multocida, Fibrobacter succinogene, Rhodospirillum rubrum, Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, Alicyclobacillus acidocaldarius acidocaldarius, Bacillus pseudomycoidespseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Lactobacillus buchneri, Treponema denticola, Microscilla marina, Burkholderiales bacteria, Polaromonas naphthalenivorans, Polaromonas species, Crocosphaera watsoni, watsonii, Cyanothece spp., Microcystis aeruginosa, Synechococcus spp., Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosumvinosum, Marinobacter spp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc spp., Arthrospira maxima, Arthrospira platensis platensis, Arthrospira spp., Lyngbya spp., Microcoleus chthonoplastes, Oscillatoria spp., Petrotoga mobilis, Thermosipho africanus, Streptococcus pasteurianus, Neisseria cinerea, Campylobacter lari, Parvibaculum lavamentivorans, Corynebacterium diphtheria or Acaryochloris marina. In some embodiments, the Cas9 protein is derived from Streptococcus pyogenes (SpCas9). In some embodiments, the Cas9 protein is derived from Streptococcus thermophilus (StCas9). In some embodiments, the Cas9 protein is derived from Neisseria meningitidis (NeisseriaIn some embodiments, the Cas9 protein is derived from Staphylococcus aureus (SaCas9). In some embodiments, the Cas9 protein is derived from Campylobacter jejuni (CjCas9).

[0298] In some embodiments, the Cas nuclease may contain multiple nuclease domains. For example, the Cas9 nuclease may contain at least one RuvC-like nuclease domain (e.g., Cpf1) and at least one HNH-like nuclease domain (e.g., Cas9). In some embodiments, the Cas9 nuclease introduces a DSB into the target sequence. In some embodiments, the Cas9 nuclease is modified to contain only one functional nuclease domain. For example, the Cas9 nuclease is modified to have one of the nuclease domains mutated or completely or partially deleted to reduce its nucleic acid cleavage activity. In some embodiments, the Cas9 nuclease is modified to not contain a functional RuvC-like nuclease domain. In other embodiments, the Cas9 nuclease is modified to not contain a functional HNH-like nuclease domain. In some embodiments where only one of the nuclease domains is functional, the Cas9 nuclease is a nickase that can introduce a single-strand break ("nick") into a target sequence. In some embodiments, conserved amino acids in the Cas9 nuclease domain are substituted to reduce or alter nuclease activity. In some embodiments, the Cas nuclease nickase comprises an amino acid substitution in the RuvC-like nuclease domain. Exemplary amino acid substitutions in the RuvC-like nuclease domain include D10A (based on S. pyogenes Cas9 nuclease). In some embodiments, the nickase comprises an amino acid substitution in the HNH-like nuclease domain. Exemplary amino acid substitutions in the HNH-like nuclease domain include E762A, H840A, N863A, H983A and D986A (based on S. pyogenes Cas9 nuclease). In some embodiments, the nuclease system described herein comprises a nickase and a pair of guide RNAs that are complementary to the sense and antisense strands of a target sequence, respectively. The guide RNA guides the nickase to target and introduce DSBs by generating nicks on opposing strands of the target sequence (i.e., double nicking).Use chimeric Cas9 nuclease, in which one domain or region of protein is replaced by a part of a different protein.For example, Cas9 nuclease domain is replaced by a domain from a different nuclease, such as Fok1.Cas9 nuclease is a modified nuclease.

[0299] In some embodiments, the Cas nuclease is from a type I CRISPR / Cas system. In some embodiments, the Cas nuclease is a component of a 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.

[0300] In some embodiments, the Cas nuclease is a Mad endonuclease. The CRISPR / Mad system is closely related to the class 2 family of Cas enzymes, type V (Cpf1-like). In some embodiments, the CRISPR-Mad system uses the Eubacterium rectale Mad7 endonuclease or a mutant thereof. The Mad7-crRNA complex cleaves the target DNA by identifying the PAM 5'-YTTN.

[0301] 2. Engineered nucleases In some embodiments, the cells described herein are genetically engineered with a site-specific nuclease, and the site-specific nuclease is an engineered nuclease. Exemplary engineered nucleases are meganucleases (e.g., homing endonucleases), ZFNs, TALENs and megaTALs.

[0302] Naturally occurring meganucleases can recognize and cleave double-stranded DNA sequences of about 12-40 base pairs and are generally classified into five families. In some embodiments, the meganuclease is selected from the LAGLIDADG family, the GIY-YIG family, the HNH family, the His-Cys box family, and the PD-(D / E)XK family. In some embodiments, the DNA binding domain of the meganuclease is engineered to recognize and bind to sequences other than its cognate target sequence. In some embodiments, the DNA binding domain of the meganuclease is fused to a heterologous nuclease domain. In some embodiments, meganucleases, such as homing endonucleases, are fused to TAL modules to create hybrid proteins, such as "megaTAL" proteins. MegaTAL proteins have improved DNA targeting specificity by recognizing the target sequence of the DNA binding domain of the meganuclease and the target sequence of the TAL module.

[0303] ZFN is a fusion protein that includes 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 triplet), and ZF repeats are combined to recognize DNA target sequences and provide sufficient affinity. Thus, engineered ZF repeats are combined to recognize even longer DNA sequences, such as 9bp, 12bp, 15bp or 18bp. In some embodiments, ZFN includes a ZF fused to a nuclease domain derived from a restriction endonuclease. For example, the restriction endonuclease is FokI. In some embodiments, the nuclease domain includes a dimerization domain, such as when the nuclease dimerizes to become active, and a pair of ZFNs that include ZF repeats, where the nuclease domain is designed to target a target sequence that includes two halves of a target sequence recognized by each ZF repeat on opposing strands of a DNA molecule, with an interconnecting sequence (sometimes referred to in the literature as a spacer) between them. For example, the interconnecting sequence is 5-7 bp long. When both ZFNs of the pair bind, the nuclease domain can dimerize and introduce a DSB within the interconnecting sequence. In some embodiments, the dimerization domain of the nuclease domain includes a knob-into-hole motif to promote dimerization. For example, the ZFN includes a knob-into-hole motif in the dimerization domain of FokI.

[0304] The DNA binding domain of TALENs usually contains a variable number of 34 or 35 amino acid repeats ("modules" or "TAL modules"), each module binds to a single DNA base pair, A, T, G or C. The adjacent residues at positions 12 and 13 of each module ("repeat-variable diresidues" or RVD) specify the single DNA base pair that the module binds to. Although the module used to recognize G may also have affinity for A, TALENs benefit from a simple recognition code (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, TALENs may contain a nuclease domain derived from a restriction endonuclease. For example, the restriction endonuclease is FokI. In some embodiments, the nuclease domains may dimerize to become active, and a pair of TALENSs is designed to target a target sequence that contains two half target sequences recognized by each DNA binding domain on opposite strands of a DNA molecule, with an interconnecting sequence between them. For example, the target sequence of each half is within 10-20 bp and the interconnecting sequence is 12-19 bp long. Upon binding of both TALENs of a pair, the nuclease domains can dimerize and introduce DSBs within the interconnecting sequence. In some embodiments, the dimerization domain of the nuclease domain can contain a knob-into-hole motif to promote dimerization. For example, the TALEN can contain a knob-into-hole motif in the dimerization domain of FokI.

[0305] 3. Target site In some embodiments, the site-specific nuclease described herein is directed to a target nucleic acid molecule (e.g., an endogenous gene) and cleaves it (e.g., introduces a DSB). In some embodiments, the target nucleic acid molecule is a housekeeping gene. In some embodiments, the housekeeping gene is eukaryotic translation elongation factor 1 alpha (EEF1A), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ubiquitin C (UBC), or actin beta (ACTB). In some embodiments, the target 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.

[0306] A target nucleic acid molecule is any DNA molecule that is endogenous or exogenous to a cell. As used herein, the term "endogenous sequence" refers to a sequence that is natural to a cell. In some embodiments, the target nucleic acid molecule is a genomic DNA (gDNA) molecule or chromosome from or within a cell. In some embodiments, the target sequence of the target nucleic acid molecule is a genomic sequence from or within 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 transcriptional control sequence of a gene, the translational 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.

[0307] In some embodiments, the target sequence may be located at a non-gene functional site within the genome that controls an aspect of chromatin organization, e.g., a scaffold site or locus control region. In some embodiments, the target sequence may be a genetic safe harbor site, i.e., a locus that facilitates safe genetic modification.

[0308] In some embodiments, the target sequence may be adjacent to a protospacer adjacent motif (PAM), a short sequence recognized by the CRISPR / Cas complex. In some embodiments, the PAM may be adjacent to or within 1, 2, 3, or 4 nucleotides at the 3' end of the target sequence. In some embodiments, the target sequence may include a PAM. The length and sequence of the PAM may depend on the Cas protein used. For example, the PAM may be selected from consensus sequences or specific PAM sequences for a particular Cas nuclease or Cas ortholog, including those disclosed in FIG. 1 of Ran et al., (2015) Nature, 520:186-191 (2015), which is incorporated herein by reference. In some embodiments, the PAM may include 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. Non-limiting exemplary PAM sequences include NGG (SpCas9 WT, SpCas9 nickase, dimeric dCas9-Fok1, SpCas9-HF1, SpCas9 K855A, eSpCas9(1.0), eSpCas9(1.1)), NGAN or NGNG (SpCas9 VQR mutant), NGAG (SpCas9 EQR mutant), NGCG (SpCas9 VRER mutant), NAAG (SpCas9 QQR1 mutant), NNGRRT or NNGRRN (SaCas9), NNNRRT (KKH SaCas9), NNNNRYAC(CjCas9), NNAGAAW(St1Cas9), NAAAAC(TdCas9), NGGNG(St3Cas9), NG(FnCas9), NAAAAN(TdCas9), NNAAAAW(StCas9), NNNNACA(CjCas9), GNNNCNNA(PmCas9), and NNNNGATT(NmCas9) (e.g., Cong et al. 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. 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 either 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.

[0309] In some embodiments, the PAM sequence recognized by a nuclease, such as Cas9, varies depending on the particular nuclease and the bacterial species from which it is derived. In some embodiments, the PAM sequence recognized by SpCas9 is the nucleotide sequence 5'-NGG-3', where "N" is any nucleotide. In some embodiments, the PAM sequence recognized by SaCas9 is the nucleotide sequence 5'-NGRRT-3' or the nucleotide sequence 5'-NGRRN-3', where "N" is any nucleotide and "R" is a purine (e.g., guanine or adenine). In some embodiments, the PAM sequence recognized by NmeCas9 is the nucleotide sequence 5'-NNNNGATT-3', where "N" is any nucleotide. In some embodiments, the PAM sequence recognized by CjCas9 is the nucleotide sequence 5'-NNNNRYAC-3', where "N" is any nucleotide, "R" is a purine (e.g., guanine or adenine), and "Y" is a pyrimidine (e.g., cytosine or thymine). In some embodiments, the PAM sequence recognized by StCas9 is the nucleotide sequence 5'-NNAGAAW-3', where "N" is any nucleotide and "W" is an adenine or thymine.

[0310] In some embodiments, the recombinant nuclease is Cas9 and the PAM sequence is the nucleotide sequence: (a) 5'-NGG-3'; (b) 5'-NGRRT-3', or 5'-NGRRN-3'; (c) 5'-NNNNGATT-3'; (d) 5'-NNNNRYAC-3'; or (e) 5'-NNAGAAW-3', where "N" is any nucleotide, "R" is a purine (e.g., guanine or adenine), "Y" is a pyrimidine (e.g., cytosine or thymine), and "W" is an adenine or thymine. In some embodiments, the recombinant nuclease is Cas9, e.g., SpCas9, and the PAM sequence is 5'-NGG-3', where "N" is any nucleotide. In some embodiments, the recombinant nuclease is Cas9, e.g., SaCas9, and the PAM sequence is 5'-NGRRT-3', or 5'-NGRRN-3', where "N" is any nucleotide and "R" is a purine, e.g., guanine or adenine. In some embodiments, the recombinant nuclease is Cas9, e.g., NmeCas9, and the PAM sequence is 5'-NNNNGATT-3', where "N" is any nucleotide. In some embodiments, the recombinant nuclease is Cas9, e.g., CjCas9, and the PAM sequence is 5'-NNNNRYAC-3', where "N" is any nucleotide, "R" is a purine, e.g., guanine or adenine, and "Y" is a pyrimidine, e.g., cytosine or thymine. In some embodiments, the recombinant nuclease is Cas9, e.g., StCas9, and the PAM sequence is 5'-NNAGAAW-3', where "N" is any nucleotide and "W" is adenine or thymine.

[0311] 4. Ribonucleoproteins In some embodiments, the site-directed polypeptide (e.g., Cas nuclease) and the genome-targeting nucleic acid (e.g., gRNA or sgRNA) can each be administered separately to a cell or subject. In some embodiments, the site-directed polypeptide can be pre-complexed with one or more guide RNAs or one or more sgRNAs. Such pre-complexed materials are known as ribonucleoprotein particles (RNPs). In some embodiments, the nuclease system comprises a ribonucleoprotein (RNP). In some embodiments, the nuclease system comprises a Cas9 RNP comprising purified Cas9 protein complexed with a gRNA. In some embodiments, the nuclease system comprises a Mad7 RNP comprising purified Mad7 protein complexed with a gRNA. The Cas9 and Mad7 proteins can be expressed and purified by any means known in the art. The ribonucleoproteins can be constructed in vitro and delivered directly to cells using standard electroporation or transfection techniques known in the art.

[0312] B. Homologous Recombination Repair (HDR) In some aspects, the embodiments provided involve targeted integration of a nucleic acid sequence, e.g., a donor template, into a target nucleic acid sequence, e.g., an endogenous gene. In some embodiments, the target nucleic acid molecule is a housekeeping gene. In some embodiments, the housekeeping gene is eukaryotic translation elongation factor 1 alpha (EEF1A), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ubiquitin C (UBC), or actin beta (ACTB). In some embodiments, the target nucleic acid is B2M, TRAC, or SIRPA. In some embodiments, the target nucleic acid is B2M.

[0313] In some embodiments, the DNA repair mechanism can be nuclease-induced after (i) two SSBs, one on each strand, thereby inducing a single-stranded overhang, or (ii) a DSB that occurs at the same break site on both strands, thereby inducing a blunt-end break.

[0314] In some embodiments, HDR is utilized for targeted integration or insertion of a nucleic acid sequence, for example, a donor template, into one or more target nucleic acid molecules (e.g., endogenous genes). In some embodiments, HDR can be used to integrate a donor template that includes a synthetic cytokine receptor (e.g., RACR) into a target nucleic acid molecule (e.g., endogenous gene). For example, HDR can be used to integrate a donor template that encodes RACR into the B2M locus.

[0315] Agents capable of inducing DSBs, such as Cas nucleases (e.g., Cas9), TALENs and ZFNs, facilitate genome editing by inducing DSBs at cleavage sites within target nucleic acid molecules, such as endogenous genes, e.g., B2M, as described in the previous section.

[0316] Agents capable of inducing SSBs, sometimes referred to as nicks, include recombinant nucleases with nickase activity, such as Cas9, such as those described in the previous section. Examples of agents with nickase activity include, for example, Cas9 from Streptococcus pyogenes that includes a mutation selected from the group consisting of D10A, H840A, H854A, and H863A.

[0317] Upon cleavage by one of these agents, a target endogenous gene bearing an SSB or DSB, e.g., B2M, undergoes one of two major pathways for DNA damage repair: (1) the error-prone non-homologous end joining (NHEJ) pathway or (2) the high-fidelity homology-directed repair (HDR) pathway.

[0318] In some embodiments, a cell in which an SSB or DSB has previously been induced by one or more agents including a nuclease is obtained, and a donor template, e.g., an ssODN, is introduced to effect HDR into a target endogenous gene, e.g., B2M, and integration of the donor template.

[0319] Generally, in the absence of a repair template, e.g., a donor template, e.g., a ssODN, the NHEJ process religates the ends of the broken DNA strand, often resulting in the deletion and insertion of nucleotides at the break site.

[0320] The nucleic acid sequence change at the target endogenous locus, for example, the B2M locus, can be caused by HDR by incorporating the exogenously provided donor template encoding synthetic cytokine receptor (for example, RACR).HDR pathway can be caused by canonical HDR pathway or alternative HDR pathway.Unless otherwise specified, the term "HDR" or "homologous recombination repair" used herein encompasses both canonical HDR and alternative HDR.

[0321] Canonical HDR or "canonical homology-directed repair" or cHDR" are used interchangeably and refer to the process of repairing DNA damage using homologous nucleic acid (e.g., endogenous homologous sequence, e.g., sister chromatid, or exogenous nucleic acid, e.g., donor template). Canonical HDR typically operates when there is significant resection at DSB, forming at least one single-stranded portion of DNA. In normal cells, canonical HDR typically involves a series of steps such as recognition of break, stabilization of break, resection, stabilization of single-stranded DNA, formation of DNA crossover intermediate, division of crossover intermediate, and ligation. The canonical HDR process requires RAD51 and BRCA2, and the homologous nucleic acid, e.g., donor template, is typically double-stranded. In canonical HDR, following excision at the break in which a double-stranded polynucleotide, e.g., a double-stranded donor template, is introduced that contains a sequence homologous to the targeted sequence in the target endogenous locus and is either directly integrated into the targeted sequence or used as a template for inserting the sequence or a portion of the sequence of the donor template into the target endogenous gene, e.g., B2M, repair can proceed by various pathways, e.g., the double Holliday junction model (also called the double-strand break repair pathway or DSBR pathway) or by the synthesis-dependent strand annealing (SDSA) pathway.

[0322] In the double Holliday junction model, strand invasion occurs through two single-stranded overhangs of a targeting sequence against a homologous sequence in a double-stranded polynucleotide, such as a double-stranded donor template, forming an intermediate with two Holliday junctions. The junction moves as new DNA is synthesized from the end of the invading strand to fill the gap resulting from resection. The end of the newly synthesized DNA is ligated to the resected end, the junction is split, and the targeting sequence, or the part of the targeting sequence that contains the gene variant, is inserted. Crossover with a polynucleotide, such as a donor template, can occur when the junction is split.

[0323] In the SDSA pathway, a single single-stranded overhang invades a polynucleotide, e.g., a donor template, and new DNA is synthesized from the end of the invading strand to fill the gap resulting from the resection. The newly synthesized DNA then anneals to the remaining single-stranded overhang, new DNA is synthesized to fill the gap, and the strands are ligated to generate a modified DNA duplex.

[0324] Alternative HDR or "alternative homology-directed repair" or "alternative HDR" are used interchangeably and in some embodiments refer to the process of using homologous nucleic acid (e.g., endogenous homologous sequence, e.g., sister chromatid, or exogenous nucleic acid, e.g., donor template) to repair DNA damage.Alternative HDR differs from canonical HDR in that this process utilizes a different pathway from canonical HDR and can be inhibited by RAD51 and BRCA2, which are mediators of canonical HDR.In addition, alternative HDR is also distinguished by the involvement of single-stranded or nicked homologous nucleic acid template, e.g., donor template, whereas canonical HDR generally involves double-stranded homologous template.In alternative HDR pathway, single-stranded template polynucleotide, e.g., donor template, is introduced. A nick, single-strand break or DSB at the desired target site, e.g., the cut site for changing the target endogenous gene, e.g., B2M, is mediated by a nuclease molecule, e.g., any of the nucleases described herein, and excision occurs at the cut to reveal a single-strand overhang.The incorporation of the sequence of a template polynucleotide, e.g., a donor template, for changing the target site of DNA typically occurs by the SDSA pathway, as described herein.

[0325] In some embodiments, HDR is carried out by introducing one or more agents capable of inducing DSB, such as any of those described herein, and donor template, such as ssODN, such as any of those described herein, into cell.Introduction can be carried out by any suitable delivery means, such as any of those described herein.The condition that can cause HDR can be any condition suitable for carrying out HDR in cell.

[0326] In some embodiments, HDR is carried out by introducing one or more agents capable of inducing SSB in each stand, such as any of those described herein, and a donor template, such as ssODN, such as any of those described herein, into cells.The introduction can be carried out by any suitable delivery means, such as any of those described herein.The condition that can cause HDR can be any condition suitable for carrying out HDR in cells.

[0327] Donor Mold In some embodiments, the method provided comprises the use of a donor template, for example, a donor template that encodes a synthetic cytokine receptor, for example, RACR, that is homologous to a portion of the targeting sequence in target gene, for example, B2M.In some embodiments, the targeting sequence is included in the sense strand.In some embodiments, the targeting sequence is included in the antisense strand.In some embodiments, the donor template is also provided for use in the method provided herein, for example, as a template for HDR-mediated integration of the nucleic acid sequence that encodes RACR.

[0328] In some embodiments, the donor template is used with one or more agents capable of inducing DNA breaks, e.g., SSBs or DSBs. In some embodiments, the donor template is used with one or more agents capable of inducing DSBs and guide RNAs, e.g., sgRNAs, to knock in a nucleic acid sequence encoding a synthetic cytokine receptor (e.g., RACR) into a target endogenous locus (e.g., B2M). In some embodiments, the donor template is used with one or more agents capable of inducing SSBs, a first guide RNA, e.g., a first sgRNA, and a second guide RNA, e.g., a second sgRNA, to knock in a nucleic acid sequence encoding a synthetic cytokine receptor (e.g., RACR) into a target endogenous locus (e.g., B2M).

[0329] In some embodiments, the donor template comprises a nucleic acid sequence that is homologous to a cleavage site in a target gene, for example, B2M. In some embodiments, the donor template comprises a nucleic acid sequence that is homologous to a cleavage site in a sense strand of a target gene, for example, B2M. In some embodiments, the donor template comprises a nucleic acid sequence that is homologous to a cleavage site in an antisense strand of a target gene, for example, B2M. In some embodiments, the target gene, for example, B2M, comprises a sense strand and an antisense strand, and the sense strand comprises a targeting sequence. In some embodiments, the target gene, for example, B2M, comprises a sense strand and an antisense strand, and the antisense strand comprises a targeting sequence.

[0330] In some embodiments, the donor template, e.g., ssODN, comprises a nucleic acid sequence that includes a PAM sequence that is homologous to a PAM sequence in the targeting sequence.

[0331] In some embodiments, the donor template is single-stranded. In some embodiments, the donor template is a single-stranded DNA oligonucleotide (ssODN). In some embodiments, the donor template is double-stranded.

[0332] In some embodiments, ssODN comprises 5'ssODN arm and 3'ssODN arm.In some embodiments, 5'ssODN arm is directly connected to 3'ssODN arm.In some embodiments, 5'ssODN arm is homologous to the sequence of target gene immediately upstream of cleavage site, for example, B2M, and 3'ssODN arm is homologous to the sequence of target gene immediately downstream of cleavage site.

[0333] In some embodiments, the 5'ssODN arm and / or the 3'ssODN arm have a length that is 250-750 nucleotides long. In some embodiments, the 5'ssODN arm has a length that is 250-750 nucleotides long. In some embodiments, the 3'ssODN arm has a length that is 250-750 nucleotides long. In some embodiments, each of the 5'ssODN arm and the 3'ssODN arm has a length that is 250-750 nucleotides long. In some embodiments, the 5'ssODN arm and / or the 3'ssODN arm have a length that is about 500 nucleotides long. In some embodiments, the 5'ssODN arm has a length that is about 500 nucleotides long. In some embodiments, the 3'ssODN arm has a length that is about 500 nucleotides long. In some embodiments, each of the 5'ssODN arm and the 3'ssODN arm has a length that is about 500 nucleotides long.

[0334] In some embodiments, the target gene is B2M, and the donor template comprises a nucleic acid sequence that is homologous to a cleavage site in the B2M gene. In some embodiments, the donor template comprises a nucleic acid sequence that is homologous to a cleavage site in the sense strand of the B2M target gene. In some embodiments, the donor template comprises a nucleic acid sequence that is homologous to a cleavage site in the antisense strand of the B2M target gene. In some embodiments, the donor template is a ssODN, and the 5' ssODN arm is homologous to a sequence of the B2M target gene immediately upstream of the cleavage site, and the 3' ssODN arm is homologous to a sequence of the B2M target gene immediately downstream of the cleavage site.

[0335] In some embodiments, the 5' ssODN arm comprises a nucleic acid sequence having at least 80%, at least 85%, 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. In some embodiments, the 5' ssODN arm comprises a nucleic acid sequence set forth in SEQ ID NO: 22.

[0336] In some embodiments, the 3' ssODN arm comprises a nucleic acid sequence having at least 80%, at least 85%, 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.

[0337] In some embodiments, the 5' ssODN arm comprises a nucleic acid sequence set forth in SEQ ID NO:22 and the 3' ssODN arm comprises a nucleic acid sequence set forth in SEQ ID NO:23.

[0338] Also provided herein is an isolated nucleic acid, for example, an isolated nucleic acid for use in a method of knocking in a synthetic cytokine receptor (e.g., RACR) into a target gene (e.g., B2M), comprising a donor template described herein, for example, a ssODN or a portion thereof, for example, or the nucleic acid sequence of either the 5'ssODN arm, or the 3'ssODN arm. In some embodiments, the 5'ssODN comprises the nucleic acid sequence set forth in SEQ ID NO:22. In some embodiments, the 3'ssODN arm comprises the nucleic acid sequence set forth in SEQ ID NO:23.

[0339] In some embodiments, the crRNA 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:18, the 5' ssODN arm comprises a nucleic acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:22, and the 3' ssODN arm 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. In some embodiments, the crRNA 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. In some embodiments, the crRNA comprises a nucleic acid sequence set forth in any one of SEQ ID NO: 18, the 5'ssODN comprises a nucleic acid sequence set forth in SEQ ID NO: 22, and the 3'ssODN comprises a nucleic acid sequence set forth in SEQ ID NO: 23.

[0340] In some embodiments, the donor template, e.g., ssODN, comprises a nucleic acid sequence that encodes a transgene sequence that encodes a synthetic cytokine receptor. In some embodiments, the synthetic cytokine receptor is a rapamycin-activated cytokine receptor (RACR) that is responsive to rapamycin or an analog (e.g., a rapalog). In some embodiments, the transgene sequence is a tandem cassette that encodes both polypeptides of the synthetic cytokine receptor.

[0341] In some embodiments, a transgene encoding a synthetic cytokine receptor (e.g., RACR) can be inserted such that its expression is driven by an endogenous promoter at the integration site, for example, the promoter that drives the expression of the endogenous B2M gene. In some embodiments where the polypeptide encoding sequence is promoterless, the expression of the integrated transgene is then ensured by the transcription driven by the endogenous promoter or other control element in the region of interest. For example, a transgene encoding a part of a synthetic cytokine receptor (e.g., RACR) can be inserted in frame with the coding sequence of an endogenous locus (e.g., B2M locus) without a promoter, so that the expression of the integrated transgene is controlled by the transcription of the endogenous promoter and / or other regulatory element at the integration site. In some embodiments, a multicistronic element, such as a ribosome skipping / self-cleaving element (e.g., a 2A element or internal ribosome entry site (IRES)), is positioned upstream of the transgene such that the multicistronic element is positioned in frame with one or more exons of an endogenous open reading frame at the endogenous locus (e.g., the B2M locus) such that expression of the transgene is operably linked to an endogenous promoter.

[0342] In some embodiments, each nucleic acid encoding a synthetic cytokine receptor polypeptide in a "tandem" cassette is independently controlled by a regulatory element or both as a multicistronic (e.g., bicistronic) expression system. In other embodiments, each nucleic acid encoding a synthetic cytokine receptor polypeptide in a "tandem" cassette can be operably linked to a promoter that can be the same or different. In some embodiments, the nucleic acid molecule can contain a promoter that drives the expression of two or more different polypeptide chains. In some embodiments, such a nucleic acid molecule can be multicistronic (bicistronic or tricistronic, see, e.g., U.S. Pat. No. 6,060,273). In some embodiments, the transcription unit can be engineered as a bicistronic unit that contains an IRES (internal ribosome entry site) that allows for co-expression of gene products by messages from a single promoter. Alternatively, in some cases, a single promoter can direct the expression of an RNA that contains two polypeptides in a single open reading frame (ORF) separated from each other by a sequence that encodes a cleavable linker as described herein. Thus, the ORF encodes a single polypeptide that is processed into individual polypeptide chains during or after translation. In some embodiments, the promoter is selected from among the human elongation factor 1 alpha (EF1α) promoter (e.g., as described in SEQ ID NO:24, 25, or 26). In some embodiments, the promoter is the MND promoter (e.g., as described in SEQ ID NO:27).

[0343] In some embodiments, the donor template, e.g., ssODN, comprises a nucleic acid sequence encoding a synthetic cytokine receptor (e.g., RACR). In some embodiments, the nucleic acid sequence encoding a synthetic cytokine receptor (e.g., RACR) is located between the 5' ssODN arm and the 3' ssODN arm. In some embodiments, the nucleic acid sequence encoding a synthetic cytokine receptor (e.g., RACR) comprises an EF1-alpha promoter (e.g., SEQ ID NO: 24, 25, or 26). In some embodiments, the nucleic acid sequence encoding a synthetic cytokine receptor (e.g., RACR) comprises an MND promoter (e.g., SEQ ID NO: 27). In some embodiments, the synthetic cytokine receptor is a rapamycin-activated cytokine receptor (RACR). The RACR can be any of those described in Section V, etc. In some embodiments, the nucleic acid molecule is a tandem cassette encoding a first polypeptide sequence of RACR and a second polypeptide sequence of RACR.

[0344] In some embodiments, the first nucleic acid sequence encoding RACR comprises a nucleic acid sequence encoding a RACR-gamma chain (e.g., SEQ ID NO:28) and a nucleic acid sequence encoding a RACR-beta chain (e.g., SEQ ID NO:33). In some embodiments, the first nucleic acid sequence encodes a RACR-gamma chain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:28. In some embodiments, the first nucleic acid sequence encodes a RACR-gamma chain sequence set forth in SEQ ID NO:28. In some embodiments, the nucleic acid sequence encoding the RACR-gamma chain further encodes a signal peptide at the N-terminus of the nascent protein to facilitate transport of the protein when expressed. In some embodiments, the signal peptide has a sequence set forth in SEQ ID NO:29. In some embodiments, the second nucleic acid sequence encodes a RACR-beta chain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:33. In some embodiments, the second nucleic acid sequence encodes a RACR-beta chain set forth in SEQ ID NO:33. In some embodiments, the nucleic acid sequence encoding the RACR-beta chain further encodes a signal peptide at the N-terminus of the nascent protein to facilitate transport of the protein when expressed. In some embodiments, the signal peptide has a sequence set forth in SEQ ID NO:34.

[0345] In some embodiments, the first nucleic acid sequence encoding the RACR-gamma chain 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. In some embodiments, the first nucleic acid sequence encoding the RACR-gamma chain has the sequence set forth in SEQ ID NO:37. In some embodiments, the second nucleic acid sequence encoding the RACR-beta chain 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 second nucleic acid sequence encoding the RACR-beta chain is set forth in SEQ ID NO:38.

[0346] In some embodiments, the nucleic acid sequence encoding the RACR-gamma chain and the nucleic acid sequence encoding the RACR-beta chain are separated by a nucleic acid sequence encoding a cleavable linker. In some embodiments, a further nucleic acid sequence encoding a cleavable linker is located downstream of the nucleic acid sequence encoding the RACR-beta chain.

[0347] In some embodiments, the linker is a protein quantification reporter linker (PQR; e.g., SEQ ID NO:42), including any described in Canadian Patent Application No. CA2970093, which is incorporated by reference in its entirety. In some embodiments, the PQR linker has a sequence set forth in SEQ ID NO:42. In some embodiments, the PQR linker is encoded by the nucleotide sequence set forth in SEQ ID NO:41.

[0348] In some embodiments, the cleavable linker is a self-cleaving peptide, such as a 2A ribosomal skip element. In some cases, the cleavable linker, for example, T2A, can cause ribosomes to skip the synthesis of the peptide bond at the C-terminus of the 2A element (ribosomal skip), resulting in a separation between the end of the 2A sequence and the next downstream peptide (see, for example, de Felipe. Genetic Vaccines and Ther. 2:13 (2004) and deFelipe et al. Traffic 5:616-626 (2004)). Many 2A elements are known. Examples of 2A sequences that can be used in the methods and nucleic acids disclosed herein include, but are not limited to, 2A sequences from foot and mouth disease virus (F2A, e.g., SEQ ID NO:43), equine rhinitis A virus (E2A, e.g., SEQ ID NO:44), Thosea asigna virus (T2A, e.g., SEQ ID NO:45 or 46) and porcine teschovirus-1 (P2A, e.g., SEQ ID NO:47 or 48), as described in U.S. Patent Application Publication No. 20070116690.

[0349] In some embodiments, due to a cleavable element located between the first and second nucleic acid sequences, expression of the nucleic acid sequence encoding RACR produces a first peptide (i.e., RACR-gamma chain) and a separate second peptide (i.e., RACR-beta chain).

[0350] In some embodiments, the transgene sequence may also include sequences required for transcription termination and / or polyadenylation signals. In some aspects, exemplary polyadenylation signals are selected from SV40, hGH, BGH and rbGlob transcription termination and / or polyadenylation signals. In some embodiments, the transgene includes an SV40 polyadenylation signal. In some embodiments, the transcription termination and / or polyadenylation signal, if present in the transgene, is typically the 3'-most sequence in the transgene and is linked to one of the homology arms. In some embodiments, the transgene sequence includes the polyadenylation sequence set forth in SEQ ID NO:39.

[0351] In some embodiments, the ssODN comprises, in order, a 5' ssODN arm, an EF1-alpha promoter, a nucleic acid sequence encoding a RACR-gamma chain, a nucleic acid sequence encoding a cleavable linker (e.g., a PQR linker), a nucleic acid sequence encoding a RACR-beta chain, a polyA sequence, and a 3' ssODN arm.

[0352] In some embodiments, the ssODN comprises a 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 ssODN is set forth in SEQ ID NO:40.

[0353] In some embodiments, after the integration of ssODN into the target gene, the target gene is knocked out. In some embodiments, the target gene is human B2M, and after the integration of ssODN into B2M, B2M is knocked out. In some embodiments, the nucleic acid sequence encoding a synthetic cytokine receptor is integrated into the B2M locus. In some embodiments, the engineered iPSCs and CILs have a modified B2M locus, in which the endogenous B2M gene is genetically disrupted by knocking out the B2M gene and knocking in the nucleic acid encoding a synthetic cytokine receptor by targeted integration. In some embodiments, the synthetic cytokine receptor is RACR encoded by a nucleic acid sequence that contains, in order, an EF1-alpha promoter, a nucleic acid sequence encoding a RACR-gamma chain, a nucleic acid sequence encoding a cleavable linker (e.g., a PQR linker), a nucleic acid sequence encoding a RACR-beta chain, and a polyA sequence. In some embodiments, the nucleic acid sequence encoding RACR integrated into the B2M locus has a sequence set forth in SEQ ID NO:32 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:32. In some embodiments, the nucleic acid encoding RACR integrated into the B2M locus is set forth in SEQ ID NO:32.

[0354] VII. Chimeric Antigen Receptors In some cases, the engineered lymphocytes, e.g., CIL cells, of the present disclosure may comprise or be transduced with a polynucleotide encoding a chimeric antigen receptor (CAR), thereby generating lymphocytes, e.g., CIL cells, that express a CAR.

[0355] In some embodiments, the present disclosure contemplates a chimeric antigen receptor (CAR) system for use in treating a subject with cancer. In some embodiments, the engineered lymphocytes of the present disclosure, such as CIL cells, contain a CAR sequence (CAR-CIL cells or CAR-iCIL cells).

[0356] In some embodiments, lymphocytes, such as CIL cells, are engineered to express a CAR construct by transfecting the cell population with an expression vector encoding the CAR construct. Illustrative examples of cell populations that can be transfected include HSCs, blood progenitor cells, common lymphoid progenitor cells, or CIL cells. Suitable means for preparing a transduced population of lymphocytes, e.g., CIL cells, expressing a selected CAR construct are well known to those skilled in the art, and include, to name a few, retroviruses, lentiviruses (viral-mediated CAR gene delivery systems), Sleeping Beauty, and Piggyback (transposon / transposase systems, including non-viral-mediated CAR gene delivery systems). In some embodiments, any of the transduction methods contemplated in this disclosure may be used to generate CAR-expressing CIL cells.

[0357] A. Targeting Agents for CAR Traditionally, CAR is generated by fusing a polynucleotide encoding VL, VH or scFv to the 5' end of a polynucleotide encoding a transmembrane domain and an intracellular domain, and then transducing the polynucleotide and corresponding VH or VL into cells as necessary. Many variations of CAR are known in the art, and the present disclosure contemplates the use of any of the known variations. Furthermore, VL / VH pairs and scFvs for a myriad of haptens are known in the art or can be routinely generated by conventional methods. Thus, the present disclosure contemplates the use of any known hapten binding domain.

[0358] Various methods of targeting CARs and CAR-expressing cells have been described in the art, including, for example, US Patent No. 2020 / 0123224, the disclosure of which is incorporated herein by reference. For example, fluorescein or fluorescein isothiocyanate (FITC) moieties can be conjugated to an agent that binds to the desired target cells (such as cancer cells), whereby CAR-CIL cells expressing anti-fluorescein / FITC chimeric antigen receptors can selectively target the target cells labeled by the conjugate. In a variant, other haptens recognized by CARs may be used instead of fluorescein / FITC. CARs can be generated using various scFv sequences known in the art, or scFv sequences generated by conventional routine methods. Further exemplary scFv sequences for fluorescein / FITC, and other haptens, are provided, for example, in International Publication WO 2021 / 076788, the disclosure of which is incorporated herein by reference.

[0359] In some embodiments, the CAR system of the present disclosure utilizes a CAR that targets a moiety that is not produced or expressed by the cells of the subject being treated. Thus, this CAR system allows for focused targeting of CIL cells to target cells, such as cancer cells. By administering small conjugate molecules with CAR-expressing cells, such as CAR-expressing CIL cells, the cellular response of the engineered cells can only target cells that express the tumor receptor, thereby reducing off-target toxicity, and the activation of engineered lymphocytes, e.g., CIL cells, can be more easily controlled due to the rapid clearance of small conjugate molecules. As an additional advantage, CAR-expressing lymphocytes, such as CAR-expressing CIL cells, can be used as "universal" cytotoxic cells to target a wide variety of tumors without the need to prepare separate CAR constructs. The target moiety recognized by the CAR can also remain constant. Only the ligand portion of the small conjugate molecule needs to be changed to allow the system to target cancer cells of different identities.

[0360] In one aspect, the present disclosure provides an illustration of this conjugate molecule / CAR system.

[0361] In some embodiments, the CAR system of the present disclosure utilizes a conjugate molecule as a bridge between CAR-expressing cells and target cancer cells.The conjugate molecule is a conjugate that includes a hapten and a cell targeting moiety, such as any suitable tumor cell-specific ligand.Exemplary haptens that can be recognized and bound by CAR include FITC (fluorescein isothiocyanate), NHS-fluorescein, and fluorescein and its derivatives, including pentafluorophenyl ester (PFP) and tetrafluorophenyl ester (TFP) derivatives, knottin, centyrin, and DARPin, as well as low molecular weight organic molecules such as DNP (2,4-dinitrophenol), TNP (2,4,6-trinitrophenol), biotin, and digoxigenin. Suitable cell targeting moieties that may themselves act as haptens for CAR include knottins (see Kolmar H. et al., The FEBS Journal. 2008. 275(11):26684-90), centrins and DARPins (see Reichert, JMMAbs 2009. 1(3):190-209).

[0362] In some embodiments, the cell targeting moiety has nanomolar affinity (K D = 14nM; see Kularatne, SA et al., Mol Pharm. 2009.6(3):780-9), a ligand bound by PSMA-positive human prostate cancer cells (DUPA-(99m)Tc). In one embodiment, the DUPA derivative can be a ligand of a small molecule ligand linked to a targeting moiety, and the DUPA derivatives are described in International Publication No. WO 2015 / 057852, which is incorporated herein by reference.

[0363] In some embodiments, the cell targeting moiety is a CCK2R ligand that is a ligand bound by CCK2R-positive cancer cells (e.g., thyroid, lung, pancreatic, ovarian, brain, gastric, gastrointestinal stromal and colon cancers; see Wayua. C. et al., Molecular Pharmaceutics. 2013. ePublication).

[0364] In some embodiments, the cell targeting moiety is folate, folic acid or an analog thereof, a ligand bound by folate receptors on cells of cancer, including ovarian, cervical, endometrial, lung, renal, brain, breast, colon and head and neck cancers. See Sega, EI et al., Cancer Metastasis Rev. 2008.27(4):655-64).

[0365] In some embodiments, the cell targeting moiety is an NK-1R ligand.Receptors for NK-1R ligand are found, for example, on colon and pancreatic cancers.In some embodiments, the NK-1R ligand can be synthesized according to the method disclosed in International Patent Application No. PCT / US2015 / 044229, which is incorporated herein by reference.

[0366] In some embodiments, the cell targeting moiety can be a peptide ligand, for example, the ligand can be a peptide ligand that is an endogenous ligand for the NK1 receptor. In some embodiments, the small conjugate molecule ligand can be a regulatory peptide that belongs to the family of tachykinins that target tachykinin receptors. Such regulatory peptides include substance P (SP), neurokinin A (substance K) and neurokinin B (neuromedin K) (see Hennig et al., International Journal of Cancer: 61, 786-792).

[0367] In some embodiments, the cell targeting moiety is a CAIX ligand. For example, the receptor for CAIX ligand found in renal, ovarian, vulvar and breast cancer. CAIX ligand may also be referred to herein as CA9.

[0368] In some embodiments, the cell targeting moiety is a ligand of gamma glutamyl transpeptidase, which is overexpressed in, for example, ovarian cancer, colon cancer, hepatoma, astroblastoma, melanoma, and leukemia.

[0369] In some embodiments, the cell targeting moiety is a CCK2R ligand, a receptor for CCK2R found in cancers of the thyroid, lung, pancreas, ovary, brain, stomach, gastrointestinal stromal and colon, among others.

[0370] In one embodiment, a cell targeting moiety may have a mass of less than about 10,000 daltons, less than about 9000 daltons, less than about 8,000 daltons, less than about 7000 daltons, less than about 6000 daltons, less than about 5000 daltons, less than about 4500 daltons, less than about 4000 daltons, less than about 3500 daltons, less than about 3000 daltons, less than about 2500 daltons, less than about 2000 daltons, less than about 1500 daltons, less than about 1000 daltons or less than about 500 daltons. In another embodiment, the small molecule ligand can have a mass of about 1 to about 10,000 daltons, about 1 to about 9000 daltons, about 1 to about 8,000 daltons, about 1 to about 7000 daltons, about 1 to about 6000 daltons, about 1 to about 5000 daltons, about 1 to about 4500 daltons, about 1 to about 4000 daltons, about 1 to about 3500 daltons, about 1 to about 3000 daltons, about 1 to about 2500 daltons, about 1 to about 2000 daltons, about 1 to about 1500 daltons, about 1 to about 1000 daltons, or about 1 to about 500 daltons.

[0371] In an exemplary embodiment, the linkage in the conjugates described herein can be a direct linkage (e.g., reaction between an isothiocyanate group of FITC and a free amine group of a small molecule ligand), or the linkage can be via an intermediate linker. In an embodiment, the intermediate linker, if present, can be any biocompatible linker known in the art, such as a bivalent linker. In an exemplary embodiment, the bivalent linker can include about 1 to about 30 carbon atoms. In another exemplary embodiment, the bivalent linker can include about 2 to about 20 carbon atoms. In other embodiments, even lower molecular weight bivalent linkers (i.e., those having an approximate molecular weight of about 30 to about 300 Da) are used. In another embodiment, suitable linker lengths include, but are not limited to, linkers having 2, 3, 4, 5, 6, 7, 8, 9. 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37. 38, 39, or 40 or more atoms.

[0372] In some embodiments, the hapten and cell targeting moiety can be directly conjugated by means such as the reaction between the isothiocyanate group of FITC and the free amine group of a small ligand (e.g., folate, DUPA, and CCK2R ligand). However, the use of a linking domain to connect the two molecules can be useful as it can provide flexibility and stability. Examples of suitable linking domains include: 1) polyethylene glycol (PEG); 2) polyproline; 3) hydrophilic amino acids; 4) sugars; 5) non-natural peptidoglycans; 6) polyvinylpyrrolidone; 7) Pluronic F-127. Suitable linker lengths include, but are not limited to, linkers having 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 or more atoms.

[0373] In some embodiments, the linker can be a bivalent linker, which can include one or more spacers.

[0374] An exemplary conjugate of the present disclosure is FITC-Folate. The file is TIFF2025500894000023.tif39128. An exemplary conjugate of the present disclosure is FITC-CA9 The file is TIFF2025500894000024.tif31128.

[0375] Exemplary conjugates of the present disclosure include the following molecules: FITC-(PEG)- 12 -Folate, FITC-(PEG)- 20 -Folate, FITC-(PEG)- 108 -Folate, FITC-DUPA, FITC-(PEG) 12 -DUPA, FITC-CCK2R ligand, FITC-(PEG) 12 -CCK2R ligand, FITC-(PEG) 11 -NK1R ligand, and FITC-(PEG)2-CA9.

[0376] The binding affinity of the ligand and the cancer cell receptor can vary, and in some cases lower affinity binding may be preferred (such as about 1 μM), but the binding affinity of the ligand and the cancer cell receptor is generally at least about 100 μM, at least about 1 nM, at least about 10 nM or at least about 100 nM, preferably at least about 1 pM or at least about 10 pM, and even more preferably at least about 100 pM.

[0377] Examples of conjugates and methods of making them are provided in U.S. Patent Applications US 2017 / 0290900, US 2019 / 0091308 and US 2020 / 0023009, all of which are incorporated by reference herein.

[0378] B. CAR Constructs In some embodiments, the CAR construct contains an extracellular binding moiety, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the intracellular signaling domain contains a costimulatory signaling domain and / or an activating signaling domain. In some embodiments, the CAR construct contains an extracellular binding moiety, a transmembrane domain, and an intracellular signaling domain comprising a costimulatory signaling domain. In some embodiments, the CAR construct contains an extracellular binding moiety, a transmembrane domain, and an intracellular signaling domain comprising an activating signaling domain. In some embodiments, the CAR construct contains an extracellular binding moiety, a transmembrane domain, and an intracellular signaling domain comprising a costimulatory signaling domain and an activating signaling domain.

[0379] In any of the embodiments described herein, the binding portion of the CAR can be, for example, a single chain fragment variable region (scFv), Fab, Fv, Fc, or (Fab')2 fragment of an antibody, etc. The use of unaltered (i.e., full-size) antibodies such as IgG, IgM, IgA, IgD, or IgE in or as a CAR is excluded from the scope of the present invention.

[0380] In some embodiments, the costimulatory domain serves to enhance lymphocyte proliferation and survival when the CAR binds to the targeting moiety. The identity of the costimulatory domain is limited only in that it has the ability to enhance cell proliferation and survival activation upon the binding of the targeting moiety by the CAR. Suitable costimulatory domains include, but are not limited to, CD28 (see, e.g., Alvarez-Vallina, L. et al., Eur J Immunol. 1996.26(10):2304-9); CD137 (4-1BB), a member of the tumor necrosis factor (TNF) receptor family (see, e.g., Imai, C. et al., Leukemia. 2004.18:676-84); and CD134 (OX40), a member of the TNFR superfamily of receptors (see, e.g., Latza, U. et al., Eur. J. Immunol. 1994.24:677). One of skill in the art will understand that sequence variants of these costimulatory domains can be used and will have the same or similar activity as the domains from which they are modeled. In various embodiments, such variants have at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity to the amino acid sequence of the domain from which they are derived.

[0381] In some embodiments of the invention, the CAR construct comprises two costimulatory domains. Specific combinations include all possible variations of the four described domains, but specific examples include 1) CD28+CD137 (4-1BB), and 2) CD28+CD134 (OX40).

[0382] In some embodiments, the activation signaling domain serves to activate cells when the CAR binds to the targeting moiety. The identity of the activation signaling domain is limited only in that it has the ability to induce activation of the selected cell upon binding of the targeting moiety by the CAR. Suitable activation signaling domains include CD3 zeta chain and Fc receptor gamma. Those skilled in the art will understand that sequence variants of these described activation signaling domains can be used without adversely affecting the invention, and the variants have the same or similar activity as the domains they are modeled on. Such variants can have at least about 80%, at least about 90%, at least about 95%. At least about 97%, at least about 98%, at least about 99% or at least about 99.5% sequence identity to the amino acid sequence of the domain from which they are derived.

[0383] In some embodiments, CARs may contain additional elements, such as a signal peptide to ensure proper transport of the fusion protein to the cell surface, a transmembrane domain to ensure that the fusion protein is maintained as an integral membrane protein, and a hinge domain that confers flexibility to the recognition region and allows for strong binding to the target moiety.

[0384] Exemplary CAR constructs suitable for CAR engineered lymphocytes, e.g., CAR-CIL cells, are provided below: (1) scFv-CD8 TM -4-1BB IC -CD3ζ (see, e.g., Liu E, Tong Y, Dotti G, et al., Leukemia. 2018;32:520-531); (2) scFv-CD28 TM+IC-CD3ζ (e.g., Han J, Chu J, Keung CW et al., Sci Rep.2015;5:11483; Kruschinski A, Moosmann A, Poschke I et al., Proc Natl Acad Sci US A.2008;105:17481-17486; and Chu J, Deng Y, Benson DM et al.,Leukemia.2014;28:917-927); (3) scFv-DAP12 TM+IC (see, e.g., Muller N, Michen S, Tietze S et al., J Immunother. 2015;38:197-210); (4) scFv-CD8 TM -2B4 IC -CD3ζ (see e.g. Xu Y, Liu Q, Zhong M et al.,J Hematol Oncol.2019;12:49); (5) scFv-2B4 TM+IC -CD3ζ (see, e.g., Altvater B, Landmeier S, Pscherer S et al., Clin Cancer Res. 2009;15:4857-4866); (6)scFv-CD28 TM+IC -4-1BB IC -CD3ζ (see, e.g., Kloss S, Oberschmidt O, Morgan M et al., Hum Gene Ther. 2017;28:897-913); (7)scFv-CD16 TM -2B4 IC -CD3ζ (see, e.g., Li Y, Hermanson DL, Moriarity BS, Kaufman DS, Cell Stem Cell. 2018;23:181-192); (8)scFv-NKp44 TM -DAP10 IC -CD3ζ (see, e.g., Li Y, Hermanson DL, Moriarity BS, Kaufman DS, Cell Stem Cell. 2018;23:181-192); (9)scFv-NKp46 TM -2B4 IC -CD3ζ (see, e.g., Li Y, Hermanson DL, Moriarity BS, Kaufman DS, Cell Stem Cell. 2018;23:181-192); (10)scFv-NKG2D TM -2B4 IC -CD3ζ (see, e.g., Li Y, Hermanson DL, Moriarity BS, Kaufman DS, Cell Stem Cell. 2018;23:181-192); (11)scFv-NKG2D TM -4-1BB IC -CD3ζ (see, e.g., Li Y, Hermanson DL, Moriarity BS, Kaufman DS, Cell Stem Cell. 2018;23:181-192); (12)scFv-NKG2D TM -2B4 IC -DAP12 IC -CD3ζ (see, e.g., Li Y, Hermanson DL, Moriarity BS, Kaufman DS, Cell Stem Cell. 2018;23:181-192); (13)scFv-NKG2D TM -2B4 IC -DAP10 IC -CD3ζ (see, e.g., Li Y, Hermanson DL, Moriarity BS, Kaufman DS, Cell Stem Cell. 2018;23:181-192); (14)scFv-NKG2D TM -4-1BB IC -2B4 IC -CD3ζS (see, e.g., Li Y, Hermanson DL, Moriarity BS, Kaufman DS, Cell Stem Cell. 2018;23:181-192); and (15)scFv-NKG2D TM-CD3ζS (see, e.g., Li Y, Hermanson DL, Moriarity BS, Kaufman DS, Cell Stem Cell. 2018;23:181-192).

[0385] In some embodiments, the binding portion of the CAR can be directed to any antigen that is desirable to target, such as because of its overexpression on a cell or its association with a disease or condition, such as cancer.

[0386] In some embodiments, the binding portion of the CAR is specific for a tumor antigen. The choice of antigen binding domain depends on the particular type of cancer to be treated. Tumor antigens are well known in the art and include, for example, glioma-associated antigen, carcinoembryonic antigen (CEA), EGFRvIII, IL-11Ra, IL-13Ra, EGFR, FAP, B7H3, Kit, CA LX, CS-1, MUC1, BCMA, bcr-abl, HER2, β-human chorionic gonadotropin, alpha fetoprotein (AFP), ALK, CD19, CD123, cyclin B1, lectin-reactive AFP, Fos-related antigen 1, ADRB3, thyroglobulin, EphA2, RAGE-1, RU1, RU2, SSX2, AKAP-4, LCK, OY-TES1, PAXS, SART3, CLL-1, fucosyl GM1, GloboH, MN-CA IX, EPCAM, EVT6-AML, TGS5, human telomerase reverse transcriptase, plysialic acid acid), PLAC1, RU1, RU2(AS), intestinal carboxylesterase, lewisY, sLe, LY6K, mut hsp70-2, M-CSF, MYCN, RhoC, TRP-2, CYPIBI, BORIS, prostase, prostate-specific antigen (PSA), PAX3, PAP, NY-ESO-1, LAGE-la, LMP2, NCAM, p53, p53 mutant, Ras mutant, gplOO, prostein, OR51E2, PANX3, PSMA, PSCA, Her2 / neu, hTERT, HMWMAA, HAVCR1, VEGFR2, PDGFR-beta, survivin and telomerase, legumain, HPV E6, E7, sperm protein 17, SSEA-4, tyrosinase, TARP, WT1, prostate cancer tumor antigen-1 (PCTA-1), ML-IAP, MAGE, MAGE-A1, MAD-CT-1, MAD-CT-2, MelanA / MART 1, XAGE1, ELF2M, ERG(TMPRSS2ETS fusion gene), NA17, neutrophil elastase, sarcoma translocation breakpoints, NY-BR-1, ephnnB2, CD20, CD22, CD24, CD30, CD33, CD38, CD44v6, CD97, CD171, CD179a, androgen receptor, FAP, insulin growth factor (IGF)-I, IGFII, IGF-I receptor, GD2, o-acetyl-GD2, GD3, GM3, GPRCSD, GPR20, CXORF61, folate receptor (FRa), folate receptor beta, ROR1, Flt3, TAG72, TN Ag, Tie 2, TEM1, TEM7R, CLDN6, TSHR, UPK2 and mesothelin. Non-limiting examples of tumor antigens include differentiation antigens such as tyrosinase, TRP-1, TRP-2, and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pi 5; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor suppressor genes such as p53, Ras, HER-2 / neu; unique tumor antigens resulting from chromosomal translocations; such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens such as Epstein-Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7. Other large protein antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, IL13Ra2, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB\70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-related protein, TAAL6, TAG72, TLP, TPS, GPC3, MUC16, LMP1, EBMA-1, BARF-1, CS1, CD319, HER1, B7H6, L1CAM, IL6, and MET.

[0387] Those skilled in the art are easily familiar with CARs for various tumor antigens. Any one of such CARs can be used as CAR. A number of CARs have been approved by FDA, including but not limited to anti-CD19 CAR T cells and anti-BCMA CAR T cells, such as tisagenlecleucel (Kymriah), axicabtagene silolucel (Yescarta), brexcabtagene autolucel (Tecartus), lysocabtagene maralucel (Breyanzi) or idecabtagene bicleucel (Abecma). It is within the level of a person skilled in the art to generate similar constructs to specifically target desired tumor antigens.

[0388] In some embodiments, the binding portion of the CAR can be directed to a universal antigen that targets a wide variety of tumors without the need to prepare separate CAR constructs. The targeting portion recognized by the CAR can also remain constant. In some embodiments, a ligand can be administered to a subject to allow interaction with target cells and with the binding portion of the CAR. Only the ligand portion of the small conjugate molecule needs to be changed to allow the system to target cancer cells of different identities. Exemplary universal CAR systems are described in the above section.

[0389] In some embodiments, the CAR is an anti-FITC CAR, and the ligand is composed of a fluorescein moiety or a fluorescein isothiocyanate (FITC) moiety conjugated to an agent that binds to a desired target cell (such as a cancer cell). Exemplary ligands are described in the above section. In some embodiments, the ligand is FITC-folate.

[0390] An exemplary CAR of the present disclosure is shown in FIG. 8, where the fusion protein is encoded by a lentiviral expression vector, "SP" is the signal peptide, the CAR is an anti-FITC CAR, the CD8α hinge is present, the transmembrane domain is present ("TM"), the costimulatory domain is 4-1BB, and the activation signaling domain is CD3ζ.

[0391] An exemplary nucleotide sequence encoding a CAR can include SEQ ID NO:13. TIFF2025500894000025.tif231146

[0392] An exemplary CAR amino acid sequence can include SEQ ID NO:14. TIFF2025500894000026.tif77147

[0393] An exemplary nucleotide insert can include SEQ ID NO:15. TIFF2025500894000027.tif230147

[0394] In some embodiments, the CAR can be encoded by a nucleic acid sequence that encodes a signal peptide for signaling the trafficking of the CAR within the cell. It is understood that the signal peptide is typically removed from the protein.

[0395] An exemplary CAR amino acid sequence without the signal peptide can include SEQ ID NO:16. TIFF2025500894000028.tif70147

[0396] An exemplary CAR amino acid sequence signal peptide can include SEQ ID NO:17. TIFF2025500894000029.tif4128

[0397] In various embodiments, a CAR expressing cell is provided that comprises a nucleic acid of SEQ ID NO:13 or 15. In some embodiments, a chimeric antigen receptor polypeptide comprising SEQ ID NO:14 is contemplated. In some embodiments, a chimeric antigen receptor polypeptide comprising SEQ ID NO:16 is contemplated. In some embodiments, a vector comprising SEQ ID NO:13 or 15 is contemplated. In some embodiments, a lentiviral vector comprising SEQ ID NO:13 or 15 is contemplated. In some embodiments, SEQ ID NO:14 may comprise or consist of a human or humanized amino acid sequence. In some embodiments, SEQ ID NO:16 may comprise or consist of a human or humanized amino acid sequence.

[0398] In some embodiments, variant nucleic acid or amino acid sequences having at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99% or at least about 99.5% sequence identity to SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15 or SEQ ID NO:16 are contemplated.

[0399] Although the affinity with which a CAR expressed by a lymphocyte binds to a target moiety can vary, and in some cases lower affinity binding may be preferred (such as about 50 nM), the binding affinity of a CAR to a target ligand is generally at least about 100 nM, at least about 1 pM or at least about 10 pM, preferably at least about 100 pM, at least about 1 fM or at least about 10 fM, and even more preferably at least about 100 fM.

[0400] VII. Characterization of iCIL cells expressing synthetic cytokine receptors In some embodiments, the CIL cells are CD3-CD5-, CD16+, CD56+, CD57+, NKp30+, NKp46+, NKG2A+, and / or NKG2D+.

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

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

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

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

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

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

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

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

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

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

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

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

[0413] K562 cells are a sensitive target for in vitro cytotoxic innate lymphoid cell cytotoxicity assay. In this assay, CIL cells are co-incubated at various ratios with K562 target tumor cells, which are known to be sensitive to CIL cell-mediated cytotoxicity. Target cells (K562) are pre-labeled with fluorescent dyes to allow differentiation from effector cells (CIL cells). After an incubation period, killed target cells are identified by a nucleic acid stain that specifically permeates dead cells. The death percentage is calculated by comparing the total number of viable cells in each experimental assay well with non-effector control wells. As used herein, the term "activity" refers to a measurement of the cytotoxicity ability of CIL cells against target cells.

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

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

[0416] In some embodiments, 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.

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

[0418] As described above, provided herein are hematopoietic stem cells or hematopoietic progenitor cells that can be differentiated into lymphoid cells using synthetic cytokine receptor complexes activated by non-physiological ligands, and differentiated cells produced from those stem cells or progenitor cells, for use in medical treatment. The differentiated cells can be, without limitation, iCIL cells. Advantages of the embodiment can include the ability to generate effector cells expressing synthetic cytokine receptor complexes activated by non-physiological ligands in relation to an abundant cell source (e.g., induced pluripotent stem cells), such that the proliferation of effector cells in a patient can be controlled by administration or cessation of administration of the non-physiological ligand. Other advantages of the embodiment include, without limitation, the ability to generate effector cells from source cells in a medium that is substantially free of cytokines, such as IL-2, IL-7 and / or IL-15, that are conventionally used in the art for CIL cell differentiation.

[0419] CIL cells may be generated from multiple sources, illustrative examples include iPSC, PBMC or UCB. In some embodiments, CIL source cells are autologous cells. In some embodiments, CIL source cells are allogeneic cells. In some embodiments, CIL source cells are xenogeneic cells. For example, allogeneic cells may be used when the subject being treated with the composition of the present disclosure is undergoing high-dose chemotherapy or radiation therapy to destroy the subject's immune system.

[0420] In some embodiments, hematopoietic stem cells can be engineered to express synthetic cytokine receptors. In some embodiments, blood progenitor (white blood) cells 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.

[0421] In some embodiments, the non-physiological ligand may induce differentiation in addition to or in place of exogenous cytokines, hi some embodiments, the non-physiological ligand may induce differentiation during one or more of mesoderm formation, hematopoietic specification, lymphoid progenitor differentiation, CIL cell differentiation.

[0422] In some embodiments, a non-physiological ligand may be contacted with differentiating cells that require an IL-7 and / or IL-15 signal.

[0423] In some embodiments, a non-physiological ligand may be contacted with cells undergoing expansion that require an IL-2 signal.

[0424] In some embodiments, engineering cells to express synthetic cytokine receptors and activating the receptors with non-physiological ligands allows for the generation of CIL cells that can be differentiated and / or expanded without the use of exogenous factors.

[0425] Because it is well known in the relevant art that differentiation and / or expansion of CIL cells is dependent on the presence of various exogenous stimuli, the differentiation and / or expansion of engineered CIL cells as described herein without one, two or all of IL-2, IL-15 and IL-7 is surprising and unexpected.

[0426] In some embodiments, the engineered common lymphoid precursors are differentiated into CIL cells without IL-15 in the differentiation medium.

[0427] In some embodiments, the engineered common lymphoid precursors are differentiated into CIL cells without IL-7 in the differentiation medium.

[0428] In some embodiments, the engineered common lymphoid precursors are differentiated into CIL cells without IL-15 or IL-7 in the differentiation medium.

[0429] In some embodiments, common lymphoid precursors are engineered to express a synthetic cytokine receptor, such as RACR, and differentiated into CIL cells with a rapalog in the differentiation medium and without IL-15 or IL-7 in the differentiation medium.

[0430] In some embodiments, the engineered CIL cells are expanded without IL-2 in the expansion medium.

[0431] In some embodiments, CIL cells are engineered to express a synthetic cytokine receptor, such as RACR, and expanded with the rapalog in expansion medium and without recombinant cytokine in expansion medium.

[0432] In some embodiments, the CIL cell expansion step is carried out in a cell culture medium that is substantially free of recombinant cytokines.

[0433] In some embodiments, the expansion step is performed in a feeder-free cell culture.

[0434] 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.

[0435] In some embodiments, a method is provided for administering to a subject an effective amount of a non-physiological ligand, wherein the non-physiological ligand differentiates common lymphoid progenitor cells into CIL cells according to any of the preceding embodiments.

[0436] In some aspects, a method is provided for administering to a subject an effective amount of a non-physiological ligand, where the non-physiological ligand causes proliferation of cells according to any of the previous aspects.

[0437] In some embodiments, the non-physiological ligand is present or provided in an amount effective to cause expansion of CIL cells ex vivo.

[0438] In some embodiments, the non-physiological ligand is present or provided in an amount effective to induce CIL cell cytokine secretion ex vivo.

[0439] In some embodiments, the non-physiological ligand is present or provided in an amount effective to induce CIL cell secretion of CD107a, interferon gamma (IFNγ) and / or tumor necrosis factor alpha (TNF-α) ex vivo.

[0440] In some embodiments, the non-physiological ligand is present or provided in an amount effective to cause tumor cell killing.

[0441] In some embodiments, the non-physiological ligand is present or provided in a therapeutically effective amount.

[0442] IX. METHODS OF TREATING SUBJECTS WITH THE DISCLOSED COMPOSITIONS The present disclosure provides a method of treating a subject in need thereof using the compositions, therapeutic compositions, cells, vectors and polynucleotides disclosed herein.In some embodiments, the present disclosure provides a method of treating cancer and / or killing cancer cells in a subject, comprising administering to the subject a therapeutically effective amount of the disclosed cells.Also provided is a method of treating tumor and / or killing tumor cells in a subject, comprising administering to the subject an effective amount of a non-physiological ligand, wherein the non-physiological ligand causes the cells to proliferate according to any of the above-mentioned embodiments.

[0443] In some embodiments, the malignancy is a solid tumor, a sarcoma, a carcinoma, a lymphoma, multiple myeloma, Hodgkin's disease, non-Hodgkin's lymphoma (NHL), primary mediastinal large B-cell lymphoma (PBMC), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), transformed follicular lymphoma, splenic marginal zone lymphoma (SMZL), chronic or acute leukemia, acute myeloid leukemia, chronic myelogenous leukemia, acute lymphoblastic leukemia (ALL) (including non-T-cell ALL), chronic lymphocytic leukemia (CLL), T-cell lymphoma, B-cell acute lymphoid leukemia ("BALL"), T-cell acute lymphoid leukemia ("TALL"). , acute lymphoid leukemia (ALL), chronic myelogenous leukemia (CML), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell follicular lymphoma or large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, myelodysplastic and myelodysplastic syndromes, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's hypergammaglobulinemia, plasma cell proliferative disorders (e.g., asymptomatic myeloma, myeloma (smoldering multiple myeloma or indolent myeloma), monoclonal gammopathy of undetermined significance (MGUS), plasmacytoma (e.g., plasma cell dysplasia, solitary myeloma, solitary plasmacytoma, extramedullary plasmacytoma, and multiple plasmacytoma), systemic amyloid light chain amyloidosis, POEMS syndrome (also known as Crow-Fukase syndrome, Takatsuki disease, and PEP syndrome), or a combination thereof.

[0444] In some embodiments, the methods disclosed herein may be used to treat cancer and / or kill cancer cells in a subject by administering a therapeutically effective amount of cells according to any of the preceding embodiments.

[0445] The present disclosure also provides a method of treating cancer and / or killing cancer cells in a subject comprising administering to the subject the system of any of the aforementioned aspects.

[0446] In some embodiments, the present disclosure provides a method of treating cancer using any of the compositions provided herein. "Cancer" has its plain and ordinary meaning when read in the light of the present specification, and may include, but is not limited to, a group of diseases involving abnormal cell proliferation with the potential to invade or spread to other parts of the body. Subjects that may be addressed using the methods described herein include subjects identified or selected as having cancer, including, but not limited to, colon cancer, lung cancer, liver cancer, breast cancer, kidney cancer, prostate cancer, ovarian cancer, skin cancer (including melanoma), bone cancer, and brain cancer. Such identification and / or selection may be performed by clinical or diagnostic evaluation. In some embodiments, tumor-associated antigens or tumor-associated molecules are known, such as melanoma, breast cancer, brain cancer, squamous cell carcinoma, colon cancer, leukemia, myeloma, and / or prostate cancer. Examples include, but are not limited to, B-cell lymphoma, breast cancer, brain cancer, prostate cancer, and / or leukemia. In some embodiments, the one or more oncogenic polypeptides are associated with renal cancer, uterine cancer, colon cancer, lung cancer, liver cancer, breast cancer, renal cancer, prostate cancer, ovarian cancer, skin cancer (including melanoma), bone cancer, brain cancer, adenocarcinoma, pancreatic cancer, chronic myelogenous leukemia or leukemia. In some embodiments, a method of treating, ameliorating or inhibiting cancer in a subject is provided. In some embodiments, the cancer is breast cancer, ovarian cancer, lung cancer, pancreatic cancer, prostate cancer, melanoma, renal cancer, pancreatic cancer, glioblastoma, neuroblastoma, medulloblastoma, sarcoma, liver cancer, colon cancer, skin cancer (including melanoma), bone cancer or brain cancer.

[0447] In some embodiments, the target cell is a tumor cell. In some embodiments, the target cell is present in the tumor microenvironment.

[0448] In some embodiments, the present disclosure provides a method of treating cancer with any of the compositions provided herein without preconditioning the subject.In some embodiments, the subject of the present disclosure does not need to undergo lymphodepleting therapy.Those skilled in the art will understand available common lymphodepleting therapy, such as chemotherapy.In some embodiments, the subject of the present disclosure has never undergone lymphodepleting therapy.In some embodiments, the differentiated cells are provided to a subject that has never undergone lymphodepleting therapy.In some embodiments, the subject has never undergone lymphodepleting therapy for 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 days before administration of the differentiated cells.

[0449] In some embodiments, the differentiated cells are provided to the subject 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 24, 36, 48, 60 or 72 hours after administration of the ligand composition, or any time within the range defined by any two of the aforementioned values. In some embodiments, the cells are provided to the subject 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 24, 36 or 48 hours before administration of the ligand composition, or any time within the range defined by any two of the aforementioned values. In some embodiments, the cells are provided to the subject within seconds or minutes, e.g., within less than an hour, of providing the composition to the subject. In some embodiments, a boost of cells and / or compositions is provided to the subject.

[0450] In some aspects, the present disclosure provides methods of treating cancer, as described in International Publication No. WO 2019 / 144095, which is incorporated by reference in its entirety.

[0451] In some embodiments, the present disclosure provides a method of treating cancer comprising administering a chimeric antigen receptor cell (e.g., an NK cell), wherein the CAR comprises an E2 anti-fluorescein antibody fragment. In some embodiments, the method of treating cancer further comprises administering a small molecule linked to a targeting moiety by a linker.

[0452] In some embodiments, the targeting moiety is determined by the type of cancer being treated in the subject.As an example, folate receptors are highly expressed on the surface of a wide variety of solid tumor cells, including breast cancer (e.g., triple-negative breast cancer), ovarian cancer, endometrial cancer, renal cancer, lung cancer, brain cancer, pancreatic cancer, gastric cancer, prostate cancer, acute myeloid leukemia and non-small cell lung cancer.Therefore, in some embodiments, the targeting moiety comprises folate, which will bind to the folate receptor expressed on cancer cells or tumor cells.In other embodiments, the folate can be folic acid, folic acid analogs, or any folate-receptor binding molecule.In some embodiments, the small molecule comprises fluorescein, fluorescein isothiocyanate (FITC), NHS-fluorescein, or any other fluorophore.In some embodiments, the small molecule linked to the targeting moiety is FITC-folate.

[0453] In some embodiments, the present disclosure contemplates the engraftment of engineered stem cells for use in treating subjects with cancer. In some embodiments, the engineered cytotoxic innate lymphoid cells derived from iPSCs are stably engrafted in subjects. In some embodiments, the engineered cytotoxic innate lymphoid cells derived from iPSCs show long-term engraftment in subjects. In some embodiments, administration of the engineered cytotoxic innate lymphoid cells derived from iPSCs allows for engraftment and further differentiation in subjects. In some embodiments, the engineered cytotoxic innate lymphoid cells derived from iPSCs are natural killer cells.

[0454] In some embodiments, additional cancer treatments are provided, such as small molecules, e.g., chemical compounds, antibody therapies, e.g., humanized monoclonal antibodies with or without conjugation to radionuclides, toxins or drugs, surgery, and / or radiation.

[0455] In some embodiments, the subject is selected to receive additional cancer therapy, which may include cancer therapeutics, radiation, chemotherapy, or drugs to treat cancer. In some embodiments, the drug is abiraterone, alemtuzumab, anastrozole, aprepitant, arsenic trioxide, atezolizumab, azacitidine, bevacizumab, bleomycin, bortezomib, cabazitaxel, capecitabine, carboplatin, cetuximab, combination chemotherapy drugs, cisplatin, crizotinib, cyclophosphamide, cytarabine, denosumab, docetaxel, doxorubicin, eribulin, erlotinib, etoposide, everolimus, exemestane, filgrastim, fluorouracil, fulvescin ... These include tradioactive agents, including tradioactive agents, gemcitabine, imatinib, imiquimod, ipilimumab, ixabepilone, lapatinib, lenalidomide, letrozole, leuprolide, mesna, methotrexate, nivolumab, oxaliplatin, paclitaxel, palonosetron, pembrolizumab, pemetrexed, prednisone, radium-223, rituximab, sipuleucel-T, sorafenib, sunitinib, talc intrapleural, tamoxifen, temozolomide, temsirolimus, thalidomide, trastuzumab, vinorelbine or zoledronic acid.

[0456] Mode of administration In some embodiments, transduced lymphocytes, such as CIL cells, engineered with synthetic cytokine receptors as described herein, can be grown under conditions suitable for the cell population to be introduced into a subject, such as a human. Specific considerations include the use of culture media lacking animal products, such as bovine serum. Other considerations include sterile conditions to avoid bacterial, fungal and mycoplasmal contamination. In some embodiments, CAR-expressing lymphocytes, such as CAR-expressing CIL cells, can be grown using any of the cell culture methods contemplated in this disclosure.

[0457] In some embodiments, after transfection, the cells can be administered to the patient immediately, or the cells can be cultured for at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10. at least about 11. at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18 days or more, or for about 5 to about 12 days, about 6 to about 13 days, about 7 to about 14 days, or about 8 to about 15 days, e.g., to allow time for the cells to recover from the transfection. Suitable culture conditions can be similar to the conditions under which the cells were cultured for activation, with or without an agent used to promote activation. In some embodiments, CAR-expressing lymphocytes, such as CAR-expressing CIL cells, may be administered to a patient using any of the methods of administering engineered lymphocytes, e.g., CIL cells, contemplated in the present disclosure.

[0458] The disclosed cells can be administered in a number of ways, depending on whether local or systemic treatment is desired.

[0459] In the case of adoptive cell therapy, methods of administering cells for adoptive cell therapy are known and can be used in connection with the provided methods and compositions.

[0460] In general, administration can be topical, parenteral or enteral. The compositions of the present disclosure are typically suitable for parenteral administration. As used herein, "parenteral administration" of a pharmaceutical composition is characterized by physical damage to the tissue of the subject and administration of the pharmaceutical composition through the tissue damage, and therefore includes any route of administration that generally results in direct administration to the bloodstream, muscle or internal organs. Thus, parenteral administration includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, application of the composition through a surgical incision, application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is intended to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrastemal, intravenous, intraarterial, intrathecal, intraventricular, intraurethral, ​​intracranial, intratumoral, intrasynovial injection or infusion, and kidney dialysis infusion techniques. In one aspect, parenteral administration of the compositions of the present disclosure includes intravenous administration.

[0461] A formulation of a pharmaceutical composition suitable for parenteral administration typically generally comprises the active ingredient in combination with a pharma- ceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as ampoules, or multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and the like. Such formulations may further comprise one or more additional components, including, but not limited to, suspending agents, stabilizing agents, or dispersing agents. In one aspect of a formulation for parenteral administration, the active ingredient is provided in a dry (i.e., powder or granular) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition. Parenteral formulations also include aqueous solutions that may contain excipients such as salts, carbohydrates, and buffers (preferably to a pH of 3-9), although for some applications they may be more suitably formulated as sterile nonaqueous solutions or as dry forms for use with a suitable vehicle such as sterile pyrogen-free water. Exemplary parenteral dosage forms include solutions or suspensions in sterile aqueous solutions, for example, aqueous propylene glycol or aqueous dextrose solutions. Such dosage forms may be suitably buffered, if desired. Other parenterally administrable formulations that are useful include those that contain the active ingredient in microcrystalline form or in a liposomal preparation. Formulations for parenteral administration may be formulated to be immediate and / or modified release. Modified release formulations include delayed, sustained, pulsed, controlled, targeted, and programmed release.

[0462] The composition of the present invention may further contain other auxiliary components that are conventionally found in pharmaceutical compositions.Thus, for example, the composition may contain additional compatible pharmacoactive materials, such as antipruritic agents, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials that are useful for physically formulating the composition of the present invention in various dosage forms, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickeners and stabilizers.However, such materials, when added, should not excessively interfere with the biological activity of the components of the composition of the present invention.The formulation can be sterilized and, if desired, mixed with auxiliary agents, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts that affect osmotic pressure, buffers, colorants, flavorings and / or aromatic substances that do not adversely interact with the nucleic acid of the formulation.

[0463] The composition of viral particles, adaptor molecules and / or immune cells can be administered in an amount effective for treating or preventing a disease or condition, e.g., a therapeutically or prophylactically effective amount. In some embodiments, therapeutic or prophylactic effectiveness is monitored by periodic evaluation of the treated subject. In the case of repeated administration over several days or longer, depending on the condition, treatment is repeated until the desired suppression of disease symptoms occurs. However, other administration regimes may be useful and can be determined. The desired dosage can be delivered by a single bolus administration of the composition, by multiple boluses of the composition, or by continuous infusion administration of the composition.

[0464] In certain embodiments, in connection with injecting differentiated or transgenic differentiated cells according to the present disclosure, a subject may be injected with about 1 million to about 100 billion cells, e.g., 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), e.g., about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the foregoing values), in some cases in the range of about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells), and / or such number of cells per kilogram of the subject's body weight. For example, in some embodiments, administration of the cells or cell populations is in the range of about 100 million cells per kg of body weight, including all integer values ​​of the number of cells within those ranges. 3 ~about 10 9 The method may include administering individual cells.

[0465] X. Illustrative Embodiments Among the aspects provided are the following: 1. A cell population comprising engineered cytotoxic innate lymphoid cells engineered for controlled expansion and / or activity, the engineered cytotoxic innate lymphoid cells comprising synthetic cytokine receptors for non-physiological ligands; Cytokine receptors, a synthetic gamma chain polypeptide comprising a first dimerization domain, a first transmembrane domain, and an interleukin-2 receptor subunit gamma (IL-2RG) intracellular domain; and A synthetic beta chain polypeptide comprising a second dimerization domain, a second transmembrane domain, and an intracellular domain selected from an interleukin-2 receptor subunit beta (IL-2RB) intracellular domain, an interleukin-7 receptor subunit beta (IL-7RB) intracellular domain, and / or an interleukin-21 receptor subunit beta (IL-21RB) intracellular domain. Including, The non-physiological ligand activates a synthetic cytokine receptor in the cytotoxic innate lymphoid cells, inducing expansion and / or activation of the engineered cytotoxic innate lymphoid cells; Cell population. 2. The cell population of embodiment 1, wherein the beta chain intracellular domain comprises the IL-2RB intracellular domain. 3. The cell population of embodiment 2, wherein the IL-2RB intracellular domain comprises a polypeptide sequence at least 95% identical to, or as set forth in, SEQ ID NO:2. 4. The cell population of embodiment 1, wherein the beta chain intracellular domain comprises an IL-7RB intracellular domain. 5. The cell population of embodiment 4, wherein the IL-7RB intracellular domain comprises a polypeptide sequence at least 95% identical to, or as set forth in, SEQ ID NO:3. 6. The cell population of embodiment 1, wherein the beta chain intracellular domain comprises the IL-21RB intracellular domain. 7. The cell population of embodiment 6, wherein the IL-21RB intracellular domain comprises a polypeptide sequence at least 95% identical to, or set forth in, SEQ ID NO:4. 8. The first dimerization domain and the second dimerization domain are extracellular domains; a synthetic gamma chain polypeptide comprising, in order from N to C terminus, a first dimerization domain, a first transmembrane domain, and an interleukin-2 receptor subunit gamma (IL-2RG) intracellular domain; The cell population of any one of aspects 1-7, wherein the synthetic beta chain polypeptide comprises, in order from N→C terminus, a second dimerization domain, a second transmembrane domain, and an intracellular domain. 9. The first dimerization domain and the second dimerization domain are heterodimerization domains selected from the FK506-binding protein (FKBP) of size 12 kD and the FKBP12-rapamycin binding (FRB) domain; and / or The non-physiological ligand is rapamycin or a rapalog; The cell population of any one of aspects 1 to 8. 10. The cell population of embodiment 9, wherein the FRB domain comprises a polypeptide sequence at least 95% identical to SEQ ID NO:6 or SEQ ID NO:7. 11. The cell population of embodiment 9, wherein the FRB domain comprises the polypeptide sequence set forth in SEQ ID NO:6 or SEQ ID NO:7. 12. The first dimerization domain and the second dimerization domain are heterodimerization domains selected from FK506-binding protein (FKBP) and calcineurin domains of size 12 kD; and / or The non-physiological ligand is FK506 or an analog thereof. The cell population of any one of aspects 1 to 8. 13. The cell population of any one of aspects 1-12, wherein the FKBP domain comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:5. 14. The cell population of any one of aspects 1-12, wherein the FKBP domain comprises the polypeptide sequence set forth in SEQ ID NO:5. 15. The first dimerization domain and the second dimerization domain are (i) FK506-binding protein (FKBP) with a size of 12 kD; (ii) cyclophilin A (CypA); or (iii) Gyrase B (CyrB) and a homodimerization domain selected from and non-physiological ligands, (i) FK1012, AP1510, AP1903 or AP20187 or an analogue thereof; (ii) cyclosporine-A (CsA) or an analogue thereof; or (iii) coumermycin or its analogues The cell population of any one of aspects 1 to 8, 16. The cell population of any one of aspects 1-15, wherein the cytotoxic innate lymphoid cells express a cytosolic polypeptide that binds to a non-physiological ligand. 17. The cell population of any one of aspects 1-15, wherein the non-physiological ligand is rapamycin or a rapalog, and the cytotoxic innate lymphoid cells express a cytosolic FRB domain or a mutant thereof. 18. The cell population of embodiment 17, wherein the cytosolic FRB domain comprises a polypeptide sequence at least 95% identical to SEQ ID NO:6 or SEQ ID NO:7. 19. The cell population of embodiment 17, wherein the cytosolic FRB domain comprises a polypeptide sequence at least 98% identical to SEQ ID NO:6 or SEQ ID NO:7. 20. The cell population of any one of aspects 1-19, wherein the cytotoxic innate lymphoid cells are primary cytotoxic innate lymphoid cells. 21. The cell population of any one of aspects 1-19, wherein the cytotoxic innate lymphoid cells are blood-derived cytotoxic innate lymphoid cells. 22. The cell population of any one of aspects 1-19, wherein the cytotoxic innate lymphoid cells are induced cytotoxic innate lymphoid (iCIL) cells. 23. The cell population of any one of aspects 1-22, wherein the engineered cells are CD3-, CD5-, CD16+, CD56+, CD57+, NKp30+, NKp46+, NKG2A+, and / or NKG2D+. 24. The cell population of any one of aspects 1-23, wherein the population of engineered cytotoxic innate lymphoid cells is at least 40% CD16+. 25. The cell population of any one of aspects 1-24, wherein the population of engineered cytotoxic innate lymphoid cells is, or is at least 40% CD56+. 26. The cell population of any one of aspects 1-23, wherein the population of engineered cytotoxic innate lymphoid cells is at least 40% CD16+CD56+. 27. The cell population of any one of aspects 1-26, wherein the engineered cytotoxic innate lymphoid cells express at least the same levels of CD16, CD56, CD57, NKG2A, NKG2D and / or CD57 as control cytotoxic innate lymphoid cells expanded with IL-2. 28. The cell population of any one of aspects 1-27, wherein the engineered cytotoxic innate lymphoid cells express NKp30 and / or NKp46 at higher levels than control cytotoxic innate lymphoid cells expanded with IL-2. 29. The cell population of any one of aspects 1-28, wherein the engineered cytotoxic innate lymphoid cells secrete CD107a in response to an antigen recognized by the engineered cytotoxic innate lymphoid cells. 30. The cell population of any one of aspects 1-29, wherein the engineered cytotoxic innate lymphoid cells secrete interferon gamma (IFNγ) and / or tumor necrosis factor alpha (TNF-α) in response to an antigen recognized by the engineered cytotoxic innate lymphoid cells. 31. Contacting a source cell with a vector comprising a polynucleotide encoding a synthetic cytokine receptor for a non-physiological ligand, thereby transducing the source cells to generate a cell population comprising the transduced cells; Here, the cytokine receptor is a synthetic gamma chain polypeptide comprising a first dimerization domain, a first transmembrane domain, and an interleukin-2 receptor subunit gamma (IL-2RG) intracellular domain; and a synthetic beta chain polypeptide comprising a second dimerization domain, a second transmembrane domain, and an intracellular domain selected from an interleukin-2 receptor subunit beta (IL-2RB) intracellular domain, an interleukin-7 receptor subunit beta (IL-7RB) intracellular domain, and / or an interleukin-21 receptor subunit beta (IL-21RB) intracellular domain. Including, expanding the engineered cytotoxic innate lymphoid cells within the cell population by contacting the cell population with a non-physiological ligand to activate the synthetic cytokine receptor; thereby causing expansion of the engineered cytotoxic innate lymphoid cells within the cell population. A method for producing engineered cytotoxic innate lymphoid cells for controlled expansion and / or activity, comprising: 32. The method of embodiment 31, wherein the source cells are primary cytotoxic innate lymphoid cells. 33. The method of embodiment 31, wherein the source cells are hematopoietic progenitor cells or hematopoietic stem cells or common lymphoid progenitor cells obtained by differentiating human pluripotent stem cells or human embryonic stem cells. 34. The method of embodiment 31, wherein the source cells are human pluripotent stem cells. 35. The method of embodiment 33 or embodiment 34, comprising a step of contacting the population of cells with a non-physiological ligand during differentiation of the transduced cells into the engineered cytotoxic innate lymphoid cells. 36. The method of any one of aspects 31 to 35, wherein the expansion step is performed in a cell culture medium that is substantially free of recombinant cytokines, the expansion step is performed in a feeder-free cell culture, and / or the expansion step is performed in a culture vessel that is not coated with a recombinant ligand for cytotoxic innate lymphoid cell expansion. 37. A cell population produced by the method of any one of aspects 31 to 36. 38. A pharmaceutical composition comprising the cell population of any one of embodiments 1 to 30 or embodiment 37. 39. A method of immunotherapy for a subject in need thereof, comprising the step of administering to the subject an effective amount of the cell population of any one of embodiments 1 to 30 or embodiment 37, or the pharmaceutical composition of embodiment 38. 40. The method of embodiment 39, comprising administering to the subject a non-physiological ligand in an amount effective to expand the engineered cytotoxic innate lymphoid cells in the subject. 41. The method of embodiment 39 or embodiment 40, comprising a step of administering to the subject a non-physiological ligand in an amount effective to cause the engineered cytotoxic innate lymphoid cells to present CD107a or secrete interferon gamma (IFNγ) and / or tumor necrosis factor alpha (TNFα). 42. The method of any one of aspects 39-41, comprising administering to the subject a non-physiological ligand in an amount effective to cause the engineered cytotoxic innate lymphoid cells to kill tumor cells. 43. A kit comprising the cell population of any one of embodiments 1 to 30 or embodiment 37, or the pharmaceutical composition of embodiment 38, and instructions for use. 44. A cell population comprising engineered cells engineered for controlled expansion and / or activity, the engineered cells comprising synthetic cytokine receptors for non-physiological ligands; Cytokine receptors, a synthetic gamma chain polypeptide comprising a first dimerization domain, a first transmembrane domain, and an interleukin-2 receptor subunit gamma (IL-2RG) intracellular domain; and a synthetic beta chain polypeptide comprising a second dimerization domain, a second transmembrane domain, and an intracellular domain selected from an interleukin-2 receptor subunit beta (IL-2RB) intracellular domain, an interleukin-7 receptor subunit beta (IL-7RB) intracellular domain, and / or an interleukin-21 receptor subunit beta (IL-21RB) intracellular domain. Including, A cell population in which a non-physiological ligand activates a synthetic cytokine receptor within the engineered cells, inducing expansion and / or activation of the engineered cells. 45. The cell population of embodiment 44, wherein the engineered cells are human pluripotent stem cells. 46. ​​The cell population of embodiment 44, wherein the engineered cells are hematopoietic stem or progenitor cells. 47. The cell population of embodiment 44, wherein the engineered cells are common lymphoid progenitor cells. 48. The cell population of any one of aspects 44 to 47, which is the cell population of any one of aspects 2 to 30 or aspect 37. 49. A pharmaceutical composition or kit comprising the cell population of any one of aspects 44 to 48. 50. A method for producing a cell population of embodiment 1 according to any one of embodiments 31 to 37. 51. Use of the cell population of embodiment 50 in a method according to any one of embodiments 38 to 42. 52. a) optionally providing an engineered cell containing a synthetic cytokine receptor by introducing a polynucleotide encoding the synthetic cytokine receptor into a source cell; and b) incubating the engineered source cells in a medium containing a non-physiological ligand; 1. A method for generating and / or expanding a population of engineered cells containing a synthetic cytokine receptor for a non-physiological ligand, comprising: Cytokine receptors, a synthetic gamma chain polypeptide comprising a first dimerization domain, a first transmembrane domain, and an interleukin-2 receptor subunit gamma (IL-2RG) intracellular domain; and a synthetic beta chain polypeptide comprising a second dimerization domain, a second transmembrane domain, and an intracellular domain selected from an interleukin-2 receptor subunit beta (IL-2RB) intracellular domain, an interleukin-7 receptor subunit beta (IL-7RB) intracellular domain, and / or an interleukin-21 receptor subunit beta (IL-21RB) intracellular domain. Including, A method in which a non-physiological ligand activates a synthetic cytokine receptor in an engineered cell to induce expansion and / or activation of the engineered cell. 53. The method of embodiment 52, wherein the source cells are induced pluripotent stem cells. 54. The method of embodiment 52, wherein the source cells are hematopoietic stem or progenitor cells. 55. The method of embodiment 52, wherein the source cells are common lymphoid progenitor cells. 56. The method of embodiment 52, wherein the source cells are cytotoxic innate lymphoid cells. 57. The method of embodiment 52, wherein the vector is a lentiviral vector. 58. The method of embodiment 52, wherein the non-physiological ligand is a rapalog. 59. An engineered cytotoxic innate lymphoid (CIL) cell engineered for controlled expansion and / or activity, the engineered CIL cell comprising a synthetic cytokine receptor for a non-physiological ligand; Cytokine receptors, a synthetic gamma chain polypeptide comprising a first dimerization domain, a first transmembrane domain, and an interleukin-2 receptor subunit gamma (IL-2RG) intracellular domain; and a synthetic beta chain polypeptide comprising a second dimerization domain, a second transmembrane domain, and an intracellular domain selected from an interleukin-2 receptor subunit beta (IL-2RB) intracellular domain, an interleukin-7 receptor subunit beta (IL-7RB) intracellular domain, and / or an interleukin-21 receptor subunit beta (IL-21RB) intracellular domain. Including, An engineered cytotoxic innate lymphoid (CIL) cell, in which binding of a non-physiological ligand to the synthetic cytokine receptor activates the synthetic cytokine receptor in the engineered CIL cell, inducing expansion and / or activation of the engineered CIL cell within the cell population. 60. The engineered CIL cell of embodiment 59, wherein the first dimerization domain and the second dimerization domain are extracellular domains. 61. The engineered CIL cell of embodiment 59 or embodiment 60, wherein the synthetic gamma chain polypeptide comprises, in N->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 beta chain polypeptide comprises, in N->C-terminal order, a second dimerization domain, a second transmembrane domain, and the intracellular domain. 62. The engineered CIL cell of any of aspects 59-61, wherein the IL-2RG intracellular domain comprises a polypeptide sequence at least 95% identical to, or set forth in, SEQ ID NO:1. 63. The engineered CIL cell of any of aspects 59-62, wherein the first transmembrane domain comprises an IL-2RG transmembrane domain. 64. The engineered CIL cell of embodiment 63, wherein the IL-2RG transmembrane domain comprises a polypeptide sequence at least 95% identical to, or set forth in, SEQ ID NO:8 or 31. 65. The engineered CIL cell of any of aspects 59-64, wherein the beta chain intracellular domain comprises an IL-2RB intracellular domain. 66. The CIL cell of embodiment 65, wherein the IL-2RB intracellular domain comprises a polypeptide sequence at least 95% identical to, or set forth in, SEQ ID NO:2. 67. The engineered CIL cell of any of aspects 59-64, wherein the beta chain intracellular domain comprises an IL-7RB intracellular domain. 68. The engineered CIL cell of any of aspects 59-64, wherein the IL-7RB intracellular domain comprises a polypeptide sequence at least 95% identical to, or set forth in, SEQ ID NO:3. 69. The engineered CIL cell of any of aspects 59-64, wherein the beta chain intracellular domain comprises the IL-21RB intracellular domain. 70. The engineered CIL cell of embodiment 69, wherein the IL-21RB intracellular domain comprises a polypeptide sequence at least 95% identical to, or set forth in, SEQ ID NO:4. 71. The engineered CIL cell of any of aspects 59-70, wherein the second transmembrane domain comprises a transmembrane domain derived from the same beta chain intracellular domain. 72. The engineered CIL cell of embodiments 59 to 66 and 71, wherein the second transmembrane domain is a transmembrane domain of IL-2RB comprising a polypeptide sequence at least 95% identical to, or set forth in, SEQ ID NO: 35 or 36. 73. A synthetic gamma chain polypeptide comprising 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; A synthetic beta chain polypeptide comprising an IL-2RB TM domain comprising the sequence set forth in SEQ ID NO:35 or 36, and an IL-2RB intracellular domain comprising the sequence set forth in SEQ ID NO:2. The engineered CIL cell of any of embodiments 59 to 66, 71 and 72. 74. The first dimerization domain and the second dimerization domain are heterodimerization domains selected from the FK506-binding protein (FKBP) of size 12 kD and the FKBP12-rapamycin binding (FRB) domain; and / or The non-physiological ligand is rapamycin or a rapalog; The engineered CIL cell of any of embodiments 59 to 73. 75. The engineered CIL cell of embodiment 74, wherein the FRB domain comprises a polypeptide sequence at least 95% identical to SEQ ID NO:6 or SEQ ID NO:7. 76. The engineered CIL cell of embodiment 74, wherein the FRB domain comprises the polypeptide sequence set forth in SEQ ID NO:6 or SEQ ID NO:7. 77. The first dimerization domain and the second dimerization domain are heterodimerization domains selected from FK506-binding protein (FKBP) and calcineurin domains of size 12 kD; and / or The non-physiological ligand is FK506 or an analog thereof. The engineered CIL cell of any of embodiments 59 to 73. 78. The engineered CIL cell of any of aspects 59-74, wherein the FKBP domain comprises a polypeptide sequence at least 95% identical to SEQ ID NO:5 or SEQ ID NO:30. 79. The engineered CIL cell of any of aspects 59 to 74, wherein the FKBP domain comprises the polypeptide sequence set forth in SEQ ID NO:5 or SEQ ID NO:30. 80. The engineered CIL cell of any of aspects 59-66 and 71-79, wherein the synthetic gamma chain polypeptide has an 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 an 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. 81. The engineered CIL cell of any of aspects 59-66 and 71-80, wherein the synthetic gamma chain polypeptide has the amino acid sequence set forth in SEQ ID NO:28, and the synthetic beta chain polypeptide has the amino acid sequence set forth in SEQ ID NO:33. 82. The first dimerization domain and the second dimerization domain are (i) FK506-binding protein (FKBP) with a size of 12 kD; (ii) cyclophilin A (CypA); or (iii) Gyrase B (CyrB) and a homodimerization domain selected from and ...

Claims

1. engineered cytotoxic innate lymphoid (CIL) cells engineered for controlled expansion and / or activity, comprising synthetic cytokine receptors for non-physiological ligands; The cytokine receptor is a synthetic gamma chain polypeptide comprising a first dimerization domain, a first transmembrane domain, and an interleukin-2 receptor subunit gamma (IL-2RG) intracellular domain; and a synthetic alpha chain polypeptide or a synthetic beta chain polypeptide comprising a second dimerization domain, a second transmembrane domain, and an intracellular domain selected from an interleukin-2 receptor subunit beta (IL-2RB) intracellular domain, an interleukin-7 receptor subunit beta (IL-7RB) intracellular domain, an interleukin-21 receptor subunit beta (IL-21RB) intracellular domain, or an interleukin-9 receptor subunit alpha (IL-9RA) intracellular domain; Including, Binding of the non-physiological ligand to the synthetic cytokine receptor activates the synthetic cytokine receptor in the engineered CIL cells, inducing expansion and / or activation of the engineered CIL cells within the cell population. The engineered cytotoxic innate lymphoid (CIL) cells.

2. The cytokine receptor a synthetic gamma chain polypeptide comprising a first dimerization domain, a first transmembrane domain, and an interleukin-2 receptor subunit gamma (IL-2RG) intracellular domain; and a synthetic beta chain polypeptide comprising a second dimerization domain, a second transmembrane domain, and an intracellular domain selected from an interleukin-2 receptor subunit beta (IL-2RB) intracellular domain, an interleukin-7 receptor subunit beta (IL-7RB) intracellular domain, and / or an interleukin-21 receptor subunit beta (IL-21RB) intracellular domain; 2. The engineered CIL cell of claim 1, comprising:

3. The engineered CIL cell of claim 1, wherein the first transmembrane domain comprises the IL-2RG transmembrane domain.

4. The engineered CIL cell of claim 1, wherein the beta chain intracellular domain comprises the IL-2RB intracellular domain.

5. the IL-2RB intracellular domain comprises a polypeptide sequence at least 95% identical to, or set forth in, SEQ ID NO:2; the IL-7RB intracellular domain comprises a polypeptide sequence at least 95% identical to, or set forth in, SEQ ID NO:3; or the IL-21RB intracellular domain comprises a polypeptide sequence at least 95% identical to, or set forth in, SEQ ID NO:4; The engineered CIL cells of claim 4.

6. the synthetic alpha chain polypeptide comprises the IL-9RA intracellular domain, optionally wherein the IL-9RA intracellular domain comprises a polypeptide sequence at least 95% identical to, or set forth in, SEQ ID NO:51; The engineered CIL cells of claim 1.

7. the second transmembrane domain is a transmembrane domain of IL-2RB comprising a polypeptide sequence at least 95% identical to, or set forth in, SEQ ID NO:35 or 36; the first transmembrane domain is the transmembrane domain of IL-2RB, comprising a polypeptide sequence at least 95% identical to, or set forth in, SEQ ID NO:8 or 31; or the synthetic gamma chain polypeptide contains an IL-2RG 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 / or the synthetic beta chain polypeptide comprises an IL-2RB transmembrane 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; 2. The engineered CIL cells of claim 1. (a) the first dimerization domain and the second dimerization domain are heterodimerization domains selected from the FK506-binding protein (FKBP) and FKBP12-rapamycin binding (FRB) domains of 12 kD in size; and / or the non-physiological ligand is rapamycin or a rapalog; or (b) the first dimerization domain and the second dimerization domain are heterodimerization domains selected from FK506-binding protein (FKBP) and calcineurin domains of 12 kD in size; and / or The non-physiological ligand is FK506 or an analog thereof. The engineered CIL cells of claim 1.

9. the FRB domain comprises a polypeptide sequence at least 95% identical to SEQ ID NO:6 or SEQ ID NO:7; the FKBP domain comprises a polypeptide sequence at least 95% identical to, or set forth in, SEQ ID NO:5 or SEQ ID NO:30; or the FRB domain comprises a polypeptide sequence at least 95% identical to, or comprises the polypeptide sequence set forth in, SEQ ID NO:6 or SEQ ID NO:7, and the FKBP domain comprises a polypeptide sequence at least 95% identical to, or comprises the polypeptide sequence set forth in SEQ ID NO:5 or SEQ ID NO:30; The engineered CIL cell of claim 8.

10. 2. The engineered CIL cell of claim 1, wherein the synthetic gamma chain polypeptide has an 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 an 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.

11. the first dimerization domain and the second dimerization domain are (i) FK506-binding protein (FKBP) with a size of 12 kD; (ii) cyclophilin A (CypA); or (iii) Gyrase B (CyrB) a homodimerization domain selected from and the non-physiological ligand is, respectively: (i) FK1012, AP1510, AP1903 or AP20187 or an analog thereof; (ii) cyclosporine-A (CsA) or its analogs; or (iii) coumermycin or its analogues 2. The engineered CIL cell of claim 1, wherein the CIL cell is

12. The engineered CIL cell of claim 1, wherein the engineered CIL cell expresses a cytosolic polypeptide that binds to the non-physiological ligand.

13. The engineered CIL cell of claim 1, wherein the non-physiological ligand is rapamycin or a rapalog, and the engineered CIL cell expresses a cytosolic FRB domain or a mutant thereof.

14. The engineered CIL cells contain a disrupted FKBP12 gene that reduces expression of FKBP12, or contain a knockout of the FKBP12 gene. The engineered CIL cells of claim 1.

15. The engineered CIL cell of claim 1, wherein the engineered CIL cell comprises a nucleotide sequence encoding the synthetic cytokine receptor inserted into the genome of the engineered CIL cell.

16. the engineered CIL cells comprise a B2M knockout, or the engineered CIL cells comprise a B2M knockout and an FKBP12 knockout; The engineered CIL cells of claim 1.

17. 2. The engineered CIL cell of claim 1, comprising a chimeric antigen receptor (CAR).

18. The engineered CIL cells of claim 1, wherein the engineered CIL cells are iPSC-derived natural killer cells.

19. A cell population comprising the engineered CIL cells of claim 1.

20. A method for producing engineered cytotoxic innate lymphoid (CIL) cells for controlled expansion and / or activity, comprising: (a) contacting human pluripotent stem cells with a vector comprising a polynucleotide encoding a synthetic cytokine receptor for a non-physiological ligand; thereby transducing said stem cells to generate a cell population comprising transduced cells; wherein the synthetic cytokine receptor is a synthetic gamma chain polypeptide comprising a first dimerization domain, a first transmembrane domain, and an interleukin-2 receptor subunit gamma (IL-2RG) intracellular domain; and a synthetic beta chain polypeptide comprising a second dimerization domain, a second transmembrane domain, and an intracellular domain selected from an interleukin-2 receptor subunit beta (IL-2RB) intracellular domain, an interleukin-7 receptor subunit beta (IL-7RB) intracellular domain, and / or an interleukin-21 receptor subunit beta (IL-21RB) intracellular domain; and (b) expanding the engineered CIL cells within the cell population by contacting the cell population with a non-physiological ligand to activate the synthetic cytokine receptor; Thereby causing expansion of the engineered cytotoxic innate lymphoid cells within the cell population.

21. The method of claim 20, wherein the engineered CIL cells are iPSC-derived natural killer cells.

22. contacting a population of CIL cells with a recombinant vector comprising (i) a guide RNA (gRNA) that targets a target site in an endogenous gene, (ii) an RNA-guided endonuclease, and (iii) a nucleotide sequence encoding a synthetic cytokine receptor for a non-physiological ligand, thereby inserting the nucleotide sequence into the endogenous gene; 1. A method of genetically engineering cytotoxic innate lymphoid (CIL) cells to express a synthetic cytokine receptor, comprising: The cytokine receptor is a synthetic gamma chain polypeptide comprising a first dimerization domain, a first transmembrane domain, and an interleukin-2 receptor subunit gamma (IL-2RG) intracellular domain; and a synthetic beta chain polypeptide comprising a second dimerization domain, a second transmembrane domain, and an intracellular domain selected from an interleukin-2 receptor subunit beta (IL-2RB) intracellular domain, an interleukin-7 receptor subunit beta (IL-7RB) intracellular domain, and / or an interleukin-21 receptor subunit beta (IL-21RB) intracellular domain; Including, The method.

23. 23. The method of claim 22, wherein the nucleotide sequence encoding the synthetic cytokine receptor comprises a first nucleic acid sequence encoding a gamma chain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:37, and a second nucleic acid sequence encoding a beta chain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:

38.

24. The nucleotide sequence encoding a synthetic cytokine receptor for a non-physiological ligand is under the functional control of a heterologous promoter; the nucleotide sequence encoding a synthetic cytokine receptor for a non-physiological ligand is under the functional control of an EF1α promoter or an MND promoter; or the recombinant vector comprises a 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; 23. The method of claim 22.

25. 23. The method of claim 22, comprising engineering the population of CIL cells to be resistant to rapamycin-mediated mTOR inhibition.

26. 26. The method of claim 25, wherein engineering the population of CIL cells to be resistant to rapamycin comprises knocking out the FKBP12 gene.

27. 23. The method of claim 22, further comprising introducing a chimeric antigen receptor (CAR) into said population of CIL cells.

28. 21. A cell population produced by the method of claim 20.

29. A pharmaceutical composition comprising the engineered CIL cells of claim 1.

30. A method for expanding engineered CIL cells, the method comprising contacting the engineered CIL cells of any one of claims 1 to 18 with the non-physiological ligand of the synthetic cytokine receptor.

31. 29. The engineered CIL cells of any one of claims 1 to 18, the cell population of claim 19 or 28, or the pharmaceutical composition of claim 29, for use in treating cancer in a subject: Here, the treatment comprises administering the engineered CIL cells, the cell population, or the pharmaceutical composition to a subject.

32. the subject has not been administered lymphodepleting therapy prior to administering the engineered CIL cells, population of CIL cells, or pharmaceutical composition; the engineered CIL cells express a CAR that targets cancer cells in the subject; or the non-physiological ligand is rapamycin or a rapamycin analog, and optionally the rapamycin analog is a rapalog; 32. The engineered CIL cell, cell population, or pharmaceutical composition for use according to claim 31.

33. The engineered CIL cells of any one of claims 1 to 18, the cell population of claim 19 or 28, or the pharmaceutical composition of claim 29 for use in killing or inhibiting the growth of cancer cells: wherein said killing or inhibiting comprises contacting cancer cells with said engineered CIL cells, said cell population, or said pharmaceutical composition.

34. Use of an engineered CIL cell described in any one of claims 1 to 18, a cell population described in claim 19 or 28, or a pharmaceutical composition described in claim 29 for the manufacture of a medicament for treating cancer in a subject.

35. A kit comprising the engineered CIL cells of any one of claims 1 to 18, the cell population of claim 19 or 28, or the pharmaceutical composition of claim 29, together with instructions for administering the same to a subject in need thereof.

Images