Cytotoxic innate lymphoid cells and uses thereof

Abstract

Compositions and methods are provided relating to a population of cells comprising engineered cytotoxic innate lymphoid cells engineered for controlled expansion and / or activity, the engineered cytotoxic innate lymphoid cells comprising synthetic cytokine receptors of non-physiological ligands. In some embodiments, the cytokine receptor can comprise a synthetic gamma chain polypeptide in 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 in the form of a second dimerization domain, a second transmembrane domain, and an intracellular domain selected from the group consisting of 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 activates the synthetic cytokine receptor in the cytotoxic innate lymphoid cell to induce expansion and / or activation of the engineered cytotoxic innate lymphoid cell.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to the following patent applications: U.S. Provisional Application No. 63 / 291,235 filed on December 17, 2021; U.S. Provisional Application No. 63 / 291,237 filed on December 17, 2021; U.S. Provisional Application No. 63 / 392,862 filed on July 27, 2022; U.S. Provisional Application No. 63 / 392,864 filed on July 27, 2022; U.S. Provisional Application No. 63 / 411,056 filed on September 28, 2022; U.S. Provisional Application No. 63 / 411,063 filed on September 28, 2022; U.S. Provisional Application No. 63 / 422,848 filed on November 4, 2022; U.S. Provisional Application No. 63 / 422,830 filed on November 4, 2022; the contents of which are incorporated by reference in their entirety. Incorporation by Reference into the Sequence Listing

[0002] This application is filed with a sequence listing in electronic form. The sequence listing is provided as a file named 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. Technical Field

[0003] The present invention generally relates to engineered lymphocytes, such as cytotoxic innate lymphoid (CIL) cells, methods for making such cells, and methods of using them in immunotherapy. Background Art

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

[0005] CIL cells (e.g., NK cells) used in immunotherapy can be obtained from an original source (such as peripheral blood or umbilical cord blood). Artificial sources of CIL cells include pluripotent stem cells (including induced pluripotent stem cells (iPSC), which are cells derived from somatic cells (usually fibroblasts or peripheral blood mononuclear cells [PBMC])), and human embryonic stem cells (hESC) (when subjected to appropriate differentiation conditions, induced to become able to proliferate indefinitely and differentiate into other cell types). CIL cells can be derived from iPSC in the following ways: sequentially, iPSC is differentiated into hematopoietic progenitor cells (HPC), also known as hematopoietic stem cells (HSC); HPC is differentiated into common lymphoid progenitor cells (CLP); and CLP is then differentiated into CIL cells, which are called iPSC-derived cytotoxic innate lymphoid cells (iPSC-CIL). Typically, iPSC-CIL cells express CD56 and have cytotoxic activity, like NK cells; but iPSC-CIL cells may be phenotypically and otherwise different from NK cells.

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

[0007] Effective methods for CIL cell expansion known for manufacturing purposes require such factors or feeder cells. Such methods can further provide CIL cells with other factors to drive expansion. For example, antigen presenting cells (APCs) can be genetically engineered to produce artificial APCs (aAPCs). Amplification is usually further enhanced by the following means: aAPCs are engineered to secrete interleukin 15 (IL-15) or interleukin 21 (IL-21); or to display recombinant membrane-bound IL-15 (mbIL-15) or membrane-bound IL-21 (mbIL-21). In short, known methods for effective CIL cell expansion usually rely on a variety of exogenous soluble factors and factors based on feeder cells.

[0008] It is known to use a culture of single-cell iPSC on embryoid bodies (EB) or feeder cells to differentiate iPSC into CD34+HPC. CD34+HPC can then be differentiated into CLP, and a combination of stem cell factor (SCF), bone morphogenetic protein 4 (BMP4) and vascular endothelial growth factor (VEGF) is routinely used. Known methods for differentiating CIL from CLP generally involve contacting CLP with: SCF, interleukin 17 (IL-7), interleukin 15 (IL-15), FMS-like tyrosine kinase 3 ligands (FLT3L) and optionally interleukin (IL-3). The iPSC-CIL cells obtained can then be amplified using the methods already described.

[0009] There remains a need in the art for compositions and methods related to engineering cytotoxic innate lymphoid cells, methods for making such cells, and methods of using them in immunotherapy. Summary of the invention

[0010] The present disclosure is based in part on the unexpected discovery of the inventors of unexpected properties of lymphocytes, such as cytotoxic innate lymphoid (CIL) cells (e.g., NK cells), engineered to express synthetic cytokine receptors. In some embodiments, lymphocytes, such as CIL cells (e.g., NK cells), engineered to express synthetic cytokine receptors can be expanded in response to cognate non-physiological ligands of the receptors and have other desirable characteristics, such as functional activity equal to or greater than that of non-engineered lymphocytes (e.g., CIL cells) from other sources.

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

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

[0013] Embodiments provided include an engineered lymphocyte or progenitor cell comprising a synthetic cytokine receptor for a non-physiological ligand, wherein the cytokine receptor comprises: (i) a synthetic γ chain polypeptide comprising a first dimerization domain, a first transmembrane domain, and an interleukin-2 receptor subunit γ (IL-2RG) intracellular domain, and (ii) a synthetic α or β chain polypeptide comprising a second dimerization domain, a second transmembrane domain, and an intracellular domain selected from the following: an interleukin-2 receptor subunit β (IL-2RB) intracellular domain, an interleukin-7 receptor subunit β (IL-7RB) intracellular domain, an interleukin-21 receptor subunit β (IL-21RB) intracellular domain, or an interleukin-9 receptor subunit α (IL-9RA) intracellular domain. In some embodiments, the engineering of the synthetic cytokine receptor in the engineered lymphocyte or progenitor cell provides controlled differentiation and / or expansion of the engineered cell. In some embodiments, binding of the non-physiological ligand to the synthetic cytokine receptor activates the synthetic cytokine receptor in the engineered lymphocytes or progenitor cells to induce differentiation and / or expansion of the engineered lymphocytes and / or progenitor cells in the cell population.

[0014] In some embodiments, the engineered cell is an engineered progenitor cell, which is a hematopoietic progenitor cell (HPC; also known as hematopoietic stem cell (HSC)) or a common lymphoid progenitor cell (CLP). In some embodiments, the engineered cell is a cytotoxic innate lymphocyte (CIL), a NK cell, or a T cell. In some embodiments, the T cell is a γδT cell or an αβT cell. In a specific embodiment, the engineered cell is a CIL, such as a NK cell.

[0015] In some embodiments, the present invention provides an engineered cytotoxic innate lymphoid (CIL) cell comprising a synthetic cytokine receptor for a non-physiological ligand, wherein the cytokine receptor comprises: a synthetic γ chain polypeptide comprising a first dimerization domain, a first transmembrane domain, and an interleukin-2 receptor subunit γ (IL-2RG) intracellular domain; and a synthetic α or β chain polypeptide comprising a second dimerization domain, a second transmembrane domain, and an intracellular domain selected from the following: an interleukin-2 receptor subunit β (IL-2RB) intracellular domain, an interleukin-7 receptor subunit β (IL-7RB) intracellular domain, an interleukin-21 receptor subunit β (IL-21RB) intracellular domain, or an interleukin-9 receptor subunit α (IL-9RA) intracellular domain. In some embodiments, the synthetic cytokine receptor for a non-physiological ligand is engineered into the engineered CIL cell for controlled expansion and / or activity of the engineered CIL. In some embodiments, binding of the non-physiological ligand to the synthetic cytokine receptor activates the synthetic cytokine receptor in the engineered CIL cells to induce expansion and / or activation of the engineered CIL cells in the cell population.

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

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

[0018] Also provided herein are pharmaceutical compositions comprising any of the provided engineered cells (eg, engineered CILs). In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

[0019] In some embodiments, the present invention also provides any provided engineered cell or engineered cell group treatment method and use. Also provided herein are any provided pharmaceutical compositions treatment method and use. Provided herein are any provided engineered cells, engineered cell groups or pharmaceutical compositions in such methods and treatments and in the preparation of medicaments for such treatment methods. In some embodiments, the method is performed by administering the engineered cell or a composition comprising it to a subject suffering from, already suffering from or suspected of having a disease or disorder or disorder. In some embodiments, the method thus treats the subject's disease or disorder or disorder. In some embodiments, the method can be used to treat any disease or disorder, wherein immunotherapy performed by adoptive cell therapy can be used to treat the disease or disorder. In some embodiments, the engineered cell expresses a recombinant receptor, such as a chimeric antigen receptor (CAR), which targets antigens on cells associated with a disease or disorder. In some embodiments, the disease or disorder is cancer. Exemplary methods and uses are described herein.

[0020] In certain embodiments, provided herein are methods of treatment, kits and pharmaceutical compositions for use in immunotherapy using lymphocytes (e.g., CIL cells, such as NK cells) produced in vitro. In certain embodiments, engineered lymphocytes as described herein, such as CIL cells, are produced and optionally expanded in vitro and then administered to a subject in need of immunotherapy.

[0021] In a preferred embodiment, the synthetic cytokine receptor is a rapamycin activated cytokine receptor (RACR). The RACR can be activated by rapamycin or a derivative thereof (rapalog).

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

[0023] The present disclosure also provides other compositions and methods, including but not limited to pharmaceutical compositions, treatment methods, kits, etc. The aforementioned advantages may be present in some but not all embodiments described herein, and other advantages of the compositions and methods described herein will become clear from the detailed description below. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1A is a diagram of an embodiment showing expansion of engineered cytotoxic innate lymphoid cells (eg, natural killer (NK) cells).

[0025] Figure 1B is a diagram of an embodiment showing differentiation of engineered stem or progenitor cells.

[0026] Figure 2A It is a set of figures, which depicts the RACR expansion of CD19 CAR blood-derived NK (bdNK) cells. The top small figure shows the timeline of the experiment. The percentage of CAR expression over time in each group is shown in the left small figure, and the total CAR+NK cells over time are shown in the right small figure.

[0027] Figure 2B It is a set of figures, which depicts the CAR expression analysis performed by flow cytometry. The CAR expression of bdNK cells amplified in the presence of IL-2 (1000U / ml) or AP21967 (20nM) is shown on the top panel. The CD56 and CD16 expression of CAR+ cells are shown on the bottom panel.

[0028] Figure 2C is a set of graphs depicting the ability of RACR-expanded blood-derived NK cells to recognize and target tumor cells. The top panel shows the timeline of the experiment. A flow cytometry graph depicting CAR expression at day 31 after transduction is shown on the upper right panel. In a 96-well plate, 1x10 5 NK cells and 1x10 5K562 cells, Nalm6 cells or Nalm6 CD19 knockout cells were incubated together for 5 hours. The CD107a (left panel) and IFNγ (middle panel) expression levels on CAR+ cells (AP-20) or untransduced controls amplified by AP21967 are shown. Death % (right panel) is calculated by comparing the total number of celltrace violet+ cells alive in each well with non-effector control wells. NK effector cells are added with different effector cells: target cells (E:T) ratios.

[0029] Figure 3A It is a diagram showing the process of generating iPSC-derived CIL cells (eg, NK cells).

[0030] Figure 3B is a graph depicting the % CD34+ cells before and after cell selection and analysis at day 12 of the differentiation process.

[0031] Figure 3C is a set of graphs depicting CD34+ selection by flow cytometry analysis at day 12 of the differentiation process.

[0032] Figure 3D Graph depicting the percentage of CD45+ cells among leukocytes and the percentage of leukocytes among progenitor cells and CIL cells (NK cells). The percentage of CD45+ cells is plotted as leukocyte %, CD7+ cells are plotted as progenitor cells, and CD56+ cells are plotted as CIL cells.

[0033] Figure 3E is a set of graphs depicting flow cytometric analysis of differentiated leukocytes (CD45+), progenitor cells (CD45+ / CD5- / CD7+), and CIL cells (CD45+ / CD5- / CD7+ / CD56+).

[0034] Figure 3F is a set of graphs depicting the % of leukocytes or CIL cells at day 40 (left panel) and flow cytometric analysis of differentiated cells at day 40 (right panel).

[0035] Figure 3G is a set of graphs depicting the percentage of iCIL cells 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 different effector:target (E:T) ratios.

[0036] Figure 4A is a diagram depicting the timeline of the experiment.

[0037] Figure 4B It is a set of figures, which depicts representative flow cytometry analysis of CD56+ cells and TagCAR (anti-fluorescein isothiocyanate (FITC) chimeric antigen receptor (CAR)) enrichment during the experiment. The x-axis is TagCAR detection by FITC-AF647, and the y-axis is side scatter. After "simulated" transduction, cells were analyzed on the 32nd day, the 35th day, the 38th day and the 42nd day after transduction.

[0038] Figure 4C is a set of graphs depicting RACR enrichment of TagCAR cells over time (left panel), total cell counts (middle panel), and the percentage of cells expressing CD45 and CD56 (right panel) for the BXS cell line over time.

[0039] Figure 4D is a set of graphs depicting RACR enrichment of TagCAR cells over time (left panel), total cell counts (middle panel), and the percentage of cells expressing CD45 and CD56 (right panel) for NHS cell lines over time.

[0040] Figure 5A Is a set of histograms depicting different activation markers of cytokine differentiated mock CIL cells (BXS line) and RACR-iCIL cells (BXS line and NH5 line). Cells were gated for CD45+CD7+CD5- and then plotted for activation markers CD56, CD16, NKp30, NKp40, and NKG2D.

[0041] Figure 5B is a graph of a cytotoxicity assay depicting killing of MDA-mCherry tumor cell lines by cytokine-differentiated mock CIL cells (BXS cell line) and RACR-iCIL cells (BXS cell line and NH5 cell line).

[0042] Fig. 6A is a diagram depicting the timeline of the experiment.

[0043] Figure 6B Figure 3 is a graph depicting the % of RACR iCIL cells over time and the 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 cocktail) or cells treated with the A / C heterodimerizer AP21967 (AP 100 nM).

[0044] Figure 6Cis a set of flow cytometry graphs depicting cells stained for the CD56 clone HCD56 and RACR-FRB positive cells detected by mCherry.

[0045] Fig.6D is a set of graphs depicting the % of FITC-CAR iCIL cells over time (top panels) and the total number of FLCAR iCIL cells over time (bottom panels) for cells transduced with viral constructs containing TagCAR-RACR-FRB(206) and FRB-RACR-TagCAR(205).

[0046] Fig. 6E is a set of flow cytometry graphs depicting cells stained for CD56 clone HCD56 and FLCAR positive cells.

[0047] Fig. 7A is a set of flow cytometry graphs depicting cells stained for CD56 and Tag-CAR.

[0048] Figure 7B is a set of histograms depicting different 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.

[0049] Figure 7C is a set of histograms depicting CD107a secretion in response to antigen in mock IL2-expanded CIL cells and TagCAR RACR-expanded iCIL cells.

[0050] Figure 8 is a diagram of an illustrative CAR of the present disclosure, wherein the fusion protein is encoded by a lentiviral expression vector, and wherein "SP" is a signal peptide, the CAR is an anti-FITC CAR, the CD8α hinge is present, the transmembrane domain is present ("TM"), the co-stimulatory domain is 4-1BB, and the activation signaling domain is CD3ζ.

[0051] Fig. 9It is a diagram depicting the differentiation factors involved in the transition from iPSC to CIL cells and a timeline. The differentiation factors involved in each stage of differentiation are depicted in an outline box; the differentiation factors illustrating the embodiments of the present disclosure are in bold. In some embodiments, starting at week 4, cells are differentiated in a culture medium (optionally with or without rapalog) comprising IL7, IL15, SCF, FLT3L and UM729. Starting from week 6, these cells are expanded in a culture medium (optionally with or without rapalog) comprising IL7, IL15, SCF, FLT3L, UM729 and CD2 / NKp46 beads. In some embodiments, starting at week 4, cells are differentiated in a culture medium (optionally with or without rapalog) comprising SCF, FLT3L and UM729. Starting from week 6, these cells are expanded in a culture medium (optionally with or without rapalog) comprising SCF, FLT3L, UM729 and CD2 / NKp46 beads. In some embodiments, starting at week 4, cells are differentiated in a culture medium comprising IL7, IL15, and UM729 (optionally with or without rapalog). Starting from week 6, these cells are expanded in a culture medium comprising IL7, IL15, UM729, and CD2 / NKp46 beads (optionally with or without rapalog). In some embodiments, starting at week 4, cells are differentiated in a culture medium comprising UM729 (optionally with or without rapalog). Starting from week 6, these cells are expanded in a culture medium comprising UM729 and CD2 / NKp46 beads (optionally with or without rapalog).

[0052] Fig.10 It is a graph depicting the percentage of RACR CD19-CAR positive cells as a function of culture days. 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. The cells were then expanded in complete medium in 100IU / mL human IL-2 or 100nM AP219667, with membrane-bound IL-21 (mbIL-21) 41BBL and K562 feeder cells added weekly. RACR (IL-2RG / IL-2RB) "RACR2" supports the highest amplification in AP219667, and RACR (IL-2RG / IL-7RB) "RACR7" and RACR (IL-2RG / IL-21RB) "RACR21" support RACR amplification to a lower degree.

[0053] Fig.11It is a set of graphs depicting RACR enrichment of CAR-expressing cells over time (left panel), fold change of progenitor cells (0-4 weeks) and RACR-iCIL cells (4-9 weeks) (middle panel), and for different effector cells: target cells (E: T) ratios, as measured by % killing of K562 cells by iCIL and RACR-iCIL cells (right panel). The top of the figure depicts a diagram of hematopoietic lineage differentiation and corresponding cell culture.

[0054] Fig. 12A is a graph depicting the % of cells containing the markers CD45+CD5-, CD45+CD5-CD7+, or CD45+CD5-CD7+CD56+ for cells thawed at day 26 or analyzed at day 29 post transduction.

[0055] Fig. 12B is a graph depicting the % of CAR+ cells containing the markers CD45+CD5-, CD45+CD5-CD7+, or CD45+CD5-CD7+CD56+ for cells thawed at day 26 and mock transduced or analyzed at day 29 following viral vector transduction.

[0056] Fig.13A is a graph depicting the % of CAR+iCIL cells after analyzing cells placed 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 rapalog.

[0057] Fig. 13B is a graph depicting the fold change (FC) of CAR+iCIL cell % after analyzing cells placed 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 rapalog.

[0058] Fig. 13C is a graph depicting the total number of CAR+iCIL cells after analyzing cells placed 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 rapalog.

[0059] Fig.13Dis a graph depicting the fold change (FC) in the total number of CAR+iCIL cells after analyzing cells placed 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 rapalog.

[0060] Fig.14A Is a set of histograms depicting different markers (including CD45, CD7 and CD56) in TagCAR+RACR-iCIL cells or unstained cells, wherein the TagCAR+RACR-iCIL cells were placed in a medium containing IL7, IL15, SCF, FLT3L and UM729 (full mixture), SCF, FLT3L and UM729 (SCF / FLT3L), IL7, IL15 and UM729 (IL7 / IL15), UM729 (none). Each medium condition was treated with or without rapalog.

[0061] Fig. 14B is a histogram depicting the CD56 marker in progenitor cells at day 26 (CD56-) and cells analyzed at day 40 placed in medium containing IL7, IL15, SCF, FLT3L and UM729 (full cocktail) (CD56+).

[0062] Fig.15A Figures depicting tumor cell growth after incubation of ovarian cancer cells with unstimulated NK cells, cytokine-stimulated NK cells, or RACR-stimulated NK cells are shown. Ovarian cancer cells not incubated with NK cells are shown in bold black lines. Tumor cells were reintroduced after 50 hours (i.e., tumor cell re-stimulation).

[0063] Fig. 15B Figures depicting tumor cell growth after incubation of bladder cancer cells with unstimulated NK cells, cytokine-stimulated NK cells, or RACR-stimulated NK cells are shown. Bladder cancer cells not incubated with NK cells are shown in bold black lines. Tumor cells were reintroduced at 50 and 100 hours (i.e., tumor cell re-challenge).

[0064] Fig. 15C Figures depicting tumor cell growth 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 bold black lines. Tumor cells are reintroduced after 40 hours (also known as tumor cell re-excitation).

[0065] Fig.15DShown are graphs depicting the growth of RACR-NK cells after incubation without rapamycin or cytokines (unstimulated NK), with rapamycin (RACR-stimulated NK), or with cytokines (cytokine-stimulated NK).

[0066] Fig.16A Figures depicting tumor cell killing in tumor cells incubated in the following situations are shown: no RACR engineered iCIL (tumor or T only), RACR engineered iCIL and no CAR antigen expression (R+NCA), RACR engineered iCIL and moderate CAR antigen expression (R+MCA), or RACR engineered iCIL and high CAR antigen expression (R+HCA). The CAR antigen is specific for FITC conjugated to folate, which binds to folate receptors on tumor cells.

[0067] Fig. 16B Diagrams depicting the function of iPSC-derived NK cells and CAR- and RACR-engineered iPSC-derived CILs are shown. CAR-RACR-iCIL cells secrete CD107a in response to antigens recognized by the cells.

[0068] Fig. 16C Shown are graphs depicting the growth of RACR-CAR-NK cells after incubation without rapamycin or cytokines (unstimulated NK), with rapamycin (RACR stimulated NK), or with cytokines (cytokine stimulated NK).

[0069] Fig.17A The timeline of the in vivo mouse model of breast cancer is shown. Five days before the start of the experiment, mice were injected with MDA-231mCherry / luciferase cells. One day before the start of the experiment, mice were subcutaneously injected with FITC-folic acid for two weeks. On the first day of the experiment, mice were injected intraperitoneally with RACR-TagCAR-NK-92 cells, followed by treatment with rapamycin three times a week or IL2 / IL15 three times a week.

[0070] Fig. 17B Shown are graphs depicting the quantification of luminescence associated with tumor growth in mice receiving various treatments over a three-week period.

[0071] Fig. 17C Shown are raw luminescent images of tumor growth in mice receiving various treatments over a three-week period.

[0072] Fig.17D RACR-NK in the blood of mice in FIG. 22C is depicted.

[0073] Fig.17E RACR-NK detection in mouse tissues in FIG. 22C is depicted.

[0074] Fig.18 Depicted is in vitro tumor cell killing in co-culture of RACR iCILs with MDA-MB-231 breast adenocarcinoma cells. Breast cancer cells were cultured alone and left untreated, treated with cytokines or rapalogs (left illustration), or co-cultured with RACR iCILs and left untreated, treated with cytokines or rapalogs (right illustration).

[0075] Fig.19A Depicts the timeline of the in vivo mouse model of breast cancer. Five days before the start of the experiment, mice were injected with MDA-231mCherry / luciferase cells. One day before the start of the experiment, mice were imaged and blood was drawn once a week during the duration of the experiment. On the first day of the experiment (D0), mice were injected with RACR-iCIL cells intraperitoneally, followed by treatment with rapamycin three times a week or IL2 / IL15 three times a week.

[0076] Fig.19B Depicted are raw luminescent images of tumor growth in mice that received various treatments over a three-week period.

[0077] Fig.19C Depicted is a timeline of an in vivo mouse model of breast cancer. Five days before the start of the experiment, mice were injected with MDA-231mCherry / luciferase cells. One day before the start of the experiment, mice were imaged once a week for the duration of the experiment. On the first day (D0) of the experiment, mice were injected with RACR-iCIL cells, followed by treatment with rapamycin three times a week or IL2 / IL15 three times a week. On the 27th day of the experiment, mice were injected with RACR-iCIL cells again.

[0078] Fig.19D Depicted are raw luminescent images of tumor growth in mice that received various treatments over a six-week period.

[0079] Fig.19E Shows Fig.19D Survival graph of mice described in .

[0080] Figures 20A-20D A set of graphs is shown, which depicts the 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 Fig. 20A The total number of RACR+Vd2+ cells over time is shown in Fig. 20B middle. Fig. 20A and Fig. 20BThe fold changes are shown in Fig. 20C and Fig.20D middle. DETAILED DESCRIPTION

[0081] Induced pluripotent stem cells (iPSCs) are a renewable, modifiable and scalable source of materials for cell therapy manufacturing. However, there are major challenges in the current iPSC-based methods for cell therapy. In particular, the current methods for differentiating iPSCs into therapeutic immune cell types require exogenous growth factors, and in some cases, the presence of feeder cells is required.

[0082] iPSCs can be prepared by reprogramming adult cells to a cell state similar to embryonic stem cells. iPSCs are believed to be able to differentiate into all cell types found in the human body and have unlimited amplification capacity, which means that they can be reproduced and proliferated indefinitely, producing an almost endless supply of starting materials. In addition, iPSCs are suitable for precise multiple genome editing, allowing the safe introduction of multiple genetic modifications. Due to these characteristics, iPSCs provide consistent starting materials derived from single cells (clones), which enables consistent genomic integrity in process intermediates and final cell products.

[0083] The current method for cell therapy manufacturing includes autologous or allogeneic cell starting materials, and the therapeutic cell products of targeted diseases are engineered from the materials. In some embodiments, allogeneic or "off-the-shelf" cell therapy has the potential to transform cell therapy from personalized medicine to conventional treatment. However, it is difficult for the current "off-the-shelf" cell therapy to show the same cell transplantation and persistence in vivo as the autologous cell therapy products that have been approved. In some aspects, this is due to the foreign nature of allogeneic elements or even engineered elements in autologous cells, which can be recognized and rejected by the host immune system. So far, cell therapy achieves cell transplantation by treating patients with highly toxic chemotherapy regimens, which are called lymphocyte clearance (LD) and are given before the cell therapy products are administered. LD basically removes the host immune system and provides many benefits for cell therapy products, first providing free "steady-state cytokines" for ex vivo cell therapy products, and reducing the host immune system's anti-graft response to foreign transplants. However, LD is a temporary solution, and the host immune system is quickly rebuilt. Therefore, in order to maintain allogeneic cell exposure, multiple rounds of LD and cell infusion are required. Furthermore, exogenous cytokines such as IL-2 are administered, and these cytokine treatments have low exposure times and high toxicity associated with their use. Finding better ways to increase cell persistence is key to achieving durable tumor remissions and has proven to be a challenge in the field of allogeneic cell therapy.

[0084] Allogeneic cells can be further divided into donor or iPSC-derived cells. Donor-derived cells are usually derived from the circulation or umbilical cord blood of healthy donors, and therapeutic cell types (e.g., natural killer or NK cells) are selected, then harvested, and amplified in a complex cell culture process that generally includes multiple cytokines, growth factors, genetic engineering and feeder cells, to produce multiple doses. Alternatively, iPSC-derived cells also require multiple complex cell culture conditions, which must be implemented in a stepwise manner to drive cells through the necessary progenitor cell stage, to ultimately obtain the expected final cell product (e.g., immune effector cells). For example, an effective method for CIL cell amplification for clinical scale purposes typically involves exogenous cytokines (including IL-2, IL-15 and / or IL-7) and antigen molecules, costimulatory molecules and / or cell adhesion molecules. CIL cells are cytotoxic lymphocytes, characterized in that they distinguish between self and non-self and directly kill non-self or infected target cells by monitoring the expression of MHC class I molecules, the release of cytokines. As known in the art, CIL cells do not represent uniform colonies. On the contrary, there are many distinguishable subsets of CIL cells. In many studies, these exogenous factors are supplemented during the ex vivo expansion of CIL cells and / or after the CIL cells are infused into the subject. Effectively expanding CIL cells using currently known methods relies on a large number of diverse exogenous factors, which usually require complex and expensive manufacturing processes. Another difficulty in this area relates to regulating the activity of endogenous CIL cells in vivo, particularly considering that cancer patients show significantly reduced NK cell activity compared to healthy patients.

[0085] In addition, the gold standard for cell therapy is autologous chimeric antigen receptor (CAR) T cell therapy. Starting with the "first generation" CAR T cell therapy in the 1990s and the FDA approval of the first CAR T cell therapy in 2017, decades of CAR T cell therapy efforts have been successful in the treatment of B-cell malignancies, with long-term remissions achieved in 30%-40% of certain patient populations. Importantly, the efficacy of CAR T cells requires lymphocyte depletion chemotherapy to eliminate the survival factor (such as IL-15) reservoir. Although CAR T cell therapy has revolutionized the treatment of malignancies (e.g., hematological malignancies), major limitations hinder its widespread application. The field of allogeneic CAR T therapy has shown promising early clinical results; however, despite the use of increasingly intensive LD regimens, the durable response spectrum is generally inferior compared to autologous CAR T cell therapy. This may be due to limitations in drug cell type, manufacturing process, and anti-allogeneic graft responses to therapeutic cells. Therefore, although the clinical efficacy of CAR T cells in hematological malignancies is promising, significant challenges remain, including patient access, complex manufacturing, and high costs. The provided engineered CIL cells and methods associated therewith provide "off-the-shelf" cancer therapies to overcome these challenges.

[0086] iPSCs can also be modified via CRISPR to express CARs, thereby overcoming challenges associated with targeting, for example, the heterogeneous solid tumor microenvironment.

[0087] Overall, iPSC-based cell therapies are often inefficient in generating the necessary intermediate progenitor cells, resulting in low initial yields of therapeutic cell types (e.g., cytotoxic innate lymphocytes (CILs)), which then require feeder cell-driven expansion. This feeder cell-driven expansion can significantly reduce the proliferation capacity of the final cell therapy product. Therefore, in order to achieve the necessary engraftment to have any therapeutic effect, high cell numbers (approximately 1 billion cells) and repeated dosing are required in addition to repeated cycles of lymphodepleting chemotherapy.

[0088] A platform is provided herein, and the platform is used to amplify immune effector cells by expressing synthetic cytokine receptors by genetically modifying lymphocytes such as CIL (for example, NK cells) in the absence of exogenous cytokines and feeder cells. In some aspects, the lymphocytes can be primary cells, or can be lymphoid progenitor cells or cells derived from or differentiated from lymphoid progenitor cells. In some embodiments, a platform is provided herein, and the platform is used to amplify immune effector cells by expressing synthetic cytokine receptors for differentiation into lymphocytes (such as CIL) by genetically modifying progenitor cells (for example, common lymphoid cells) in iPSC sources in the absence of exogenous cytokines and feeder cells. Synthetic small molecule ligands (for example, rapamycin) activate the receptor to drive the amplification of immune effector cells. In some embodiments, the synthetic cytokine receptor is a cytokine receptor (RACR) activated by rapamycin, which can be engaged by rapamycin or analogs (for example, rapalog). In some embodiments, the lymphocytes induced by RACR are lymphocyte types driven by RACR during differentiation and / or amplification. Examples of such "RACR-induced" cells include cytotoxic innate lymphocytes (RACR-iCIL), NK cells (RACR-iNK), or T cells (RACR-iT), including γδT cells or αβT cells. In certain embodiments, the RACR-induced cells (such as RACR-iCIL) are cells that are differentiated or expanded using RACR instead of the normal cytokine pathway (such as replacing the normal cytokine pathway from a progenitor cell (e.g., a common lymphoid progenitor cell or a pre-NK cell)).

[0089] The compositions and methods provided herein include lymphocytes engineered to express synthetic cytokine receptors, such as CIL cells (e.g., NK cells). The non-limiting advantages of the engineered lymphocytes such as CIL cells (e.g., NK cells) include excellent and controllable expansion when administered to a subject, similar cytotoxic activity compared to natural lymphocytes (e.g., CIL cells), improved iPSC-derived cell manufacturing and enhanced anti-tumor activity.

[0090] Improved cell manufacturing. In the aspect provided, 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 because RACR activation makes it unnecessary to add expensive growth factors, cytokines and other raw materials. In certain embodiments, the method disclosed herein can be further enhanced by the ability of amplification without using or using less exogenous factors (such as without using IL-2, IL-15 and / or IL-7) of engineered lymphocytes (such as CIL cells) described herein. The RACR engineering platform increases the yield of high-purity intermediates and final cell products. The RACR engineering platform increases the patient compatibility of cells because the manufacturing process is completely free of feeder cells and xenogeneic cells. The RACR engineering platform is also compatible with suspended cells, promoting the scalability of cell production.

[0091] Enhanced anti-tumor activity. In the aspect provided, the RACR engineering platform provided herein improves the anti-tumor activity of engineered lymphocytes such as CIL (including cells derived from iPSC) by increasing cell transplantation, persistence and effector function. The RACR engineering platform provided herein also improves the anti-tumor activity of engineered lymphocytes such as CIL (including cells derived from iPSC) by suppressing host immune response (which further enables transplantation of cells (e.g., CIL)) via rapamycin administration. The RACR engineering platform provided herein also improves the anti-tumor activity of engineered lymphocytes such as CIL (including cells derived from iPSC) by activating the RACR system to selectively support RACR cell expansion and survival without requiring toxic lymphocyte removal (LD).

[0092] In some aspects, the advantages of the RACR system for lymphocytes such as CIL (e.g., NK cells) (including engineering synthetic cytokine receptors and activating them with rapamycin or rapalog) include: the ability to engineer unlimited starting materials, which are effective in generating direct progenitor cells and characterized by minimizing the expansion requirements for the final cell type; the ability to effectively edit cells; no feeder cells are required, thereby minimizing complex raw materials; no lymphocyte clearance is required in subjects receiving RACR engineered cells; low to no cytokine release syndrome (CRS) or immune effector cell-related neurotoxicity syndrome (ICAN); and the administration of rapamycin or rapalog to promote transplantation, expansion and persistence. In some embodiments, engineered lymphocytes (such as RACR-iCIL) provide a cell source for allogeneic cell therapy, which can be achieved while minimizing or eliminating low immune engineering requirements in some aspects.

[0093] Provided herein are synthetic cytokine receptors (e.g., RACRs) that can be applied to support the derivation 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 distinguishing cell surface markers and functional attributes as described herein. As used herein, the term "induced cytotoxic innate lymphoid cells" or "iCIL" refers to CIL prepared by inducing the differentiation of progenitor cells. As disclosed herein, iCIL can be prepared and / or amplified by expressing synthetic cytokine receptors in stem cells or progenitor cells and acting on the synthetic cytokine receptors by non-physiological ligands. Such a process can involve differentiation of progenitor cells (e.g., lymphoid progenitor cells) engineered to express the synthetic cytokine receptors by activating synthetic cytokine receptors. The process may also involve or alternatively involve amplifying the progenitor cells or the CIL by activating the synthetic cytokine receptors.

[0094] In some embodiments, synthetic cytokine receptors (e.g., RACR) are provided herein, which can be derived to support the derivation or expansion of NK cells. NK cells derived from endogenous NK cells are referred to herein as primary NK cells. NK cells can also be derived from stem cells or progenitor cells, and such cells are referred to herein as natural killer (iNK) cells derived from iPSC. Primary NK cells and iNK cells share distinctive cell surface markers and functional attributes, but are not necessarily the same.

[0095] In a preferred embodiment, the present disclosure provides CIL cells (such as NK cells), which are engineered to express rapamycin-activated cytokine receptors (RACR), a synthetic cytokine receptor activated by small molecule rapamycin or rapalog. CIL cells comprising RACR and activated with rapamycin or rapalog are referred to herein as "RACR-iCIL" cells, for example, RACR-iNK cells. RACR has been shown to support differentiation and / or amplification of RACR-iCIL cells in the manufacturing process without feeders. RACR-iCIL cells express a variety of innate tumor targeting receptors, and when engineered to express chimeric antigen receptors (CAR), the cytolytic activity guided by CAR can be exerted. Therefore, RACR-iCIL cells provide "ready-made" allogeneic cell therapy.

[0096] The present disclosure relates in part to the unexpected discovery of the inventors that lymphocytes, such as cytotoxic innate lymphoid (CIL) cells (e.g., NK cells), engineered to express synthetic cytokine receptors can be expanded in response to cognate non-physiological ligands of the receptors. Engineered CIL cells can be produced in large quantities and have functional activities equal to or greater than CIL cells from other sources.

[0097] The present disclosure relates in part to the unexpected discovery of the inventors that common lymphoid progenitor (CLP) cells engineered to express synthetic cytokine receptors can differentiate into CIL cells in response to cognate non-physiological ligands of the receptors in the absence of exogenous factors, and the resulting CIL cells exhibit a cytotoxic phenotype. It is well known in the relevant art that the expansion of CIL cells generally relies on the use of various exogenous stimuli. The engineered CIL cells described herein have the ability to expand without or with fewer exogenous factors (e.g., without IL-2, IL-15, and / or IL-7).

[0098] The engineered CIL cells and related compositions described herein can be used for immunotherapy using ex vivo expansion.

[0099] like Figure 1A As shown in the figure, isolated lymphocytes such as CIL cells (e.g., NK cells) (including such primary cells isolated from peripheral blood) can be transduced with a vector comprising at least one polynucleotide encoding a synthetic cytokine receptor (e.g., RACR). When the transduced lymphocytes (e.g., CIL cells) are contacted with a non-physiological ligand, the extracellular domain of the cytokine receptor dimerizes by the mutual binding of the non-physiological ligand. This dimerization generates an amplification signal in the cell (e.g., CIL cell), thereby generating a phenotypically enriched and functionally active engineered cell (e.g., engineered CIL cell) group.

[0100] like Figure 1B As shown in the figure, stem cells or progenitor cells can be transduced with a vector comprising 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 domain of the cytokine receptor dimerizes. The dimerization generates a differentiation signal in the stem cell or progenitor cell, which induces continuous differentiation to become a CIL cell, such as a NK cell.

[0101] The CIL cells may be derived from iPSCs, common lymphoid progenitor cells (CLPs) or other stem cells or progenitor cells. Further provided herein are stem cells or progenitor cells engineered to express synthetic cytokine receptors, and methods for differentiating engineered stem cells or progenitor cells into CIL cells by contacting the stem cells or progenitor cells with the cognate non-physiological ligands of the cytokine receptors.

[0102] In certain embodiments, provided herein is a lymphocyte produced in vitro. In some embodiments, the lymphocyte or progenitor cell (common lymphoid progenitor cell or former NK cell) is engineered with a provided synthetic cytokine receptor (e.g., RACR). In such aspects, the non-physiological ligand of the synthetic cytokine receptor can be used for the lymphocyte or progenitor cell to amplify and / or differentiate in vitro to produce a lymphocyte population produced in vitro. The lymphocyte includes a T cell, an innate lymphoid cell (ILC) (including a cytotoxic innate lymphoid (CIL) cell, such as a natural killer (NK) cell). In some embodiments, the T cell is an αβT cell. In some embodiments, the T cell is a γδT cell.

[0103] In certain embodiments, provided herein are CIL cells produced in vitro. In some embodiments, the CIL cells or common lymphoid progenitor cells are engineered with provided synthetic cytokine receptors (e.g., RACR). In such aspects, the non-physiological ligands of the synthetic cytokine receptors can be used to amplify or differentiate the cells to produce CIL cells produced in vitro.

[0104] Engineered cells and related compositions described herein can be used for immunotherapy using ligand-controlled ex vivo expansion. Further provided herein is a method for expanding lymphocytes (e.g., CIL cells) engineered with the synthetic cytokine receptor (e.g., RACR), by contacting the cells with the homologous non-physiological ligands of the synthetic cytokine receptor. In addition, the engineered lymphocytes (e.g., engineered CIL cells) disclosed herein can be further engineered to express chimeric antigen receptors (CAR), so that the engineered lymphocytes (e.g., engineered CIL cells) can be targeted to express or be marked with cells of antigens recognized by the CAR.

[0105] In some embodiments, provided engineered lymphocytes (such as engineered CIL cells) and the methods provide improved immunotherapy compared to existing strategies.Although chimeric antigen receptor (CAR) T cell therapy has revolutionized the treatment of hematological malignancies, major limitations have hindered their widespread use.

[0106] In some embodiments, the engagement of synthetic cytokine receptors (e.g., RACR) can increase cell growth and promote expansion by the engagement of the synthetic cytokine receptors 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 the cell growth and in vivo expansion of engineered cell therapy by administering to patients (e.g., rapamycin administration of patients) with non-physiological ligands of synthetic cytokine receptors after cell therapy products. In some embodiments, the non-physiological ligands (e.g., rapamycin) simultaneously expand and protect the cells. In some embodiments, amplification is achieved by JAK / STAT signal activation, and protection is achieved by suppressing host-versus-graft response by rapamycin. In some embodiments, the need for lymphocyte removal and exogenous cytokine administration is not necessary. In some embodiments, the administration and treatment methods of the provided engineered lymphocytes (e.g., engineered iCIL cells) can be performed without lymphocyte removal (e.g., lymphocyte removal therapy, such as cyclophosphamide and / or fludarabine, is not required). In some embodiments, provided administration and treatment methods using engineered lymphocytes (such as engineered iCIL cells) can be performed without the administration of exogenous cytokines (eg, without the need for administration of IL-2 and / or IL-15).

[0107] Thus, provided embodiments employing synthetic cytokine receptor systems such as rapamycin-activated cytokine receptors (RACRs) that can be engaged by rapamycin or an analog (e.g., rapalog) both protect and expand cells with a single technique. Furthermore, additional inclusion of gene disruptions such as knockout of certain immune genes such as beta-2-microglobulin (B2M) can also generate "stealth" cells, which have additional advantages for allogeneic cell therapy.

[0108] All publications (including patent documents, scientific articles, and databases) mentioned in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication was individually incorporated by reference. If definitions set forth herein are contrary to or otherwise inconsistent with definitions set forth in patents, applications, published applications, and other publications incorporated herein by reference, the definitions set forth herein take precedence over definitions incorporated herein by reference.

[0109] The section headings used herein are only for organizational purposes and should not be construed as limiting the subject matter described. Those skilled in the art will recognize that several embodiments may be provided within the scope and spirit of the present disclosure. The following description illustrates the present disclosure and certainly should not be construed as limiting the scope of the invention as described herein in any way. I. Definitions

[0110] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0111] Unless the context dictates otherwise, the various features described herein may be used in any combination with any feature or combination of features shown herein, and each feature may be excluded or omitted from a combination.

[0112] As used herein, the singular forms "a", "an" and "the" include the plural forms as well, unless the context indicates otherwise. The conjunction "and / or" refers to all possible combinations of one or more of the listed items.

[0113] As used herein, "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.

[0114] As used herein, "treat," "treating," or "treatment" refers to any type of action or administration that results in a benefit to a subject suffering from a disease or disorder, including improvement of the patient's condition (i.e., improvement, reduction, or alleviation of one or more symptoms, as well as partial or complete response to treatment).

[0115] The term "effective amount" refers to an amount effective to produce a desired biochemical, cellular or physiological response. The term "therapeutically effective amount" refers to an amount, dosage or dosage regimen of a therapy effective to induce a desired therapeutic effect.

[0116] As used herein, "polynucleotide" refers to a biopolymer composed of two or more nucleotide monomers covalently bonded by an ester bond between the phosphoryl group of one nucleotide and the hydroxyl group of the sugar component of the next nucleotide in the chain. DNA and RNA are non-limiting examples of polynucleotides.

[0117] As used herein, "polypeptide" refers to a polymer composed of amino acid residues chained together by peptide bonds, which forms part (or all) of a protein.

[0118] It will be appreciated by the skilled artisan that due to the degeneracy of the genetic code, many different polynucleotides and nucleic acids can encode the same polypeptide. In addition, it will be appreciated that the skilled artisan can use conventional 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 will be expressed.

[0119] Nucleic acids can include DNA or RNA. They can be single-stranded or double-stranded. They can also be polynucleotides including synthetic or modified nucleotides. Many different types of modifications to oligonucleotides are known in the art. These modifications include methylphosphonate and phosphorothioate backbones, adding acridine or polylysine chains at the 3' and / or 5' ends of the molecule. For the purposes of use as described herein, it should be understood that the polynucleotides can be modified by any method available in the art. Such modifications can be performed to enhance the in vivo activity or lifespan of the related polynucleotides.

[0120] The term "variant" means a polynucleotide or polypeptide having at least one substitution, insertion or deletion in the sequence compared to a reference polynucleotide or polypeptide. A "functional variant" is a variant that retains one or more functions of a reference polynucleotide or polypeptide.

[0121] As used herein, the term "sequence identity" or "identity" related to a polynucleotide or polypeptide sequence refers to the degree to which two optimally aligned polynucleotide or polypeptide sequences match at each position in an alignment across the full length of a reference sequence."Percentage identity" is the sum of the number of matching positions in the optimal alignment divided by 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 maximum percentage identity. Sequence alignments for determining percentage identity can be accomplished by many well-known methods, including, for example, by using mathematical algorithms, such as those in the BLAST suite or Clustal Omega sequence analysis program. Unless otherwise indicated, the term "sequence identity" in the claims refers to sequence identity calculated using default parameters by BLAST version 2.12.0. And, unless otherwise indicated, alignment is the alignment of all or part of the related polynucleotide or polypeptide sequence over the full length of the reference sequence.

[0122] As used herein, "small molecule" refers to an organic compound of low molecular weight (<1000 Daltons). Small molecules can bind to specific biomacromolecules and can have a variety of biological functions or applications, including but not limited to acting as cell signaling molecules, drugs, secondary metabolites, or various other modes of action.

[0123] The term "analog" in relation to small molecules refers to a compound that is similar in structure and / or function to another compound, but differs therefrom with respect to a certain component. Analogs may differ in that one or more atoms, functional groups or substructures are replaced by other atoms, groups or substructures. Despite having a high degree of structural and / or functional similarity, analogs may have different physical, chemical, physiochemical, biochemical or pharmacological properties.

[0124] The term "rapalog" refers to a group of art-recognized analogs of rapamycin that share structural and functional similarities with rapamycin. Some rapalogs are known to share some, but not all, of the functional properties of rapamycin. For example, some rapalogs are suitable for use as non-physiological ligands because they promote dimerization but have substantially no immunosuppressive activity (e.g., AP21967, AP23102, or iRAP).

[0125] An illustrative rapalog of the present disclosure is AP21967

[0126] An illustrative rapalog of the present disclosure is AP23102

[0127] An illustrative rapalog of the present disclosure is iRAP

[0128] The term "cell population" refers to a mixture of cells suspended in solution, attached to a matrix, or stored in a container. The characteristics of the cell population as a whole can be studied by whole-body measurements of a sample size with multiple cells. Flow cytometry methods can be used to reduce the background fluorescence problems encountered in whole-body cell population measurements.

[0129] As used herein, the term "cytotoxic innate lymphoid cells" or "CIL cells" is used to refer to a class of cytotoxic lymphocytes that constitute the main component of the innate immune system. In humans, cytotoxic innate lymphoid cells typically express surface markers CD16 (FCyRIII), CD56, and may express CD127. They may express one or more of CD45, CD94, CD122, KIR, NKG2A, NKG2D, NKp30, NKp44, NKP46, NKp80. Cytotoxic innate lymphoid cells typically do not express CD3, or express lower levels of CD3 than CD3+T cells. CIL cells are cytotoxic and contain small granules in their cytoplasm that contain special proteins, such as perforin and proteases called granzymes. CIL cells provide a rapid response to virus-infected cells and respond to transformed cells. When released very close to cells intended to be killed, perforin forms holes in the cell membrane of the target cell, through which granzymes and related molecules can enter, thereby inducing apoptosis. CIL cells can act as effectors of lymphocyte populations in anti-tumor and anti-infective immunity. In some embodiments, the CIL cells are NK cells. In some embodiments, the 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" does not include adaptive immune cells and their progenitor cells, and does not include common lymphoid progenitor cells (CLP). However, CIL can be derived from CLP.

[0130] As used herein, the term "engineered" refers to cells that have been stably transduced with a heterologous polynucleotide or have undergone gene editing to introduce, delete or modify a polynucleotide in the cell, or cells that have been transiently transduced with a polynucleotide in a manner that causes a stable phenotypic change in the cell.

[0131] As used herein, the term "stem cell" is used to describe a cell with an undifferentiated phenotype that is capable of differentiating into, for example, a common lymphoid progenitor cell, a cytotoxic innate lymphoid cell, and / or a NK cell.

[0132] As used herein, the term "pluripotent" means that a stem cell is capable of forming essentially all differentiated cell types of an organism, at least in culture. For example, embryonic stem cells are a type of pluripotent stem cell that is capable of forming cells from each of the three germ layers (ectoderm, mesoderm, and endoderm).

[0133] As used herein, the terms "induced pluripotent stem cells" and "iPSCs" are used to refer to cells derived from somatic cells that have been reprogrammed back to a pluripotent state and are capable of proliferation, selective differentiation, and maturation. iPSCs are stem cells produced from differentiated adult, neonatal, or fetal cells that have been induced or altered (i.e., reprogrammed) to cells capable of differentiating into tissues of all three germ layers or cortex (mesoderm, endoderm, and ectoderm). The iPSCs produced do not refer to cells found in nature.

[0134] As used herein, the term "hematopoietic stem cell" refers to a stem cell capable of generating mature myeloid and lymphoid cell types, including natural killer cells, T cells, and B cells. Hematopoietic stem cells are typically characterized as CD34+.

[0135] The term "progenitor cell" refers to a cell that is partially differentiated into a desired cell type. Progenitor cells retain a certain degree of multipotency and can differentiate into a variety of cell types.

[0136] As used herein, the term "hematopoietic progenitor cell" refers to a cell of an intermediate cell type that is capable of down-differentiating into a blood cell lineage, wherein the hematopoietic progenitor cell can differentiate into a common myeloid progenitor cell or a common lymphoid progenitor cell. Hematopoietic progenitor cells are typically characterized by CD34+ and CD45+. CD38 is also considered a marker for hematopoietic progenitor cells. CD45 is considered a hematopoietic lineage marker.

[0137] As used herein, the term "lymphoid progenitor cell" or "lymphoblast" or "common lymphoid progenitor cell" refers to a cell that is a precursor of a lymphoid cell (e.g., CIL and NK cells). Lymphoid progenitor cells are the first stage of differentiation of hematopoietic stem cells that follow the lymphoid differentiation lineage. As used herein, the term "lymphoid progenitor cell" refers to a cell that is capable of hematopoietic transformation into a hematopoietic cell type. Lymphoid progenitor cells may be characterized by CD45+CD7+CD5+ / lo CD3-CD56-. Lymphoid progenitor cells may be characterized by CD45+CD5+ / lo CD7+.

[0138] As used herein, "differentiation" or "differentiated" is used to refer to the process and conditions that cause undifferentiated or immature (e.g., unspecialized) cells to acquire the characteristics of mature (specialized) cells, thereby acquiring a specific form and function. Stem cells (unspecialized) are often exposed to different conditions (e.g., growth factors and morphogenetic factors) to induce the specified lineage commitment (commitment) or differentiation of the stem cells.

[0139] As used herein, "expand" or "expansion" refers to increasing the number and / or purity of a cell type within a cell population by mitosis of cells with limited proliferation capacity (eg, CIL cells).

[0140] As used herein, "activity," "activate," or "activation" refers to stimulation of activating receptors on cytotoxic innate lymphoid cells, leading to cell division, cytokine (e.g., IFNγ and / or TNFα) secretion, and / or cytolytic granule release to modulate or assist an immune response. II. Source Cells and Synthetic Cytokine Receptor (eg, RACR) Engineered Cells

[0141] In some aspects, provided herein are engineered cells, the engineered cells comprising synthetic cytokine receptors of non-physiological ligands such as synthetic small molecule ligands (e.g., rapamycin), wherein the ligand can activate the synthetic cytokine receptors to drive the 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, lymphocytes include T cells, innate lymphoid cells (ILC) (including cytotoxic innate lymphoid (CIL) cells, such as natural killer (NK) cells). In some embodiments, the T cells are αβT cells. In some embodiments, the T cells are γδT cells. In some embodiments, the engineered cells are progenitor cells, such as common lymphoid progenitor cells, which can differentiate into lymphocytes, such as CIL or NK cells. Also provided are the resulting cell groups induced to differentiate or expand from such engineered lymphocytes or progenitor cells. In some embodiments, the cell groups are iCIL.

[0142] In some embodiments, the synthetic cytokine receptor is composed of: a synthetic γ chain polypeptide comprising a first dimerization domain, a first transmembrane domain, and an interleukin-2 receptor subunit γ (IL-2RG) intracellular domain; and a synthetic α or β chain polypeptide comprising a second dimerization domain, a second transmembrane domain, and an intracellular domain selected from the following: an interleukin-2 receptor subunit β (IL-2RB) intracellular domain, an interleukin-7 receptor subunit β (IL-7RB) intracellular domain, an interleukin-21 receptor subunit β (IL-21RB) intracellular domain, and / or an interleukin-9 receptor subunit α (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. Also provided herein are methods for preparing and / or amplifying engineered cell populations.

[0143] In some embodiments, engineered cell populations are obtained by introducing polynucleotides encoding synthetic cytokine receptors or vectors comprising polynucleotides encoding synthetic cytokine receptors into source cells. Source cells can be obtained from original sources, including but not limited to peripheral blood mononuclear cells (PBMC) or umbilical cord blood (UCB). The cell sources that can be engineered can also include cells differentiated from stem cells, such as pluripotent stem cells (iPSC) (which are cells derived from somatic cells (usually fibroblasts or peripheral blood mononuclear cells [PBMC])), and human embryonic stem cells (hESC). In some embodiments, the iPSC is human iPSC. In some embodiments, when undergoing appropriate differentiation conditions, stem cells can be differentiated into other cell types, and then engineered with synthetic cytokine receptors. In some embodiments, the differentiation conditions include suitable differentiation factors for making cells differentiate into lymphoid progenitor cells or lymphocytes (such as ILC, including CIL).

[0144] In some embodiments, the engineered cell is an ILC. ILC is a recently discovered family of innate immune cells derived from common lymphoid progenitor cells (CLP). ILC can be a cytotoxic innate lymphoid (CIL) cell or non-cytotoxic. CIL includes natural killer (NK) cells. In some aspects, the features that distinguish ILC from other immune cells include its 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 cells or dendritic cells (Spits and Cupedo, Annual Review of Immunology. (2012) 20: 647-75).

[0145] In some aspects, provided herein is a pharmaceutical composition comprising any engineered cell or cell group (e.g., lymphocyte, such as ILC, such as CIL) provided herein. In even other aspects, provided herein is a method for amplifying 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 engineered cell provided herein.

[0146] In some embodiments, the cells are engineered from peripheral blood cells. As used herein, the term "peripheral blood cells" is used to refer to cells derived from circulating blood and comprising hematopoietic stem cells capable of proliferation, selective differentiation and maturation. Therefore, peripheral blood NK cells can alternatively be referred to as differentiated blood-derived NK cells (bdNK).

[0147] In some embodiments, lymphocytes, such as those used to produce engineered CIL cells, can be obtained from a donor or subject (for autologous therapy) by various means well known in the art. For example, lymphocytes can be obtained in the following manner: peripheral blood containing peripheral blood mononuclear cells (PBMC) is collected from a patient, and the blood is subjected to Ficoll density gradient centrifugation and / or leukocyte apheresis, and then a lymphocyte population is separated from the peripheral blood using a separation kit. In an illustrative embodiment, the lymphocyte population does not need to be pure for the selected cell type, and other cell types may be contained, such as T cells, monocytes, macrophages, natural killer cells, and B cells. In some embodiments, the collected cell population may include at least about 90% of the selected cell type, at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the selected cell type.

[0148] In some embodiments, NK cells can be produced by 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 the production of larger amounts of cells without having to amplify a small amount of NK cells derived from a subject. Engineered cell lines also have the advantage of being well characterized.

[0149] 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 lymphoma. In some embodiments, the NK cell line is NK-92, NK-YS, KHYG-1, NKL, NKG, SNK-6 or IMC-1.

[0150] NK-92 cell line is an NK-like cell line, which was initially isolated from the blood of a subject with large granular lymphoma, and subsequently propagated in cell culture. NK-92 cell line has been described (Gong et al., 1994; Klingemann, 2002). NK-92 cells have a CD3- / CD56+ phenotype that is a feature 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 (expressed at low levels). In addition, NK-92 is a clonal cell line that is different from polyclonal NK cells isolated from blood, expressing these receptors in a consistent manner in terms of type and cell surface concentration. Similarly, when therapeutically administered to human subjects, NK-92 cells have no immunogenicity and do not cause immune rejection. In fact, NK-92 cells are well tolerated in the human body and have no known adverse effects on normal tissues.

[0151] Vectors for engineering cells include 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 (HSC), characterized by CD34+ and / or CD45+; common lymphoid progenitor cells (CLP), characterized by CD45+CD7+CD56-; CIL progenitor cells, characterized by CD45+CD5-CD7+; and / or CIL cells, characterized by CD45+CD56+CD3- and optionally CD5- and / or CD7+.

[0152] In some embodiments, the source cell can be a human embryonic stem cell (hESC) or an induced pluripotent stem cell (iPSC), which can be differentiated into lymphocytes or lymphoid progenitor cells, including any of the above. Lymphocytes or lymphoid progenitor cells can then be engineered with synthetic cytokine receptors (e.g., RACR) to replace the normal cytokine pathways from precursor cells (common lymphoid progenitor cells or former NK cells) to drive expansion and / or differentiation. In immunotherapy, the source cell is allogeneic or autologous, meaning from a donor or from a subject, respectively.

[0153] 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 known as hematopoietic stem cells (HSCs); differentiating HPCs into common lymphoid progenitor cells (CLPs); and then differentiating CLPs into CIL cells, referred to as iPSC-derived cytotoxic innate lymphoid cells (iPSC-CILs or iCILs).

[0154] iPSC is a pluripotent stem cell type derived from adult somatic cells (e.g., fibroblasts or PBMCs), which have been genetically reprogrammed to an embryonic stem cell-like state by the forced expression of genes and factors important for maintaining the defining characteristics of embryonic stem cells. iPSC can be produced from tissues (including but not limited to skin, dental tissue, peripheral blood and urine) with somatic cells. In order to produce iPSC, somatic cells can be reprogrammed by including but not limited to the following methods: transient expression of reprogramming factors, virus-free methods, adenovirus, plasmid, mini-circle vector, episomal vector, Sendai virus, synthetic mRNA, self-replicating RNA, retrovirus, slow virus, PhiC31 integrase, excisable transposon, gene editing or recombinant protein based on CRISPR.

[0155] Although the cell therapy industry has demonstrated the transformative potential of using genetically engineered patient-derived cells to treat specific disease indications, there are still many challenges in using patient-derived materials, including limited amplification capacity and scalability, manufacturing complexity, high cost, variability between patients, and patient channels. In contrast, iPSC is a pluripotent stem cell, a cell that can theoretically differentiate into any other cell type (including cytotoxic innate lymphoid (CIL) cells suitable for cancer treatment). Using iPSC, scalable and simplified manufacturing of antagonistic target cells (e.g., anti-cancer) cell therapies such as CIL cells can be provided, thus reducing the cost of cell therapy and improving the access of patients to the cell therapy. iPSC has unlimited amplification capacity, which means that they can be infinitely propagated and proliferated, potentially producing an almost endless supply of differentiated immune cells for therapy (such as cancer therapy). iPSC is also suitable for precise multiple genome editing, allowing the introduction of multiple genetic modifications to enhance their disease targeting capabilities and the safety of the immune cells they eventually become. iPSCs can similarly be engineered with the goal of protecting them from allogeneic rejection by the patient's own immune system, thereby improving their initial expansion and duration of transplantation. Furthermore, while patient-derived or donor-derived blood material is inconsistent, iPSCs provide consistent starting material derived from single-cell clones, which can allow for genomic consistency and integrity in the final cell product.

[0156] Various differentiation protocols for CIL cells are known in the art. Examples of methods for differentiating stem cells (e.g., iPSCs) into multipotent hematopoietic progenitor cells are provided in the following documents: U.S. Patent No. 9,624,470, U.S. Patent Application No. 2020 / 0080059 and Mesquitta et al., Sci. Rep. 9: 6622 (2019), the disclosure of which is incorporated herein by reference in its entirety. 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 Nos. 9,260,696 and 10,626,372, and Zhu and Kaufman, Methods Mol. Biol. 2048: 107-119 (2019) and Matsubara et al., Biochem. Biophys. Res. Commun. 515(1): 1-8 (2019), the disclosures of which are incorporated herein by reference in their entirety.

[0157] Broadly, the technology for differentiated cells involves using methods based on polynucleotides, polypeptides and / or small molecules to directly or indirectly regulate specific cell pathways. The developmental effectiveness of the cell can be regulated, for example, by contacting the cell with one or more regulators. In some embodiments, cells are cultured to induce cell differentiation in the presence of one or more medicaments (such as, for example, small molecules, proteins, peptides, etc.). In some embodiments, one or more differentiation agents are introduced into cells during in vitro culture. Cells can be maintained in a culture medium comprising one or more medicaments for a period of time sufficient to enable the cell to achieve a desired differentiation phenotype.

[0158] In some embodiments, stem cells are suitable for feeder-free culture. As used herein, a "feeder-free" (FF) environment refers to an environment such as culture conditions, cell culture or culture medium that is substantially free of feeders or stromal cells, and / or has not yet been preconditioned by the cultivation of feeder cells. "Preconditioned" culture medium refers to a culture medium harvested after feeder cells have been cultivated in the culture medium for a period of time (such as at least one day). The preconditioned culture medium comprises a number of media substances, including growth factors and cytokines secreted by the feeder cells cultivated in the culture medium.

[0159] In some embodiments, the culture platform comprises one or more of the following: nutrients, extracts, growth factors, hormones, cytokines, and culture medium additives. Exemplary nutrients and extracts may include, for example, DMEM / F-12 (Dulbecco's Modified Eagle's Medium / Nutrient Mixture F-12), which is a widely used basal medium for supporting the growth of many different mammalian cells; KOSR (knockout serum replacement); L-glut; NEAA (non-essential amino acids). Culture medium additives may include, but are not limited to, MTG, ITS, ME, antioxidants (e.g., ascorbic acid). In some embodiments, the culture medium of the present invention comprises one or more of the following cytokines or growth factors: epidermal growth factor (EGF), acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), leukemia inhibitory factor (LIF), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), insulin-like growth factor 2 (IGF-2), keratinocyte growth factor (KGF), nerve growth factor (NGF), platelet-derived growth factor (PDGF), transforming growth factor β (TGF-β), bone morphogenetic protein (BMP4), vascular endothelial growth factor (VEGF), transferrin, various interleukins (such as IL-1 to IL-18), various colony stimulating factors (such as granulocyte / macrophage colony stimulating factor (GM-CSF)), various interferons (such as IFNγ), and other cytokines (such as stem cell factor (SCF) and erythropoietin (EPO)).

[0160] These cytokines are commercially available and may be natural or recombinant. In some other embodiments, the culture medium of the present disclosure includes one or more of the following: bone morphogenetic protein (BMP4), insulin-like growth factor-1 (IGF-1), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), hematopoietic growth factor (e.g., SCF, GMCSF, GCSF, EPO, IL3, TPO, EPO), Fms-related tyrosine kinase 3 ligand (FLT3L); and one or more cytokines from leukemia inhibitory factor (LIF), IL3, IL6, IL7, IL11, IL15. In some embodiments, the concentrations of growth factors, mitogens, and cytokines are stage and / or cell type specific, determined empirically or guided by established cytokine technology. Examples of exogenous cell culture medium additives and supplements and cell selection kit components are provided in WO 2020 / 124256, the disclosure of which is incorporated herein by reference in its entirety.

[0161] In some other embodiments, the culture medium of the present disclosure comprises Roswell Park Memorial Institute (RPMI) medium, cRPMI1640 medium, fetal bovine serum (FBS), Glutamax, penicillin, streptomycin, rosuvastatin, BX795, protamine sulfate, brefeldin A, monensin, UM729, IL-2, IL-15, IL-21, IL-18, IL-7, or any combination thereof.

[0162] In some embodiments, 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.

[0163] In some embodiments, the differentiation medium contains supplements, such as serum, extracts, growth factors, hormones, cytokines, and the like.

[0164] Cell differentiation for producing CIL cells requires changing the culture system, such as changing the stimulant in the culture medium or the physical state of the cell. 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 of three-dimensional structures / interactions with three-dimensional structures). The formation of embryoid bodies induces cells to differentiate cells into mesoderm specialization.

[0165] The pluripotent stem cell aggregates are transferred to a differentiation medium that provides a cue for a selected lineage (e.g., lymphoid). In some embodiments, EBs are dissociated immediately after formation and then cultured in a culture medium to induce the mesodermal specificity of the cells.

[0166] In some aspects of the invention, the above-described platform for EB derivation and differentiation into hematopoietic progenitor cells can be performed under serum-free conditions. Examples of commercially available serum-free culture media suitable for cell attachment and / or induction include: mTeSR from StemCell Technologies (Vancouver, Canada); TM 1 or TeSR TM 2. Primate ES / iPS cell culture medium from ReproCELL (Boston, MA), From Invitrogen hESC SFM and X-VIVO from Lonza (Basel, Switzerland) TM .

[0167] In some embodiments, the culture medium for inducing mesoderm specificity step comprises one or more of the following: 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 culture medium for inducing mesoderm specificity step comprises one or more of the following: 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.

[0168] In some embodiments, the method includes differentiating the mesodermal specialized cells into hematopoietic stem cells in a first differentiation medium.

[0169] In some embodiments, the hematopoietic stem cells in the first differentiation medium contain one or more of the following: bone morphogenetic protein 4 (BMP4), fibroblast growth factor 2 (FGF2), vascular endothelial growth factor (VEGF), stem cell factor (SCF), Fms-related tyrosine kinase 3 ligand (FLT3L), thrombopoietin (TPO), interleukin-6 (IL-6), interleukin-3 (IL-3), TGF-β inhibitor, PI3K inhibitor, or any combination thereof.

[0170] In some embodiments, the TGF-β inhibitor is GW788388. In some embodiments, the PI3K inhibitor is LY294002.

[0171] In some embodiments, stem cells are differentiated to the mesodermal lineage in a differentiation medium comprising BMP4, FGF2, and VEGF.

[0172] In some embodiments, mesodermal cells are differentiated into hematopoietic stem cells in a differentiation medium comprising BMP4, FGF2, VEGF, and SCF.

[0173] In some embodiments, hematopoietic stem cells are differentiated into lymphoid progenitor cells in a differentiation medium comprising BMP4, FGF2, VEGF, and SCF.

[0174] In some embodiments, lymphoid progenitor cells are differentiated into cytotoxic innate lymphoid cells in a differentiation medium comprising a non-physiological ligand, SCF, FLT3L, and UM729. In some embodiments, the non-physiological ligand is rapalog. In some embodiments, the non-physiological ligand is rapamycin.

[0175] In some embodiments, the mesoderm specialized cells are differentiated into hematopoietic stem cells in the first differentiation medium for a period of time sufficient to allow the mesoderm specialized cells to become CD34+ hematopoietic stem cells.

[0176] In some embodiments, the method includes differentiating the hematopoietic stem cells into lymphoid progenitor cells in a second differentiation medium.

[0177] In some embodiments, the lymphoid progenitor cell differentiation medium comprises one or more of the following: bone morphogenetic protein 4 (BMP4), fibroblast growth factor 2 (FGF2), vascular endothelial growth factor (VEGF), stem cell factor (SCF), Fms-related tyrosine kinase 3 ligand (FLT3L), thrombopoietin (TPO), interleukin-6 (IL-6), interleukin-3 (IL-3), TGF-β inhibitor, PI3K inhibitor, or any combination thereof.

[0178] In some embodiments, the hematopoietic stem cells are differentiated into lymphoid progenitor cells in the second differentiation medium for a period of time sufficient to allow the hematopoietic stem cells to become Lin-CD34+CD38- / loCD45RA+CD90- lymphoid progenitor cells.

[0179] In some embodiments, the first differentiation medium further comprises a non-physiological ligand of the disclosure and / or the second differentiation medium further comprises a non-physiological ligand of the disclosure.

[0180] 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).

[0181] In some embodiments, the differentiation medium comprises one or more of the following: stem cell factor (SCF), interleukin-7 (IL-7), interleukin-15 (IL-15), Fms-related tyrosine kinase 3 ligand (FLT3L), pyrimido-indole derivatives, or any combination thereof. The pyrimido-indole derivative is UM 729 (pyrimido-[4,5-b]-indole derivative).

[0182] In some embodiments, lymphoid progenitor cells are differentiated into CIL cells in a differentiation medium for a period of time sufficient to make lymphoid progenitor cells CD3-CD56+CD45+CD94+CD122+ / IL-2Rβ+CD127 / IL-7Rα-FcγRIII / CD16+KIR+NKG2A+NKG2D+NKp30+NKp44+NKP46+NKp80+. In some embodiments, lymphoid progenitor cells are differentiated into CIL cells in a differentiation medium for a period of time sufficient to make lymphoid progenitor cells CD45+CD56+. In some embodiments, lymphoid progenitor cells are differentiated into CIL cells in a differentiation medium for a period of time sufficient to make lymphoid progenitor cells CD45+CD5-CD7+CD56+.

[0183] In some embodiments, the lymphoid progenitor cells are differentiated into CIL cells in the differentiation medium for about 8 to about 18 days. In some embodiments, the lymphoid progenitor cells are differentiated into CIL cells in the differentiation medium for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 days.

[0184] 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.

[0185] In some embodiments, after differentiation, the CIL cells are expanded in a medium comprising non-physiological ligands and CD2 / NKp46 stimulation. 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.

[0186] 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-activated feeder cells. Induced cytotoxic innate lymphoid cells (iCIL)

[0187] In some embodiments, CIL cells can be derived from iPSCs by: sequentially, differentiating iPSCs into hematopoietic progenitor cells (HPCs); differentiating HPCs into common lymphoid progenitor cells (CLPs); and then differentiating CLPs into CIL cells, referred to as "iCIL" cells. In a variant, CIL cells can be derived from HPCs by: sequentially, differentiating HPCs into CLPs; and then differentiating CLPs into iCIL cells. In another variant, CIL cells can be obtained by differentiating CLPs into iCIL cells. Engineering cells to express synthetic cytokine receptors can be performed at the HPC, CLP, or iCIL cell step of the differentiation process.

[0188] In some embodiments, the iCIL cells are characterized by 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 by CD3-CD56+CD45+ cells. In some embodiments, the iCIL cells are characterized by CD3-CD56+ cells. In some embodiments, the iCIL cells comprise one or more cell markers selected from the following: CD56+, CD45+, CD94+, CD122+ / IL-2Rβ+, FcγRIII / CD16+, KIR+, NKG2A+, NKG2D+, NKp30+, NKp44+, NKP46+, NKp80+, or any combination thereof. The iCIL cells may be CD45+; CD45+CD5-; or CD45+CD5-CD56+.

[0189] In some embodiments, the iCIL cells are characterized by being CD45+CD7+CD56 + / lo of.

[0190] In some embodiments, after the iCIL cells have been transduced with a CAR, the cells are cultured under conditions that promote cell activation.

[0191] The culture conditions can be such that the cells can be administered to a patient without concern for reactivity to culture medium components. For example, the culture conditions can omit bovine serum products, such as bovine serum albumin. In an illustrative aspect, the activation can be achieved by introducing a known activator into the culture medium. In one aspect, the iCIL cell population can be cultured for about 1 to about 4 days under conditions that promote activation. In one embodiment, the appropriate level of activation can depend on the cell size, proliferation rate, or activation markers determined by flow cytometry. In some embodiments, any culture method disclosed herein can be used to promote the activation of iCIL cells.

[0192] In some embodiments, provided herein are methods for preparing and / or expanding engineered cell populations comprising synthetic cytokine receptors for non-physiological ligands. In some embodiments, the methods include: a. providing an engineered cell comprising a synthetic cytokine receptor, optionally by introducing into a source cell a polynucleotide encoding a synthetic cytokine receptor, and b. incubating the engineered cells in a culture medium comprising a non-physiological ligand.

[0193] In this embodiment, the cytokine receptor comprises the following: a synthetic γ chain polypeptide, which comprises a first dimerization domain, a first transmembrane domain and an interleukin-2 receptor subunit γ (IL-2RG) intracellular domain; and a synthetic α chain or β chain polypeptide, which comprises a second dimerization domain, a second transmembrane domain and an intracellular domain selected from the following: an interleukin-2 receptor subunit β (IL-2RB) intracellular domain, an interleukin-7 receptor subunit β (IL-7RB) intracellular domain, an interleukin-21 receptor subunit β (IL-21RB) intracellular domain and / or an interleukin-9 receptor subunit α (IL-9RB) intracellular domain; wherein the non-physiological ligand activates the synthetic cytokine receptor in the engineered cells to induce expansion and / or activation of the engineered cells.

[0194] In this embodiment, the cytokine receptor comprises the following: a synthetic γ chain polypeptide, which comprises a first dimerization domain, a first transmembrane domain and an interleukin-2 receptor subunit γ (IL-2RG) intracellular domain; and a synthetic β chain polypeptide, which comprises a second dimerization domain, a second transmembrane domain and an intracellular domain selected from the following: an interleukin-2 receptor subunit β (IL-2RB) intracellular domain, an interleukin-7 receptor subunit β (IL-7RB) intracellular domain and / or an interleukin-21 receptor subunit β (IL-21RB) intracellular domain; wherein the non-physiological ligand activates the synthetic cytokine receptor in the engineered cells to induce expansion and / or activation of the engineered cells.

[0195] In some embodiments, iPSCs are gene edited using lentivirus.

[0196] In some embodiments, HSCs are gene-edited using a lentivirus. In some embodiments, blood progenitor cells are gene-edited using a lentivirus. In some embodiments, common lymphoid progenitor cells are gene-edited using a lentivirus. In some embodiments, NK precursor cells are gene-edited using a lentivirus. In some embodiments, common lymphoid progenitor (CLP) cells are gene-edited using a lentivirus. III. Retroviral Particles

[0197] 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. Slow viruses are complex retroviruses that contain other genes with regulatory or structural functions in addition to the common retroviral genes gag, pol and env. The higher complexity enables the virus to regulate its life cycle, such as during latent infection. Some examples of slow viruses include human immunodeficiency virus (HIV-1 and HIV-2) and simian immunodeficiency virus (SIV). Slow virus vectors have been produced by repeatedly weakening HIV virulence genes, such as by deleting genes env, vif, vpr, vpu and nef, so that the vector is biologically safe.

[0198] Illustrative 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. Pat. No. 6,013,516; and U.S. Pat. No. 5,994,136, each of which is incorporated herein by reference in its entirety. Typically, these vectors are configured to carry the necessary sequences for selecting cells containing the vector, incorporating foreign nucleic acids into lentiviral particles, and transferring nucleic acids into target cells.

[0199] Commonly used lentiviral vector systems are so-called third generation systems. The third generation lentiviral vector system includes four plasmids. "Transfer plasmid" encodes a polynucleotide sequence delivered to a target cell by a lentiviral vector system. The transfer plasmid usually has one or more target transgenic sequences flanked by long terminal repeat (LTR) sequences, which promote the integration of the transfer plasmid sequence into the host genome. For safety reasons, transfer plasmids are usually designed to make the resulting vector unable to replicate. For example, the transfer plasmid lacks the genetic elements necessary for producing infectious particles in the host cell. In addition, the transfer plasmid can be designed to lack 3'LTR, making the virus "self-inactivated" (SIN). See Dull et al. (1998) J.Virol.72:8463-71; Miyoshi et al. (1998) J.Virol.72:8150-57. Viral particles can also include 3' untranslated regions (UTR) and 5'UTR. The UTR contains retroviral regulatory elements that support packaging, reverse transcription, and integration of the proviral genome into cells following contact of the cells with retroviral particles.

[0200] The third generation system also typically contains two "packaging plasmids" and one "envelope plasmid". The "envelope plasmid" typically encodes an Env gene operably linked to a promoter. In an illustrative third generation system, the Env gene is VSV-G and the promoter is a CMV promoter. The third generation system uses two packaging plasmids, one encoding gag and pol, and the other encoding rev as another safety feature; an improvement over the single packaging plasmid of the so-called second generation system. Although safer, the third generation system can be more cumbersome to use and result in lower viral titers due to the addition of additional plasmids. Illustrative packaging plasmids include, but are not limited to, pMD2.G, pRSV-rev, pMDLG-pRRE, and pRRL-GOI.

[0201] Many retroviral vector systems rely on the use of a "packaging cell line". Typically, a packaging cell line is a cell line whose cells are capable of producing infectious retroviral particles when a transfer plasmid, one or more packaging plasmids, and an envelope plasmid are introduced into the cells. Various methods for introducing plasmids into cells can be used, including transfection or electroporation. In some cases, a packaging cell line is suitable for efficiently packaging a retroviral vector system into retroviral particles.

[0202] As used herein, the term "retroviral vector" or "lentiviral vector" is intended to mean a nucleic acid encoding a retroviral or lentiviral cis nucleic acid sequence required for genome packaging and one or more polynucleotide sequences to be delivered to a target cell. Retroviral particles and lentiviral particles typically include an RNA genome (derived from a transfer plasmid), a lipid bilayer envelope embedded with Env protein, and other auxiliary proteins including integrase, protease, and matrix protein. As used herein, the terms "retroviral particles" and "lentiviral particles" refer to viral particles that include an envelope, have one or more characteristics of a lentivirus, and are capable of invading a target host cell. Such features include, for example, infection of non-dividing host cells, transduction of non-dividing host cells, infection or transduction of host immune cells, containing retroviral or lentiviral particles (which include one or more of the gag structural polypeptides), containing retroviral or lentiviral envelopes (which include one or more of the glycoproteins encoded by env), containing genomes including one or more retroviral or lentiviral cis-acting sequences that function in replication, proviral integration or transcription, containing genomes encoding retroviral or lentiviral proteases, reverse transcriptases or integrases, or containing genomes encoding regulatory activities (such as Tat or Rev). The transfer plasmid may contain a cPPT sequence, as described in U.S. Pat. No. 8,093,042.

[0203] The efficiency of the system is an important issue in vector engineering. The efficiency of retroviral or lentiviral vector systems can be evaluated in a variety of ways 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 units of infection units / ml (IU / mL). For example, titer can be evaluated using a functional assay performed on a cultured tumor cell line HT1080, as described in the following literature: 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 titer is assessed for a cell line cultured for continuous division, stimulation is not required, so the titer measured is not affected by the surface engineering of retroviral particles. Other methods for assessing the efficiency of retroviral vector systems are provided in Comparison of retroviral vector titration methods. BMC Biotechnol. 6:34 (2006).

[0204] In some embodiments, the retroviral particles and / or lentiviral particles of the present disclosure comprise a polynucleotide comprising a sequence encoding a receptor that specifically binds to a gated adapter. In some embodiments, the sequence encoding a receptor that specifically binds to a gated adapter is operably linked to a promoter. Illustrative promoters include, but are not limited to, a cytomegalovirus (CMV) promoter, a CAG promoter, a SV40 promoter, a SV40 / CD43 promoter, and a MND promoter.

[0205] In some embodiments, the retroviral particle comprises a transduction enhancer. In some embodiments, the retroviral particle comprises a marker protein.

[0206] 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 to a ligand, and (iv) a 3' LTR or UTR.

[0207] In some embodiments, the retroviral particles comprise cell surface receptors that bind to surface markers on the target host cells, thereby allowing host cell transduction. The viral vector may comprise a heterologous viral envelope glycoprotein that gives a pseudotyped viral vector. For example, the viral envelope glycoprotein may be derived from RD114 or one of its variants, VSV-G, gibbon ape leukemia virus (GALV), or an amphibious envelope, measles envelope or baboon retrovirus envelope glycoprotein. In some embodiments, the cell surface receptor is a VSV G protein from a Cocal strain or a functional variant thereof.

[0208] Various fusion glycoproteins can be used to pseudotype lentiviral vectors. Although the most commonly used example is the envelope glycoprotein (VSVG) from vesicular stomatitis virus, many other viral proteins have also been used for pseudotyping of lentiviral vectors. See Joglekar et al. Human Gene Therapy Methods 28:291-301 (2017). The present disclosure contemplates the replacement of various fusion glycoproteins. Notably, some fusion glycoproteins result in higher vector efficiency.

[0209] In some embodiments, pseudotyping of the fusion glycoprotein or its functional variant 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 its functional variant is one or more full-length polypeptides, one or more functional fragments, one or more homologs, or one or more functional variants of the following: 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, ecotropic retrovirus (Eco) gp70, baboon simian leukemia virus (BaEV) gp70, measles virus (MV) H and F, Nipah virus (NiV) H and F, rabies virus (RabV) G, Mokola virus (MOKV) G, Ebola 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 HA, fowl plague virus (FPV) HA, vesicular stomatitis virus VSV-G, or Chandipura virus and Persian virus CNV-G and PRV-G.

[0210] In some embodiments, the fusion glycoprotein or its functional variant is a full-length polypeptide, functional fragment, homologue or functional variant of the G protein of the following viruses: Vesicular Stomatitis Alagoas virus (VSAV), Vesicular Stomatitis Caracas virus (CJSV), Chandipura Vesicular Stomatitis Virus (CHPV), Cocal Vesicular Stomatitis Virus (COCV), Vesicular Stomatitis India Virus (VSIV), Isfahan Vesicular Stomatitis Virus (ISFV), Maraba Vesicular Stomatitis Virus (MARAV), Vesicular Stomatitis New Jersey Virus (VSNJV), Bas-Congo Virus (BASV). In some embodiments, the fusion glycoprotein or its functional variant is Cocal virus G protein.

[0211] In some embodiments, the fusion glycoprotein or its functional variant is a full-length polypeptide, functional fragment, homologue or functional variant of the G protein of the following viruses: Vesicular Stomatitis Alagoas virus (VSAV), Vesicular Stomatitis Caracas virus (CJSV), Chandipura Vesicular Stomatitis Virus (CHPV), Cocal Vesicular Stomatitis Virus (COCV), Vesicular Stomatitis India Virus (VSIV), Isfahan Vesicular Stomatitis Virus (ISFV), Maraba Vesicular Stomatitis Virus (MARAV), Vesicular Stomatitis New Jersey Virus (VSNJV), Bas-Congo Virus (BASV). In some embodiments, the fusion glycoprotein or its functional variant is Cocal virus G protein.

[0212] The present disclosure further provides various retroviral vectors, including but not limited to γ-retroviral vectors, α-retroviral vectors and lentiviral vectors. In some embodiments, the vector can be a viral vector, a retroviral vector, a lentiviral vector, a γ-retroviral vector. In some embodiments, the viral vector comprises VSV G protein or a functional variant thereof. In some embodiments, the viral vector comprises Cokar G protein or a functional variant thereof. IV. Nucleic Acid Vectors

[0213] As used herein, the term "nucleic acid vector" is intended to mean any nucleic acid that functions to carry, bear or express a target nucleic acid. Nucleic acid vectors can have specialized functions, such as expression, packaging, pseudotyping, transduction or sequencing. Nucleic acid vectors can also have, for example, manipulation functions, such as cloning or shuttle vectors. The structure of the vector can include any desired form that makes manufacturing feasible and is desirable for a specific purpose. Such forms include, for example, circular forms such as plasmids and phagemids, and linear or branched forms. Nucleic acid vectors can be composed of, for example, DNA or RNA, and contain nucleotide derivatives, analogs and mimetics in part or in whole. Such nucleic acid vectors can be obtained from natural sources, recombinantly produced or chemically synthesized.

[0214] Non-limiting examples of vector systems of the present disclosure include retroviruses, lentiviruses, foamy viruses, and Sleeping Beauty transposons. V. Synthetic Cytokine Receptor Complexes and Systems

[0215] In some embodiments, provided herein are synthetic cytokine receptors for engineered cells (including cells that can be used in immunotherapy). In some embodiments, the synthetic cytokine receptor is composed of two polypeptide chains that can dimerize in response to the non-physiological ligands of the synthetic cytokine receptor to initiate signal transduction. In a specific embodiment, the synthetic cytokine receptor is a rapamycin activated cytokine receptor (RACR).

[0216] Any synthetic receptor system that provides a cytokine source or cytokine signal (e.g., JAK / STAT) without adding exogenous cytokines can be used. In some embodiments, the synthetic cytokine receptor or system induces a common cytokine receptor γ chain signaling. The common cytokine receptor γ chain is the signal transduction chain of the receptor complex of cytokines IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21. Exemplary examples of cytokines sharing a common γ cytokine chain include but are not limited to IL-2, IL-7, IL-15, IL-21 and IL-9. Various synthetic cytokine receptor systems are known and can be used, including orthogonal receptors for common γ chain receptors (e.g., Kalbasi et al. Nature, 2022607:360; Zhang et al. Sci. Transl. Med., 2021 13(625):eabg6986); drug-inducible systems of engineered cytokine fusions with drug response domains (DRDs) for controlled or inducible expression of cytokines, as described in published US20200172879; and synthetic receptors with dimerization domains that respond to chemical inducers of dimerization (CIDs).

[0217] In some embodiments, the synthetic cytokine receptor comprises a dimerization domain of a CID ligand. In some embodiments, the synthetic cytokine receptor is a heterodimer of a common cytokine receptor gamma signaling chain and an alpha or beta cytokine receptor signaling chain, each of which is fused to a dimerization domain. In some embodiments, the dimerization domain may be 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 a portion or derivative thereof. Examples of CIDs and corresponding dimerization domains are known in the art (see, e.g., U.S. Patent Publication No. 2016 / 0046700, Clackson et al. (1998) Proc Natl Acad Sci U SA. 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 an estrogen, a glucocorticoid, vitamin D, a steroid, a tetracycline, a cyclosporin, a rapamycin, a coumarin, a gibberellin, FK1012, FK506, FKCsA, a rimiducid or HaXS or an analog or derivative thereof. The dimerization domain can be extracellular (the N-terminal end of 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 a rapamycin or an analog thereof. Exemplary synthetic receptor dimerization systems are described below.

[0218] The non-physiological ligand can activate the synthetic cytokine receptor in the engineered lymphocyte (such as cytotoxic innate lymphoid cell) to induce the expansion and / or activation of the engineered cell (such as cytotoxic innate lymphoid cell). In a preferred embodiment, the non-physiological ligand is rapamycin or rapalog, and this synthetic cytokine receptor is called rapamycin activated cytokine receptor (RACR).

[0219] In some embodiments, non-physiological ligands activate synthetic cytokine receptors in engineered lymphocytes (e.g., CIL cells) to induce expansion of the engineered lymphocytes (e.g., CIL cells). In some embodiments, compared to uninduced cells, activation of synthetic cytokine receptors causes the number of lymphocytes (e.g., CIL cells) to increase by at least about 10 times, at least about 50 times, at least about 100 times, at least about 200 times, at least about 300 times, at least about 400 times, at least about 500 times, at least about 1000 times, at least about 1500 times, at least about 2000 times, at least about 2500 times, at least about 3000 times, at least about 3500 times, or at least about 4000 times.

[0220] In some embodiments, the lymphocytes (e.g., CIL cells) increase by about 10 times to about 100 times, about 50 times to about 200 times, about 100 times to about 300 times, about 200 times to about 400 times, about 300 times to about 500 times, about 400 times to about 1000 times, about 500 times to about 1500 times, about 1000 times to about 2000 times, about 1500 times to about 2500 times, about 2000 times to about 3000 times, about 2500 times to about 3500 times, about 3000 times to about 4000 times, or any value between these ranges. A. Exemplary Synthetic Cytokine Receptors (e.g., RACR)

[0221] In some embodiments, the synthetic cytokine receptor of the present disclosure comprises a first transmembrane receptor protein, which is a first synthetic cytokine chain (e.g., a synthetic γ chain comprising a co-receptor γ signaling chain); and a second transmembrane receptor protein, which is a synthetic cytokine chain (e.g., a synthetic α chain or β chain comprising an intracellular signaling chain of an α or β chain of a cytokine receptor, respectively), wherein the first transmembrane receptor protein and the second transmembrane receptor protein each contain a dimerization domain. The dimerization domain is capable of controllably dimerizing in the presence of a non-physiological ligand, thereby activating the signaling of the synthetic cytokine receptor.

[0222] In some embodiments, the first cytokine chain is a synthetic γ chain polypeptide comprising a first dimerization domain, a first transmembrane domain, and an intracellular domain of an interleukin-2 receptor subunit γ (IL-2RG). In some embodiments, the second cytokine chain comprises a second dimerization domain, a second transmembrane domain, and an intracellular domain of an α or β chain of a cytokine receptor. In some embodiments, the second cytokine chain is a synthetic α chain comprising an intracellular domain of an interleukin-9 receptor subunit α (IL-9RA) intracellular domain. In some embodiments, the second cytokine chain is a synthetic β chain comprising an intracellular domain selected from the following: an intracellular domain of an interleukin-2 receptor subunit β (IL-2RB), an intracellular domain of an interleukin-7 receptor subunit β (IL-7RB), or an intracellular domain of an interleukin-21 receptor subunit β (IL-21RB). The dimerization domain can be extracellular (N-terminal to the transmembrane domain) or intracellular (C-terminal to the transmembrane domain and either the N- or C-terminal to the intracellular domain).

[0223] In some embodiments, the synthetic cytokine receptors of the present disclosure comprise a synthetic γ chain and a synthetic α chain, each containing a dimerization domain. The dimerization domain is capable of controllably dimerizing in the presence of a non-physiological ligand, thereby activating the signaling of the synthetic cytokine receptor. In some embodiments, the synthetic γ chain polypeptide comprises a first dimerization domain, a first transmembrane domain, and an intracellular domain of interleukin-2 receptor subunit γ (IL-2RG). The dimerization domain may be extracellular (N-terminal of the transmembrane domain) or intracellular (C-terminal of the transmembrane domain and N or C-terminal of the IL-2G intracellular domain). The synthetic α chain polypeptide comprises a second dimerization domain, a second transmembrane domain, and an intracellular domain selected from the intracellular domain of interleukin-9 receptor subunit α (IL-2RA). The dimerization domain may be extracellular (N-terminal of the transmembrane domain) or intracellular (C-terminal of the transmembrane domain and N or C-terminal of the IL-9RA intracellular domain).

[0224] In some embodiments, the synthetic cytokine receptor of the present disclosure comprises a synthetic γ chain and a synthetic β chain each containing a dimerization domain. The dimerization domain is capable of controllably dimerizing in the presence of a non-physiological ligand, thereby activating the signal transduction of the synthetic cytokine receptor. In some embodiments, the synthetic γ chain polypeptide comprises a first dimerization domain, a first transmembrane domain, and an interleukin-2 receptor subunit γ (IL-2RG) intracellular domain. The dimerization domain may be extracellular (N-terminal of the transmembrane domain) or intracellular (C-terminal of the transmembrane domain and N or C-terminal of the IL-2G intracellular domain). The synthetic β chain polypeptide comprises a second dimerization domain, a second transmembrane domain, and an intracellular domain selected from the following: an interleukin-2 receptor subunit β (IL-2RB) intracellular domain, an interleukin-7 receptor subunit β (IL-7RB) intracellular domain, or an interleukin-21 receptor subunit β (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 either the N- or C-terminal to the IL-2RB, IL-7RB or IL-21RB intracellular domain).

[0225] In some embodiments, the synthetic gamma chain polypeptide is encoded by a nucleic acid sequence encoding a signal peptide. In some embodiments, the synthetic alpha chain polypeptide is encoded by a nucleic acid sequence encoding a signal peptide. In some embodiments, the synthetic beta chain polypeptide is encoded by a nucleic acid sequence encoding a signal peptide. The skilled artisan is readily familiar with signal peptides that can provide a signal to transport nascent proteins in cells. Any of a variety of signal peptides can be used.

[0226] In some embodiments, the signal peptide is the CD8a signal sequence shown as SEQ ID NO: 12: MALPVTALLLPLAL LLHAARP.

[0227] In some embodiments, the signal peptide is the signal sequence shown in SEQ ID NO: 29: MPLGLLWLGLALLG ALHAQA. 1. Intracellular domain

[0228] 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 shown in SEQ ID NO: 1. In some embodiments, the IL2Rg common gamma chain intracellular domain has at least 80% amino acid identity, at least 85% amino acid identity, at least 90% amino acid identity, at least 95% amino acid identity, or 100% amino acid identity with SEQ ID NO: 1.

[0229] The sequence of the IL2RG common gamma chain intracellular domain is shown in SEQ ID NO: 1: ERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGAL GEGPGASPCNQHSPYWAPPCYTLKPET.

[0230] In some embodiments, the synthetic cytokine receptor comprises a first transmembrane receptor protein (comprising an IL-2RG intracellular domain, a first dimerization domain), a second transmembrane receptor protein (comprising an IL-2RB intracellular domain and a second dimerization domain).

[0231] In some embodiments, the synthetic β chain comprises an interleukin-2 receptor subunit β (IL2RB) intracellular domain. IL2RB is also referred to as IL15RB or CD122. Therefore, when referred to herein, IL2RB may also refer to IL15RB. That is, these terms are used interchangeably in the present disclosure. In some embodiments, the IL2RB intracellular domain comprises the sequence shown in SEQ ID NO: 2. In some embodiments, the IL2RB intracellular domain has at least 80% amino acid identity, at least 85% amino acid identity, at least 90% amino acid identity, at least 95% amino acid identity, or 100% amino acid identity with SEQ ID NO: 2.

[0232] The sequence of the IL2RB intracellular domain is shown in SEQ ID NO: 2: NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISP LEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFT YDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPST APGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVP DAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV

[0233] In some embodiments, the synthetic cytokine receptor comprises a first transmembrane receptor protein (comprising an IL-2RG intracellular domain and a first dimerization domain), a second transmembrane receptor protein (comprising an IL-7RB intracellular domain and a second dimerization domain).

[0234] 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 shown in SEQ ID NO: 3. In some embodiments, the IL7RB intracellular domain has at least 80% amino acid identity, at least 85% amino acid identity, at least 90% amino acid identity, at least 95% amino acid identity, or 100% amino acid identity with SEQ ID NO: 3.

[0235] The sequence of the IL7RB intracellular domain is shown in SEQ ID NO: 3: KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGF LQDTFPQQLEESEKQRLGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILS SSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQPILTSLGS NQEEAYVTMSSFYQNQ

[0236] In some embodiments, the synthetic cytokine receptor comprises a first transmembrane receptor protein (comprising an IL-2RG intracellular domain and a first dimerization domain), a second transmembrane receptor protein (comprising an IL-21RB intracellular domain and a second dimerization domain).

[0237] In some embodiments, the synthetic β chain comprises an interleukin-21 receptor subunit β (IL21RB) intracellular domain. In some embodiments, the IL21RB intracellular domain comprises the sequence shown in SEQ ID NO: 4. In some embodiments, the IL21RB intracellular domain has at least 80% amino acid identity, at least 85% amino acid identity, at least 90% amino acid identity, at least 95% amino acid identity, or 100% amino acid identity with SEQ ID NO: 4.

[0238] The sequence of the IL21RB intracellular domain is shown in SEQ ID NO:4: SLKTHPLWRLWKKIWAVPSPERFFMPLYKGCSGDFKKWVGAPFFTGSSLELGPWSPEVPSTLEVYSCHPPRSPAKRLQLTELQEPAELVESDGVPKPSFWPTAQNSGGSAYSEERDRPYGLVSIDTVLDAEGPCTWPCSCE DDGYPALDLDAGLEEPSPGLEDPLLDAGTTVLSCGCVSAGSPGLGGPLGSLLDRLKPPLADGEDWAGGLPWGGRSPGGVSESEAGSPLAGLDMDTFDSGFVGSDCSSPVECDFTSPGDEGPPRSYLRQWVVIPPPLSSPGPQAS

[0239] In some embodiments, the synthetic cytokine receptor comprises a first transmembrane receptor protein comprising an IL-2RG intracellular domain and a first dimerization domain, a second transmembrane receptor protein comprising an IL-9RA intracellular domain and a second dimerization domain.

[0240] In some embodiments, the synthetic α chain comprises an interleukin-9 receptor subunit α (IL9RA) intracellular domain. In some embodiments, the IL9RA intracellular domain comprises the sequence shown 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 with SEQ ID NO: 51.

[0241] The sequence of the IL9RA intracellular domain is shown in SEQ ID NO:51: KLSPRVKRIFYQNVPSPAMFFQPLYSVHNGNFQTWMGAHGAGVLLSQDCAGTPQGALEPCVQEATALLTCGPARPWKSVALEEEQEGPGTRLPGNLSSEDVLPAGCTEWRVQTLA YLPQEDWAPTSLTRPAPPDSEGSRSSSSSSNNNNYCALGCYGGWHLSALPGNTQSSGPIPALACGLSCDHQGLETQQGVAWVLAGHCQRPGLHEDLQGMLLPSVLSKARSWTF 2. Dimerization domain

[0242] The dimerization domain can be a heterodimerization domain, including but not limited to a FK506 binding protein (FKBP) of 12 kD in size and a FKBP12-rapamycin binding (FRB) domain, which is known in the art to dimerize in the presence of rapamycin or rapalog. In some embodiments, the FRB domain may include a polypeptide sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 6 or SEQ ID NO: 7. In some embodiments, the FKBP domain may include a polypeptide sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 5. In some embodiments, the FKBP domain may comprise a polypeptide sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 48. In some embodiments, the FKBP domain may comprise a polypeptide sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 30. In some embodiments, the FKBP domain may comprise a polypeptide sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 49.

[0243] In some embodiments, the sequence of an illustrative FKBP domain is as shown in SEQ ID NO:5: GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE

[0244] In some embodiments, the sequence of an illustrative FKBP domain is as shown in SEQ ID NO:48: GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKL

[0245] In some embodiments, the sequence of an illustrative FKBP domain is as shown in SEQ ID NO:30: GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLGE

[0246] In some embodiments, the sequence of an illustrative FKBP domain is as shown in SEQ ID NO:49: GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLGE

[0247] In some embodiments, the sequence of an illustrative FRB domain is as shown in SEQ ID NO:6: ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISK

[0248] In some embodiments, the sequence of the variant FRB domain (FRB mutant domain) is as shown in SEQ ID NO:7: ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK

[0249] In some embodiments, the first dimerization domain is as shown in SEQ ID NO:5, and the second dimerization domain is as shown in SEQ ID NO:6.

[0250] In some embodiments, the first dimerization domain is as shown in SEQ ID NO:48, and the second dimerization domain is as shown in SEQ ID NO:6.

[0251] In some embodiments, the first dimerization domain is as shown in SEQ ID NO:30, and the second dimerization domain is as shown in SEQ ID NO:6.

[0252] In some embodiments, the first dimerization domain is as shown in SEQ ID NO:5, and the second dimerization domain is as shown in SEQ ID NO:7.

[0253] In some embodiments, the first dimerization domain is as shown in SEQ ID NO:48, and the second dimerization domain is as shown in SEQ ID NO:7.

[0254] In some embodiments, the first dimerization domain is as shown in SEQ ID NO:30, and the second dimerization domain is as shown in SEQ ID NO:7.

[0255] Alternatively, the first dimerization domain and the second dimerization domain may be a 12 kD FK506 binding protein (FKBP) and a calcineurin domain, both of which are known in the art to dimerize in the presence of FK506 or an analog thereof.

[0256] In some embodiments, the dimerization domain is a homodimerization domain selected from: i) FK506 binding protein (FKBP) with a size of 12 kD; ii) cyclophilin A (CypA); or iii) gyrase B (CyrB); The corresponding non-physiological ligands are i) FK1012, AP1510, AP1903 or AP20187; ii) cyclosporine-A (CsA); or iii) coumermycin or an analog thereof.

[0257] In some embodiments, the first and second dimerization domains of the transmembrane receptor protein are a FKBP domain and a cyclophilin domain.

[0258] In some embodiments, the first and second dimerization domains of the transmembrane receptor protein are a FKBP domain and a bacterial dihydrofolate reductase (DHFR) domain.

[0259] In some embodiments, the first and second dimerization domains of the transmembrane receptor protein are a calcineurin domain and a cyclophilin domain.

[0260] In some embodiments, the first and second dimerization domains of the transmembrane receptor protein are PYR1-like 1 (PYL1) and abscisic acid-insensitive protein 1 (ABI1). 3. Transmembrane domain

[0261] The transmembrane domain is the sequence of a synthetic cytokine receptor that spans the membrane. The transmembrane domain may comprise a hydrophobic alpha helix. In some embodiments, the transmembrane domain is derived from a human protein.

[0262] The sequence of the transmembrane (TM) domain is shown as SEQ ID NO:8: VVISVGSMGLIISLLCVYFWL

[0263] The sequence of the TM domain is shown as SEQ ID NO:9: VAVAGCVFLLISVLLLSGL

[0264] The sequence of the TM domain is shown as SEQ ID NO: 10: PILLTISILSFFSVALLVILACVLW

[0265] The sequence of the TM domain is shown as SEQ ID NO: 11: GWNPHLLLLLLLVIVFIPAFW

[0266] The sequence of the TM domain is shown as SEQ ID NO:36: IPWLGHLLVGLSGAFGFIILVYLLI.

[0267] In some embodiments, the TM domain and the intracellular signaling domain are from the same cytokine receptor. In some embodiments, the synthetic γ chain polypeptide comprises an IL-2RG TM domain and an IL-2RG intracellular domain. In some embodiments, the synthetic β chain polypeptide comprises an IL-2RB TM domain and an IL-2RB intracellular domain. In some embodiments, the synthetic β chain polypeptide comprises an IL-7RB TM domain and an IL-7RB intracellular domain. In some embodiments, the synthetic β chain polypeptide comprises an IL-21RB TM domain and an IL-21RB intracellular domain. In some embodiments, the synthetic α chain polypeptide comprises an IL-9RA TM domain and an IL-9RA intracellular domain.

[0268] In some embodiments, one or more additional continuous amino acids of the extracellular domain directly adjacent to the TM domain of the cytokine receptor may also be included as part of the polypeptide sequence of the chain of the synthetic cytokine receptor. In some embodiments, 1-20 continuous amino acids of the extracellular domain adjacent to the TM domain of the cytokine receptor are included as part of the polypeptide sequence of the chain of the synthetic cytokine receptor. The portion of the extracellular domain can be a continuous sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids directly adjacent to the TM sequence (e.g., at its N-terminus).

[0269] In some embodiments, the synthetic cytokine receptor is composed of a synthetic γ chain polypeptide containing a FKBP12 dimerization domain, an IL-2RG transmembrane domain, and an IL-2RG intracellular domain, and a synthetic β chain polypeptide containing a 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 γ chain polypeptide containing a FRB dimerization domain, an IL-2RG transmembrane domain, and an IL-2RG intracellular domain, and a synthetic β chain polypeptide containing a FKBP12 dimerization domain, an IL-2RB transmembrane domain, and an IL-2RB intracellular domain.

[0270] In some embodiments, the synthetic gamma chain polypeptide comprises an IL-2RG TM domain comprising the sequence set forth in SEQ ID NO: 8 and an IL-2RG intracellular domain comprising the sequence set forth in SEQ ID NO: 1. In some embodiments, the synthetic gamma chain polypeptide comprises an IL-2RG TM domain comprising the sequence set forth in SEQ ID NO: 31 and an IL-2RG intracellular domain comprising the sequence set forth in SEQ ID NO: 1.

[0271] In some embodiments, the synthetic β chain polypeptide comprises 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 β chain polypeptide comprises 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.

[0272] In some embodiments, the synthetic cytokine receptor is composed of a synthetic γ chain polypeptide containing a FKBP12 dimerization domain and an IL-2RG intracellular domain and a synthetic β chain polypeptide containing a FRB dimerization domain and an IL-2RB intracellular domain. In some embodiments, the synthetic γ chain polypeptide has the amino acid sequence shown in SEQ ID NO: 28 or an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100% identical to SEQ ID NO: 28. In some embodiments, the synthetic β chain polypeptide has the amino acid sequence shown in SEQ ID NO: 33 or an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100% identical to SEQ ID NO: 33.

[0273] In some embodiments, the synthetic cytokine receptor is composed of a synthetic γ chain polypeptide containing a FKBP12 dimerization domain and an IL-2RG intracellular domain and a synthetic β chain polypeptide containing a FRB dimerization domain and an IL-2RB intracellular domain. In some embodiments, the synthetic γ chain polypeptide has the amino acid sequence shown in SEQ ID NO: 28 or an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100% identical to SEQ ID NO: 28. In some embodiments, the synthetic β chain polypeptide has the amino acid sequence shown in SEQ ID NO: 33 or an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100% identical to SEQ ID NO: 33.

[0274] In some embodiments, the synthetic cytokine receptor is composed of the synthetic gamma chain polypeptide set forth in SEQ ID NO:28 and the synthetic beta chain polypeptide set forth in SEQ ID NO:33.

[0275] In some embodiments, the synthetic cytokine receptor is composed of a synthetic γ chain polypeptide containing a FKBP12 dimerization domain, an IL-2RG transmembrane domain, and an IL-2RG intracellular domain, and a synthetic β chain polypeptide containing a 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 γ chain polypeptide containing a FRB dimerization domain, an IL-2RG transmembrane domain, and an IL-2RG intracellular domain, and a synthetic β chain polypeptide containing a FKBP12 dimerization domain, an IL-7RB transmembrane domain, and an IL-7RB intracellular domain.

[0276] In some embodiments, the synthetic cytokine receptor is composed of a synthetic γ chain polypeptide containing a FKBP12 dimerization domain, an IL-2RG transmembrane domain, and an IL-2RG intracellular domain, and a synthetic β chain polypeptide containing a 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 γ chain polypeptide containing a FRB dimerization domain, an IL-2RG transmembrane domain, and an IL-2RG intracellular domain, and a synthetic β chain polypeptide containing a FKBP12 dimerization domain, an IL-21RB transmembrane domain, and an IL-21RB intracellular domain.

[0277] In some embodiments, the synthetic cytokine receptor is composed of a synthetic γ chain polypeptide containing a FKBP12 dimerization domain, an IL-2RG transmembrane domain, and an IL-2RG intracellular domain, and a synthetic β chain polypeptide containing a 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 γ chain polypeptide containing a FRB dimerization domain, an IL-2RG transmembrane domain, and an IL-2RG intracellular domain, and a synthetic β chain polypeptide containing a FKBP12 dimerization domain, an IL-9RA transmembrane domain, and an IL-9RA intracellular domain.

[0278] In some embodiments, the synthetic cytokine receptor is capable of being bound by a non-physiological ligand, rapamycin or a rapamycin analog. In some embodiments, the synthetic cytokine receptor is responsive to a non-physiological ligand, rapamycin or a rapamycin analog, wherein binding of the non-physiological ligand to the dimerization domain of the synthetic cytokine receptor induces cytokine receptor-mediated signaling in the cell, such as signaling via the JAK / STAT pathway. 4. Non-physiological ligands

[0279] In various embodiments of the compositions and methods of the present disclosure, the system comprises a non-physiological ligand. Illustrative small molecules that can be used as ligands include, but are not limited to, rapamycin, fluorescein, fluorescein isothiocyanate (FITC), 4-[(6-methylpyrazin-2-yl)oxy]benzoic acid (aMPOB), folic acid, rhodamine, acetazolamide, and CA9 ligand.

[0280] In some embodiments, the synthetic cytokine receptor is activated by a ligand. In some embodiments, the ligand is a non-physiological ligand.

[0281] In some embodiments, the non-physiological ligand is a rapalog.

[0282] In some embodiments, the non-physiological ligand is rapamycin.

[0283] In some embodiments, the non-physiological ligand is AP21967.

[0284] In some embodiments, the non-physiological ligand is FK506.

[0285] 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.

[0286] In some embodiments, the synthetic cytokine receptor complex is activated by: folic acid, fluorescein, aMPOB, acetazolamide, CA9 ligand, tacrolimus, rapamycin, rapalog (rapamycin analog), CD28 ligand, poly (his) tag, Strep tag, FLAG tag, VS tag, Myc tag, HA tag, NE tag, biotin, digoxigenin, dinitrophenol or its derivatives.

[0287] In some embodiments, the non-physiological ligand may be an inorganic or organic compound less than 1000 Daltons.

[0288] In some embodiments, the ligand can be rapamycin or a rapamycin analog (rapalog). In some embodiments, rapalog includes a variant of rapamycin having one or more of the following modifications relative to rapamycin: demethylation, elimination or substitution of a methoxy group at C7, C42 and / or C29; elimination, derivatization or substitution of a hydroxyl group at C13, C43 and / or C28; reduction, elimination or derivatization of a ketone at C14, C24 and / or C30; substitution of a 6-membered pipecolic acid ring with a 5-membered prolyl ring; and substitution of an alternative on a cyclohexyl ring or substitution of a cyclohexyl ring with a substituted cyclopentyl ring.

[0289] Thus, in some embodiments, the rapalog is everolimus, norvolimus, pimecrolimus, lirolimus, tacrolimus, trolimus, usololimus, zotarolimus, temsirolimus (CCI-779), C20-methallyl rapamycin, C16-(S)-3-methylindorapamycin, C16-(S)-3-methylindorapamycin (C16-iRap), AP21967 (A / C heterodimerizer, Takara ), mycophenolate sodium, benidipine hydrochloride, rapamine, AP23573 (Ridaforolimus), AP1903 (Rimidase), or their metabolites, derivatives and / or combinations.

[0290] In some embodiments, the ligand comprises FK1012 (a semisynthetic dimer of FK506), tacrolimus (FK506), FKCsA (a complex of FK506 and cyclosporine), rapamycin, coumermycin, gibberellin, HaXS dimerizer (a chemical dimerizer for HaloTag and SNAP tags), TMP-HTag (trimethoprim halogenase protein dimerizer), or ABT-737 or a functional derivative thereof.

[0291] In some embodiments, the non-physiological ligand is present or provided in an amount of 0 nM to 1000 nM, such as, for example, 0.05 nM, 0.1 nM, 0.5.nM, 1.0 nM, 5.0 nM, 10.0 nM, 15.0 nM, 20.0 nM, 25.0 nM, 30.0 nM, 35.0 nM, 40.0 nM, 45.0 nM, 50.0 nM, 55.0 nM, 60.0 nM, 65.0 nM, 70.0 nM, 75.0 nM, 80.0 nM, 90.0 nM, 95.0 nM, 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM or 1000 nM, or an amount within a range defined by any two of the foregoing amounts.

[0292] 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.

[0293] 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.

[0294] 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.

[0295] In some embodiments, the non-physiological ligand is present or provided at 1 nM.

[0296] In some embodiments, the non-physiological ligand is present or provided at 10 nM.

[0297] In some embodiments, the non-physiological ligand is present or provided at 100 nM.

[0298] In some embodiments, the non-physiological ligand is present or provided at 1000 nM. B. Cytoplasmic FRB

[0299] In some embodiments, engineered cells (such as lymphocytes or progenitor cells thereof, including iCIL cells) can be contacted with free cytoplasmic FRBs (i.e., soluble FRBs). As described in more detail elsewhere herein, rapamycin generally binds to FKBP12, and then the FKBP12-rapamycin complex binds to the FRB subunit of mTOR and blocks mTOR signaling. Therefore, in some cases, contacting cells with rapamycin can inhibit or reduce cell growth and expansion. Compared with the expansion of NK cells, certain drugs are known to have an inhibitory effect on NK cells. The following literature has reported that small molecule rapamycin inhibits NK cell function: Wai et al. Transplantation. 85 (1): 145–149 (2008). In some embodiments, cells can be made "rapamycin-resistant" by providing free cytoplasmic FRBs to cells for complexing with rapamycin, thereby eliminating or reducing rapamycin-mediated growth inhibition of source cells or engineered cells (such as engineered lymphocytes, such as iCILs).

[0300] In some embodiments, soluble FRBs can be microinjected into cells as described (including cells engineered with synthetic cytokine receptors, such as 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 vectors containing soluble FRBs to eliminate or reduce rapamycin-mediated growth inhibition. In some embodiments, soluble FRBs can be added to cell culture media to eliminate or reduce rapamycin-mediated growth inhibition.

[0301] 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.

[0302] The FRB domain is a domain of approximately 100 amino acids derived from the mTOR protein kinase. It can be expressed in the cytosol as a freely diffusible soluble protein. Advantageously, the FRB domain reduces the inhibitory effect of rapamycin on mTOR in transduced cells and promotes consistent activation of transduced cells, thereby giving the cells a proliferative advantage over native cells.

[0303] In some embodiments, the synthetic cytokine receptor complex comprises a cytoplasmic polypeptide bound to a ligand or a complex comprising a ligand.

[0304] In some embodiments, the cytoplasmic polypeptide comprises a FRB domain. In some embodiments, the cytoplasmic polypeptide comprises a FRB domain and the ligand is rapamycin. The cytoplasmic FRB domain may comprise a polypeptide sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 6 or SEQ ID NO: 7. The FRB domain may be a naked FRB domain that is essentially composed of a polypeptide having a polypeptide sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 6 or SEQ ID NO: 7. Advantageously, the cytoplasmic FRB confers resistance to the immunosuppressive effects of non-physiological ligands (e.g., rapamycin or rapalog). In some embodiments, the cytoplasmic FRB domain can comprise a polypeptide sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 50. In some embodiments, the FRB domain can be a naked FRB domain that consists essentially of a polypeptide having a polypeptide sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 50.

[0305] In some embodiments, the cell is contacted with a FRB domain protein having the sequence shown in SEQ ID NO:6.

[0306] In some embodiments, the cell is contacted with a FRB domain protein having the sequence shown in SEQ ID NO:7.

[0307] In some embodiments, the cell is contacted with a FRB domain protein having the sequence shown in SEQ ID NO:50. VI. Gene Editing

[0308] In some embodiments, the cells described herein (e.g., iPSC or CIL) can be modified by gene editing. In some embodiments, the gene-edited iPSC as described can be used as a source cell for differentiation into CIL.

[0309] Genome editing generally refers to the process of editing or changing the nucleotide sequence of a genome, preferably in an accurate, desired and / or predetermined manner. Examples of the compositions, systems and methods of genome editing described herein use site-directed nucleases to cut (cut or cleave) DNA at precise target positions in the genome, thereby producing double-strand breaks (DSBs) in the DNA. Such breaks can be repaired by endogenous DNA repair pathways, such as homology-directed repair (HDR) and / or non-homologous end joining (NHEJ) repairs (see, for example, Cox et al., (2015) Nature Medicine 21 (2): 121-31).

[0310] In some embodiments, cells as described herein (e.g., stem cells, CIL) are genetically modified. In some embodiments, the modification involves knocking out one or more endogenous genes and / or knocking in one or more exogenous target genes using a DNA targeting protein and a nuclease or RNA-guided nuclease. In some embodiments, the target gene is knocked into a specific target locus. In some embodiments, the target gene is a synthetic cytokine receptor complex. In some embodiments, the synthetic cytokine receptor complex is activated by rapamycin. In some embodiments, the synthetic cytokine receptor complex is a cytokine receptor (RACR) activated by rapamycin. In some embodiments, RACR is knocked into the target locus. In some embodiments, the target gene is a chimeric antigen receptor.

[0311] In some embodiments, the modification comprises contacting the cell with a DNA targeting protein and a nuclease or an RNA-guided nuclease. In some embodiments, the DNA targeting protein and the nuclease or the RNA-guided nuclease 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.

[0312] Rejection of cell therapy agents (e.g., CAR T cells) is at least due to the mismatch of human leukocyte antigen (HLA) between donor and recipient. A recently identified solution is to destroy 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). Therefore, in some embodiments, cells described herein (e.g., iPSC, CIL) are genetically engineered to knock out B2M locus, TRAC locus and / or SIRPA locus. In some embodiments, cells described herein are genetically engineered to knock out B2M locus. In some embodiments, cells described herein are genetically engineered to knock out TRAC locus. In some embodiments, cells described herein are genetically engineered to knock out SIRPA locus.

[0313] In some embodiments, the cells described herein are genetically engineered to have rapamycin resistance. Rapamycin is a small molecule drug that inhibits the mTOR pathway, which is a pathway necessary for cell growth and expansion. Therefore, in some cases, contacting cells with rapamycin can inhibit or reduce cell growth and expansion. In some embodiments, in order to eliminate or reduce rapamycin-mediated growth inhibition of source cells or CIL using the provided method, the endogenous genes involved in rapamycin function in the provided cells are destroyed, so that such cells have "rapamycin resistance". It should be understood that when referring to "rapamycin-resistant" cells, it refers to the ability of the endogenous mTOR pathway of the cell not to be affected or impacted by the presence of rapamycin or rapamycin analogs. However, it should be further understood that "rapamycin-resistant" cells can still respond to rapamycin via pathways that do not involve mTOR, such as due to the activation of synthetic RACR as described herein.

[0314] In some embodiments, the cell is genetically engineered to destroy genes associated with rapamycin recognition. In some embodiments, the cell is genetically engineered to destroy the mTOR gene. In some embodiments, the mTOR gene is FKBP-12 (also referred to as FKBP-1A, FKBP1, FKBP12, PKC12, PKCI2, PPIASE). FKBP12 is an essential binding agent for rapamycin and is required for its function. In some embodiments, the cell is genetically engineered to destroy the FKBP12 gene. In some embodiments, the cell is genetically engineered to knock out the FKB12 ​​gene, thereby inducing rapamycin resistance. In some embodiments, the destruction of the endogenous FKBP12 gene of a source stem cell (e.g., iPSC) is carried out by knocking out the gene using a CRISPR-Cas system. In normal cells where the gene destruction of FKBP12 is not carried out, FKBP12 is the main binding agent for rapamycin, and then the FKBP12-rapamycin complex binds to the FRB subunit of mTOR and blocks mTOR signaling. By destroying the expression of the FKBP12 gene (such as knocking out by FKBP12), the results herein demonstrate successful rapamycin inhibitory activity, because rapamycin has no function when not first compounded with FKBP1A. Therefore, the gene destruction of FKBP12 (such as knocking out by gene) makes stem cells (e.g., iPSC) highly resistant to rapamycin-mediated mTOR inhibition, so that stem cells (e.g., iPSC) can grow robustly even in the presence of high doses of rapamycin. In some embodiments, the ability to make cells resistant to rapamycin growth inhibition allows RACR to engage with rapamycin during cell differentiation without harmful effects. In addition, the knockout of FKBP12 avoids FKBP12 from competing with RACR for binding to rapamycin. Therefore, in some embodiments, the ability to make cells resistant to rapamycin growth by knocking out FKBP12 also allows activating cells containing RACR in vivo, and suppresses the potential allogeneic anti-graft response caused by mTOR inhibition by the host immune system.

[0315] In some embodiments, the cells described herein are genetically engineered to include a nucleotide sequence encoding a synthetic cytokine receptor in an endogenous gene. In some embodiments, the synthetic cytokine receptor is engineered into a gene so that the expression of the endogenous gene is not disrupted. In some embodiments, the synthetic cytokine receptor is engineered into a safe-harbor locus.

[0316] In some embodiments, the cells described herein are 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).

[0317] In some embodiments, the target gene inserted into the endogenous locus is a synthetic cytokine receptor complex. In some embodiments, an endogenous promoter of a specific locus is used. In some embodiments, additional one or more promoters may be included so that two or more promoters drive the expression of an exogenous target gene.

[0318] In some embodiments, the cells described herein are genetically engineered to include a nucleotide sequence encoding a synthetic cytokine receptor complex in a disrupted gene. For example, in some embodiments, the cells include a disrupted B2M gene and a nucleotide sequence encoding a synthetic cytokine receptor in the disrupted B2M gene.

[0319] In some embodiments, the cells described herein (e.g., iPSCs, CILs) comprise (i) a disrupted B2M locus, and (ii) a nucleotide sequence encoding a synthetic cytokine receptor (e.g., RACR) complex under the control of an endogenous B2M promoter and an EEF1A promoter.

[0320] In some embodiments, the cells described herein (e.g., iPSCs, CILs) comprise (i) a disrupted B2M locus, and (ii) a nucleotide sequence encoding a synthetic cytokine receptor (e.g., RACR) complex inserted into an endogenous B2M gene and under the control of an endogenous B2M promoter and an EEF1A promoter.

[0321] In some embodiments, a cell comprising (i) a disrupted B2M locus and (ii) a nucleotide sequence encoding a synthetic cytokine receptor (eg, RACR) complex is generated by any of the methods described below. A. Systems for Genome Editing

[0322] In some embodiments, the system for editing the cells described herein comprises a site-directed 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-directed nuclease. In some embodiments, the site-directed nuclease comprises a CRISPR / Cas nuclease system. In some embodiments, the Cas nuclease is Cas9. In some embodiments, the nuclease is Mad7. In some embodiments, the guide RNA comprising the CRISPR / Cas system is a single guide RNA (sgRNA). 1. CRISPR / Cas nuclease system

[0323] The naturally occurring CRISPR / Cas system is a genetic defense system that provides a form of acquired immunity in prokaryotes. CRISPR is a clustered regularly interspaced short palindromic repeat (CRISPR). C lustered R egularly Interspaced ShortPalindromic R CRISPR / Cas systems are a family of DNA sequences found in the genomes of bacteria and archaea that contain DNA fragments (spacer DNA) that are similar to foreign DNA previously exposed to cells (e.g., by viruses that have infected or attacked prokaryotes). These DNA fragments are used by prokaryotes to detect and destroy similar foreign DNA that is reintroduced (e.g., similar viruses from subsequent attacks). Transcription of the CRISPR locus results in the formation of RNA molecules containing spacer sequences that associate and target Cas (CRISPR-associated) proteins that can recognize and cut foreign exogenous DNA. Many types and categories of CRISPR / Cas systems have been described (see, e.g., Koonin et al., (2017) Curr Opin Microbiol 37: 67-78).

[0324] Engineered versions of the CRISPR / Cas system have been developed in various forms to mutate or edit the genomic DNA of cells from other species. The general method of using the CRISPR / Cas system involves heterologous expression of a site-directed nuclease (e.g., Cas nuclease) combined with a guide RNA (gRNA) or introduction into a cell, resulting in a DNA cleavage event (e.g., forming a single-strand or double-strand break (SSB or DSB)) at a precise targetable position in the backbone of the cell's genomic DNA. The cell repairs the DNA cleavage event in a manner that provides the opportunity to edit the genome by adding, removing or modifying (replacing) one or more DNA nucleotides or sequences (e.g., genes).

[0325] In some embodiments, the system for editing cells described herein includes a nuclease capable of inducing DNA breaks in an endogenous target gene in a cell. In some embodiments, the DNA break includes a double-strand break (DSB), which is induced by a nuclease capable of inducing DSB by cutting two chains of double-stranded DNA at a cleavage site. In some embodiments, the DNA break includes a single-strand break (SSB) at the cleavage site in the sense strand or antisense strand of the endogenous target gene. In some embodiments, the DNA break includes an SSB at the cleavage site in the sense strand, and an SSB at the cleavage site in the antisense strand, thereby producing a DSB. In some embodiments, the DSB is induced by a pair of recombinant nucleases (e.g., nickases), each of which can induce a single-strand break (SSB) at different cleavage sites in opposite DNA chains (e.g., at a cleavage site upstream of a gene variant in one chain of a target gene, and at a cleavage site downstream of a gene variant in another chain). In some embodiments, the first nickase in the nickase pair forms a complex with a first guide RNA (e.g., a first sgRNA) for targeting cleavage to one strand (e.g., the sense strand), and the second nickase in the nickase pair forms a complex with a second guide RNA (e.g., a second sgRNA) for targeting cleavage to the other strand (e.g., the antisense strand). In some embodiments, DSBs are induced by SSBs on each strand of the opposite strands (i.e., the sense strand and the antisense strand) of an endogenous target gene in a cell.

[0326] Typically, a gene is located in a double-stranded DNA comprising a sense strand and an antisense strand that are complementary to each other. The sense strand is also called the coding strand because its sequence is the DNA version of the RNA sequence being transcribed. The antisense strand is also called the template strand because its sequence is complementary to the RNA sequence being transcribed. i. Guide RNA (gRNA)

[0327] The engineered CRISPR / Cas system comprises at least two components: 1) guide RNA (gRNA) molecules and 2) Cas nucleases, which interact to form gRNA / Cas nuclease complexes. gRNA at least includes a user-defined targeting domain (referred to as "spacer"), which includes a nucleotide sequence and CRISPR repeats. In the engineered CRISPR / Cas system, the gRNA / Cas nuclease complex is targeted to a specific target sequence of interest in a target nucleic acid (e.g., a genomic DNA molecule) by generating a gRNA comprising a spacer, the spacer having a nucleotide sequence that can be combined with 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)). Therefore, the spacer provides the targeting function of the gRNA / Cas nuclease complex.

[0328] In naturally occurring type II CRISPR / Cas systems, "gRNA" consists of two RNA chains: 1) CRISPR RNA (crRNA) containing spacers and CRISPR repeats, and 2) trans-activating CRISPR RNA (tracrRNA). In type II CRISPR / Cas systems, a portion of the crRNA containing CRISPR repeats hybridizes with a portion of tracrRNA to form a crRNA:tracrRNA duplex that interacts with Cas nucleases (e.g., Cas9). As used herein, the term "split gRNA" or "modular gRNA" refers to a gRNA molecule comprising two RNA chains, wherein the first RNA chain incorporates one or more crRNA functions and / or structures, and the second RNA chain incorporates one or more tracrRNA functions and / or structures, and wherein the first and second RNA chains partially hybridize.

[0329] Therefore, in some embodiments, the gRNA comprises two RNA molecules. In some embodiments, the gRNA comprises CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA). In some embodiments, the gRNA is a split gRNA. In some embodiments, the gRNA is a modular gRNA. In some embodiments, the split gRNA comprises: a first strand, the first strand comprising a spacer and a first complementary region from 5' to 3'; and a second strand, the second strand comprising a second complementary region from 5' to 3'; and optionally a tail domain.

[0330] In some embodiments, the crRNA comprises a spacer containing a nucleotide sequence complementary to and hybridizing with the following sequence: complementary to a target sequence on a target nucleic acid (e.g., a genomic DNA molecule). In some embodiments, the crRNA comprises a region complementary to and hybridizing with a portion of the tracrRNA.

[0331] In some embodiments, the target nucleic acid (e.g., an endogenous gene) is B2M. In some embodiments, the crRNA comprises the nucleotide sequence shown in SEQ ID NO: 18, or a nucleotide sequence having at least 80%, 85%, 90% or 95% sequence identity to the nucleotide sequence shown in SEQ ID NO: 18. In some embodiments, the crRNA comprises the nucleotide sequence shown in SEQ ID NO: 18.

[0332] In some embodiments, the target nucleic acid (e.g., an endogenous gene) is FKBP12. In some embodiments, the crRNA comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 19, 20, and 21, or a nucleotide sequence having at least 80%, 85%, 90%, or 95% sequence identity with the nucleotide sequence as shown in any one of SEQ ID NOs: 19, 20, and 21. In some embodiments, the crRNA comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 19, 20, and 21. In some embodiments, the crRNA comprises a nucleotide sequence as shown in SEQ ID NO: 19, or a nucleotide sequence having at least 80%, 85%, 90%, or 95% sequence identity with the nucleotide sequence as shown in SEQ ID NO: 19. In some embodiments, the crRNA comprises a nucleotide sequence as shown in SEQ ID NO: 19. In some embodiments, the crRNA comprises the nucleotide sequence shown in SEQ ID NO: 20, or a nucleotide sequence having at least 80%, 85%, 90% or 95% sequence identity to the nucleotide sequence shown in SEQ ID NO: 20. In some embodiments, the crRNA comprises the nucleotide sequence shown in SEQ ID NO: 20. In some embodiments, the crRNA comprises the nucleotide sequence shown in SEQ ID NO: 21, or a nucleotide sequence having at least 80%, 85%, 90% or 95% sequence identity to the nucleotide sequence shown in SEQ ID NO: 21. In some embodiments, the crRNA comprises the nucleotide sequence shown in SEQ ID NO: 21.

[0333] In some embodiments, tracrRNA may include all or part of a wild-type tracrRNA sequence from a naturally occurring CRISPR / Cas system. In some embodiments, tracrRNA may include a truncated or modified variant of a wild-type tracr RNA. The length of tracr RNA may depend on the CRISPR / Cas system used. In some embodiments, the length of tracrRNA may include 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 or more than 100 nucleotides. In certain embodiments, the length of the tracrRNA is at least 26 nucleotides. In other embodiments, the length of the tracrRNA is at least 40 nucleotides. In some embodiments, tracrRNA may include certain secondary structures, such as, for example, one or more hairpins or stem-loop structures, or one or more protruding structures. a. Single guide RNA (sgRNA)

[0334] The engineered CRISPR / Cas nuclease system usually combines crRNA and tracrRNA into a single RNA molecule, referred to herein as "single guide RNA" (sgRNA), by adding a joint between these components. Not bound by theory, similar to duplex crRNA and tracrRNA, sgRNA will form a complex with Cas nuclease (e.g., Cas9), guide Cas nuclease to the target sequence and activate Cas nuclease for cutting target nucleic acid (e.g., genomic DNA). Therefore, in some embodiments, gRNA may include operably connected crRNA and tracrRNA. In some embodiments, sgRNA may include crRNA covalently linked to tracrRNA. In some embodiments, the crRNA and the tracrRNA are covalently linked via a joint. In some embodiments, sgRNA can include a stem-loop structure via base pairing between crRNA and tracrRNA. In some embodiments, sgRNA includes a spacer, a first complementary region, a connecting domain, a second complementary region, and optionally a tail domain from 5' to 3'.

[0335] The sgRNA can be unmodified or modified. For example, a modified sgRNA can comprise one or more 2'-O-methyl phosphorothioate nucleotides.

[0336] As an illustration, the guide RNA or other smaller RNA used in the CRISPR / Cas system can be easily synthesized by chemical means, as described herein and described in the art. Although chemical synthesis procedures are constantly expanding, it is often more challenging to purify such RNA by procedures such as high performance liquid chromatography (HPLC, avoiding the use of gels such as PAGE), because the polynucleotide length is significantly increased by more than one hundred or so nucleotides. One method for producing RNA of greater length is to produce two or more molecules connected together. Significantly longer RNA, such as those encoding Cas9 endonucleases, are more easily produced enzymatically. Various types of RNA modifications can be introduced during or after chemical synthesis and / or enzymatic production of RNA, such as enhancing stability, reducing the possibility or degree of innate immune response and / or enhancing modifications of other properties, as described in the art. b. Spacer

[0337] In some embodiments, the gRNA comprises a spacer sequence. The spacer sequence is a sequence that defines the target site of a 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 and is referred to as a "PAM strand", and the second strand is referred to as a "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. Therefore, in some embodiments, the spacer sequence corresponding to the target sequence adjacent to the PAM sequence is identical to the PAM strand. The gRNA spacer sequence hybridizes with a complementary strand (e.g., a non-PAM strand of a target nucleic acid / target site). In some embodiments, the spacer is fully complementary to the complementary strand of the target sequence (e.g., a non-PAM strand) to target the Cas nuclease to the target nucleic acid. In some embodiments, the spacer is at least 80%, 85%, 90%, or 95% complementary to the non-PAM strand of the target nucleic acid. In some embodiments, the spacer is 100% complementary to the non-PAM strand of the target nucleic acid. In some embodiments, the spacer comprises 1, 2, 3, 4, 5, 6 or more nucleotides that are not complementary to the non-PAM strand of the target nucleic acid. In some embodiments, the spacer comprises 1 nucleotide that is not complementary to the non-PAM strand of the target nucleic acid. In some embodiments, the spacer comprises 2 nucleotides that are not complementary to the non-PAM strand of the target nucleic acid.

[0338] In some embodiments, the most 5' nucleotide of the gRNA includes the most 5' nucleotide of the spacer. In some embodiments, the spacer is located at the 5' end of the crRNA. In some embodiments, the spacer is located at the 5' end of the sgRNA. In some embodiments, the length of the spacer is about 15-50, about 20-45, about 25-40, or about 30-35 nucleotides. In some embodiments, the length of the spacer is about 19-22 nucleotides. In some embodiments, the length of the spacer is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, the length of the spacer is 19 nucleotides. In some embodiments, the length of the spacer is 20 nucleotides, and in some embodiments, the length of the spacer is 21 nucleotides.

[0339] In some embodiments, the nucleotide sequence of the spacer is designed or selected using a computer program. The computer program can use variables such as predicted melting temperature, secondary structure formation, predicted annealing temperature, sequence identity, genomic context, chromatin accessibility, GC%, (e.g., identical or similar but different sequences at one or more points due to mismatches, insertions or deletions) genomic frequency, methylation status and / or the presence of SNPs.

[0340] In some embodiments, the spacer comprises at least one or more modified nucleotides, such as those described herein. The present disclosure provides gRNA molecules comprising a spacer that may contain the nucleobase uracil (U), and any DNA encoding a gRNA comprising a spacer containing the nucleobase uracil (U) will include the nucleobase thymine (T) in one or more corresponding positions. ii. Method for preparing gRNA

[0341] Methods for preparing gRNA are known to those skilled in the art and include, but are not limited to, in vitro transcription (IVT), synthesis and / or chemical synthesis methods or combinations thereof. Enzymatic (IVT), solid phase, liquid phase, combined synthesis methods, small area synthesis and ligation methods are utilized. In one embodiment, gRNA is prepared using IVT enzymatic synthesis methods. Methods for preparing polynucleotides by IVT are known in the art and are described in International Application PCT / US2013 / 30062. Therefore, the present disclosure also includes polynucleotides (e.g., DNA), constructs, and vectors for in vitro transcription of the gRNA described herein.

[0342] In some embodiments, non-natural modified nucleobases are introduced into polynucleotides (e.g., gRNA) during or after synthesis. In certain embodiments, the modification is on an internucleoside link, a purine or pyrimidine base, or a sugar. In some embodiments, the modification is introduced at the end of a polynucleotide; using chemical synthesis or using a polymerase. 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).

[0343] In some embodiments, enzymatic or chemical ligation methods are used to conjugate polynucleotides or regions thereof to various functional moieties such as targeting or delivery agents, fluorescent markers, liquids, nanoparticles, etc. Conjugates of polynucleotides and modified polynucleotides are reviewed in Goodchild, Bioconjugate Chemistry, Vol. 1(3), 165-187 (1990).

[0344] In some embodiments, the disclosure provides nucleic acids encoding gRNA described herein, for example, vectors. 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 crRNA. In some embodiments, the nucleotide sequence encoding crRNA comprises a spacer flanked by all or part of a repetitive sequence from a naturally occurring CRISPR / Cas system. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding 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 opposite 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.

[0345] In some embodiments, the gRNA provided by the present disclosure is chemically synthesized by any means described in the art (see, for example, WO / 2005 / 01248). Although chemical synthesis procedures are expanding, it is often more challenging to purify such RNAs by procedures such as high performance liquid chromatography (HPLC, avoiding the use of gels such as PAGE), because the polynucleotide length increases significantly by more than one hundred or so nucleotides. One method for producing RNA of greater length is to produce two or more molecules connected together.

[0346] In some embodiments, more than one guide RNA can be used with the CRISPR / Cas nuclease system. Each guide RNA can contain different targeting sequences so that the CRISPR / Cas system cuts more than one target nucleic acid. In some embodiments, one or more guide RNAs can have the same or different properties, such as activity or stability within the Cas9 RNP complex. In the case of using more than one guide RNA, each guide RNA can be encoded on the same or different vectors. The promoter used to drive the expression of more than one guide RNA is the same or different.

[0347] The guide RNA can target any sequence of interest via the targeting sequence of crRNA (e.g., spacer sequence). In some embodiments, the degree of complementarity between the targeting sequence of the guide RNA and the target sequence on the target nucleic acid molecule is about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 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 1-6 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.

[0348] The length of the targeting sequence can depend on the CRISPR-Cas system and components used. For example, different Cas9 proteins from different bacterial species have different optimal targeting sequence lengths. Therefore, the length of the targeting sequence can include 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 or more than 50 nucleotides. In some embodiments, the length of the targeting sequence can include 18-24 nucleotides. In some embodiments, the length of the targeting sequence can include 19-21 nucleotides. In some embodiments, the length of the targeting sequence can include 20 nucleotides.

[0349] In some embodiments of the present disclosure, the CRISPR / Cas nuclease system includes at least one guide RNA. In some embodiments, the guide RNA and the Cas protein can form a ribonucleoprotein (RNP), such as a CRISPR / Cas complex. The guide RNA can guide the Cas protein to a target sequence on a target nucleic acid molecule (e.g., a genomic DNA molecule), wherein the Cas protein cuts 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 2-type V-type CRISPR system. In some embodiments, the endonuclease is Mad7. iii. Cas nuclease

[0350] In some embodiments, the present disclosure provides compositions and systems (e.g., engineered CRISPR / Cas systems) comprising a site-directed nuclease, wherein the site-directed nuclease is a Cas nuclease. The Cas nuclease may include at least one domain that interacts with a guide RNA (gRNA). In addition, the Cas nuclease is guided to a target sequence by a guide RNA. The guide RNA interacts with the Cas nuclease and the target sequence so that once it is guided to the target sequence, the Cas nuclease is able to cut the target sequence. In some embodiments, the guide RNA provides specificity for target sequence cutting, and the Cas nuclease is universal and paired with different guide RNAs to cut different target sequences.

[0351] In some embodiments, the CRISPR / Cas system comprises components derived from type I, type II or type III systems. The updated classification scheme of CRISPR / Cas loci defines class 1 and class 2 CRISPR / Cas systems, which have types I to V or VI (Makarova et al., (2015) Nat Rev Microbiol, 13 (11): 722-36; Shmakov et al., (2015) Mol Cell, 60: 385-397). Class 2 CRISPR / Cas systems have a single protein effector. Cas proteins of types II, V and VI are single protein, RNA-guided endonucleases, referred to herein as "class 2 Cas nucleases". Class 2 Cas nucleases include, for example, Cas9, Cpf1, C2c1, C2c2 and C2c3 proteins. Cpf1 nuclease (Zetsche et al., (2015) Cell 163: 1-13) is homologous to Cas9 and comprises a RuvC-like nuclease domain.

[0352] In some embodiments, the Cas nuclease is from a type II CRISPR / Cas system (e.g., the Cas9 protein is from a CRISPR / Cas9 system). In some embodiments, the Cas nuclease is from a class 2 CRISPR / Cas system (a single protein Cas nuclease, such as a Cas9 protein or a Cpf1 protein). Proteins of the Cas9 and Cpf1 families are enzymes with DNA endonuclease activity, and they can be guided to cleave a desired nucleic acid target by designing appropriate guide RNAs, as further described herein.

[0353] Type II CRISPR / Cas system components are from Type IIA, Type IIB, or Type IIC systems. Cas9 and its orthologs are contemplated. Non-limiting exemplary species from which the Cas9 nuclease or other components are derived include Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp.), Staphylococcus aureus, Listeria innocua, Lactobacillus gasseri, Francisella novicida, Wolinella succinogenes, Sutterella wadsworthensis, Gamma proteobacterium, Neisseria meningitidis, Campylobacter jejuni, Pasteurella multocida, Fibrobacter succinogene, Rhodospirillum rubrum, Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces chlororaphis viridochromogenes), Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Lactobacillus buchneri, Treponema denticola, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii), Cyanothece sp., Microcystis aeruginosa, Synechococcus sp.), Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas halophilus haloplanktis), Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleuschthonoplastes, Oscillatoria sp.), Petrotoga mobilis, Thermosipho africanus, Streptococcus pasteurianus, Neisseria cinerea, Campylobacter lari, Parvibaculum lavamentivorans, Corynebacterium diphtheria, or Acaryochloris marina. In some embodiments, the Cas9 protein (SpCas9) is from Streptococcus pyogenes. In some embodiments, the Cas9 protein (StCas9) is from Streptococcus thermophilus. In some embodiments, the Cas9 protein (NmCas9) is from Neisseria meningitidis. In some embodiments, the Cas9 protein (SaCas9) is from Staphylococcus aureus. In some embodiments, the Cas9 protein (CjCas9) is from Campylobacter jejuni. .

[0354] In some embodiments, the Cas nuclease may include more than one nuclease domain. For example, the Cas9 nuclease may include at least one RuvC-like nuclease domain (e.g., Cpf1) and at least one HNH-like nuclease domain (e.g., Cas9). In some embodiments, the Cas9 nuclease introduces DSB in 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 so that one of the nuclease domains is mutated or completely or partially deleted to reduce its nucleic acid cleavage activity. In some embodiments, the Cas9 nuclease is modified to contain no functional RuvC-like nuclease domain. In other embodiments, the Cas9 nuclease is modified to contain no functional HNH-like nuclease domain. In some embodiments in which only one nuclease domain is functional, the Cas9 nuclease is a nickase capable of introducing single-strand breaks ("nicks") into the target sequence. In some embodiments, the conservative amino acids in the Cas9 nuclease domain are replaced to reduce or change the nuclease activity. In some embodiments, the Cas nuclease nickase is included in the amino acid replacement in the RuvC-like nuclease domain. Exemplary amino acid replacements in the RuvC-like nuclease domain include D10A (based on Streptococcus pyogenes Cas9 nuclease). In some embodiments, the nickase is included in the amino acid replacement in the HNH-like nuclease domain. Exemplary amino acid replacements in the HNH-like nuclease domain include E762A, H840A, N863A, H983A and D986A (based on Streptococcus pyogenes Cas9 nuclease). In some embodiments, the nuclease system described herein includes a nickase and a pair of guide RNAs, which are complementary to the sense strand and antisense strand of the target sequence, respectively. The guide RNA guides the nickase to the target and introduces DSB by producing a nick (i.e., double nick) on the opposite strand of the target sequence. Using a chimeric Cas9 nuclease, a domain or region of a protein is replaced by a part of a different protein. For example, the Cas9 nuclease domain is replaced by a domain from different nucleases (such as Fok1). Cas9 nuclease is a modified nuclease.

[0355] 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 derived from a type III CRISPR / Cas system. In some embodiments, the Cas nuclease is derived from a type IV CRISPR / Cas system. In some embodiments, the Cas nuclease is derived from a type V CRISPR / Cas system. In some embodiments, the Cas nuclease is derived from a type VI CRISPR / Cas system.

[0356] In some embodiments, the Cas nuclease is a Mad endonuclease. The CRISPR / Mad system is closely related to type V (Cpf1-like) of the class 2 family of Cas enzymes. In some embodiments, the CRISPR-Mad system employs Eubacterium rectale Mad7 endonuclease or a variant thereof. The Mad7-crRNA complex cuts the target DNA by recognizing PAM 5'-YTTN. 2. Engineered Nucleases

[0357] In some embodiments, the cells described herein are genetically engineered with a site-directed nuclease, wherein the site-directed nuclease is an engineered nuclease. Exemplary engineered nucleases are meganucleases (e.g., homing endonucleases), ZFNs, TALENs, and megaTALs.

[0358] Naturally occurring meganucleases can recognize and cut double-stranded DNA sequences of about 12 to 40 base pairs, and are generally divided into five families. In some embodiments, the 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 homologous target sequence. In some embodiments, the DNA binding domain of the meganuclease is fused to a heterologous nuclease domain. In some embodiments, a meganuclease (such as a homing nuclease) is fused to a TAL module to produce a hybrid protein (such as a "megaTAL" protein). The megaTAL protein has improved DNA targeting specificity by recognizing the target sequence of both the DNA binding domain of the meganuclease and the TAL module.

[0359] ZFN is a fusion protein comprising 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 the ZF repeats are combined to recognize the DNA target sequence and provide sufficient affinity. Therefore, engineered ZF repeats are combined to recognize longer DNA sequences, such as, for example, 9bp, 12bp, 15bp or 18bp, etc. In some embodiments, ZFN comprises a ZF fused to a nuclease domain from a restriction endonuclease. For example, the restriction endonuclease is FokI. In some embodiments, the nuclease domain comprises a dimerization domain (such as when the nuclease dimerizes to have activity), and a pair of ZFNs comprising ZF repeats and nuclease domains are designed for targeting a target sequence, the target sequence comprising two half-target sequences recognized by each ZF repeat on the opposite strand of the DNA molecule, with an interconnecting sequence (which is sometimes referred to as a spacer in the literature) between the half-target sequences. For example, the length of the interconnecting sequence is 5 to 7 bp. When the two 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 comprises a knob-in-hole motif to promote dimerization. For example, the ZFN comprises a knob-in-hole motif in the dimerization domain of FokI.

[0360] The DNA binding domain of TALEN typically contains a variable number of 34 or 35 amino acid repeats ("modules" or "TAL modules"), each of which binds to a single DNA base pair (A, T, G, or C). The adjacent residues at positions 12 and 13 of each module ("repeated variable diresidues" or RVDs) specify a single DNA base pair bound to the module. Although the module for recognizing G can also have an affinity for A, TALEN benefits from a simple recognition code, i.e., there is one module for each of the 4 bases, which greatly simplifies the customization of the DNA binding domain that recognizes a specific target sequence. In some embodiments, TALEN may include a nuclease domain from a restriction endonuclease. For example, the restriction endonuclease is FokI. In some embodiments, the nuclease domain can dimerize to be active, and a pair of TALENS is designed for targeting a target sequence comprising two half-target sequences recognized by each DNA binding domain on the opposite strands of the DNA molecule, with an interconnecting sequence between the half-target sequences. For example, the length of each half target sequence is in the range of 10 to 20 bp, and the length of the interconnecting sequence is 12 to 19 bp. When the two TALENs of the pair are bound, the nuclease domain can dimerize and introduce a DSB within the interconnecting sequence. In some embodiments, the dimerization domain of the nuclease domain can include a knob-in-hole motif to promote dimerization. For example, the TALEN can include a knob-in-hole motif in the dimerization domain of FokI. 3. Target site

[0361] In some embodiments, the site-directed nuclease described herein is directed to a target nucleic acid molecule (e.g., an endogenous gene) and cut (e.g., introducing 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 1α (EEF1A), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ubiquitin C (UBC), or actin β (ACTB). In some embodiments, the target nucleic acid molecule is a blood lineage gene. In some embodiments, the blood lineage gene is a C-type protein tyrosine phosphatase receptor (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.

[0362] The 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 a cell or in a cell. In some embodiments, the target sequence of the target nucleic acid molecule is a genomic sequence from a cell or in a cell. In some embodiments, the target sequence can be located in a non-coding sequence between a coding sequence of a gene, an intron sequence of a gene, a transcription control sequence of a gene, a translation control sequence of a gene, or a gene. 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.

[0363] In some embodiments, the target sequence can be located at a non-gene functional site in the genome that controls aspects of chromatin organization, such as a scaffold site or a locus control region. In some embodiments, the target sequence can be a gene safe harbor site, i.e., a locus that is conducive to safe genetic modification.

[0364] In some embodiments, the target sequence may be adjacent to a pre-spacer adjacent motif (PAM, a short sequence recognized by a CRISPR / Cas complex). In some embodiments, the PAM may be adjacent to the 3' end of the target sequence or within 1, 2, 3, or 4 nucleotides of the 3' end. 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 a common or specific PAM sequence of a specific Cas nuclease or Cas ortholog, including those disclosed in Figure 1 of Ran et al., (2015) Nature, 520: 186-191 (2015), which is incorporated herein by reference. In some embodiments, the length of the PAM may include 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.Non-limiting exemplary PAM sequences include NGG (SpCas9 WT, SpCas9 nickase, dimeric dCas9-Fok1, SpCas9-HF1, SpCas9K855A, eSpCas9 (1.0), eSpCas9 (1.1)), NGAN or NGNG (SpCas9 VQR variant), NGAG (SpCas9 EQR variant), NGCG (SpCas9 VRER variant), NAAG (SpCas9 QQR1 variant), NNGRRT or NNGRRN (SaCas9), NNNRRT (KKH variant). (SaCas9), NNNNRYAC (CjCas9), NNAGAAW (St1Cas9), NAAAC (TdCas9), NGGNG (St3Cas9), NG (FnCas9), NAAAAN (TdCas9), NNAAAAW (StCas9), NNNNACA (CjCas9), GNNNCNNA (PmCas9), and NNNNGATT (NmCas9) (see, e.g., Cong et al., (2013) Science 339:819-823; Kleinstiver et al., (2015) Nat Biotechnol 33:1293-1298; Kleinstiver et al., (2015) Nature 523:481-485; Kleinstiver et al., (2016) Nature 529:490-495; Tsai et al., (2014) Nat Biotechnol 32:569-576; Slaymaker et al., (2016) Science 351:84-88; Anders et al., (2016) Mol Cell 61:895-902; Kim et al., (2017) Nat Comm 8:14500; Fonfara et al., (2013) Nucleic Acids Res 42:2577-2590; Garneau et al., (2010) Nature 468:67-71; Magadan et al., (2012) PLoS ONE 7:e40913; Esvelt et al., (2013) Nat Methods 10(11):1116-1121 (wherein N is defined as any nucleotide, W is defined as A or T, R is defined as a purine (A) or (G), and Y is defined as a pyrimidine (C) or (T)). In some embodiments, the PAM sequence is NGG. In some embodiments, the PAM sequence is NGAN. In some embodiments, the PAM sequence is NGNG. In some embodiments, the PAM is NNGRRT. In some embodiments, the PAM sequence is NGGNG.In some embodiments, the PAM sequence can be NNAAAAW.

[0365] In some embodiments, the PAM sequence recognized by a nuclease (e.g., Cas9) is different depending on the specific nuclease and the bacterial species from which it comes. In some embodiments, the PAM sequence recognized by SpCas9 is the nucleotide sequence 5'-NGG-3', wherein "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', wherein "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', wherein "N" is any nucleotide. In some embodiments, the PAM sequence recognized by CjCas9 is the nucleotide sequence 5'-NNNNRYAC-3', wherein "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 adenine or thymine.

[0366] In some embodiments, the recombinant nuclease is Cas9 and the PAM sequence is the following 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'; wherein "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 adenine or thymine. In some embodiments, the recombinant nuclease is Cas9 (e.g., SpCas9), and the PAM sequence is 5'-NGG-3', wherein "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', wherein "N" is any nucleotide and "R" is a purine (such as guanine or adenine). In some embodiments, the recombinant nuclease is Cas9 (e.g., NmeCas9), and the PAM sequence is 5'-NNNNGATT-3', wherein "N" is any nucleotide. In some embodiments, the recombinant nuclease is Cas9 (e.g., CjCas9), and the PAM sequence is 5'-NNNNRYAC-3', wherein "N" is any nucleotide, "R" is a purine (such as guanine or adenine), and "Y" is a pyrimidine (such as cytosine or thymine). In some embodiments, the recombinant nuclease is Cas9 (eg, StCas9), and the PAM sequence is 5'-NNAGAAW-3', wherein "N" is any nucleotide and "W" is adenine or thymine. 4. Ribonucleoprotein

[0367] In some embodiments, the site-directed polypeptide (e.g., Cas nuclease) and the genome-targeting nucleic acid (e.g., gRNA or sgRNA) can be administered separately to a cell or subject. In some embodiments, the site-directed polypeptide can be pre-compounded with one or more guide RNAs or one or more sgRNAs. Such a pre-composite material is referred to as a ribonucleoprotein particle (RNP). In some embodiments, the nuclease system comprises a ribonucleoprotein (RNP). In some embodiments, the nuclease system comprises a Cas9 RNP, which comprises a purified Cas9 protein compounded with a gRNA. In some embodiments, the nuclease system comprises a Mad7 RNP, which comprises a purified Mad7 protein compounded with a gRNA. Cas9 and Mad7 proteins can be expressed and purified by any means known in the art. Ribonucleoproteins are assembled in vitro and can be delivered directly to cells using standard electroporation or transfection techniques known in the art. B. Homology-directed repair (HDR)

[0368] In some aspects, the embodiments provided relate to the targeted integration of a nucleic acid sequence (such as a donor template) at 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 a eukaryotic translation elongation factor 1α (EEF1A), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ubiquitin C (UBC) or actin β (ACTB). In some embodiments, the target nucleic acid is B2M, TRAC or SIRPA. In some embodiments, the target nucleic acid is B2M.

[0369] In some embodiments, the DNA repair mechanism can be induced by the nuclease following: (i) two SSBs, one on each strand, thereby inducing a single-stranded overhang; or (ii) a DSB occurring at the same cleavage site on both strands, thereby inducing a blunt-end break.

[0370] In some embodiments, HDR is used to target integration or insertion of one or more nucleic acid sequences (e.g., donor templates) in one or more target nucleic acid molecules (e.g., one or more endogenous genes). In some embodiments, HDR can be used to integrate a donor template comprising 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 encoding RACR into a B2M locus.

[0371] 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 (e.g., endogenous genes, such as B2M), as discussed in the preceding sections.

[0372] Agents capable of inducing SSB (also sometimes referred to as nicking) include recombinant nucleases (e.g., Cas9) with nickase activity, such as those described in the preceding section. Examples of agents with nickase activity include, for example, Cas9 from Streptococcus pyogenes comprising mutations selected from D10A, H840A, H854A, and H863A.

[0373] After cleavage by one of these agents, the target endogenous gene (e.g., B2M) with an SSB or DSB undergoes one of two major pathways for DNA damage repair: (1) the error-prone non-homologous end joining (NHEJ), or (2) the high-fidelity homology-directed repair (HDR) pathway.

[0374] In some embodiments, cells in which SSBs or DSBs were previously induced by one or more agents comprising a nuclease are obtained, and a donor template (e.g., ssODN) is introduced to result in HDR and integration of the donor template into a target endogenous gene (e.g., B2M).

[0375] Typically, in the absence of a repair template (e.g., a donor template such as ssODN), the NHEJ process rejoins the ends of the cleaved DNA strands, which often results in deletions and insertions of nucleotides at the cleavage site.

[0376] The change of the nucleic acid sequence at the target endogenous locus (such as the B2M locus) can be carried out by HDR by integrating an exogenously provided donor template encoding a synthetic cytokine receptor (e.g., RACR). The HDR approach can be carried out by means of a canonical HDR approach or an alternative HDR approach. Unless otherwise indicated, the term "HDR" or "homologous directed repair" as used herein covers both canonical HDR and alternative HDR.

[0377] Canonical HDR or "canonical homology-directed repair" or "cHDR" are used interchangeably and refer to the process of repairing DNA damage using homologous nucleic acids (e.g., endogenous homologous sequences, such as sister chromatids; or exogenous nucleic acids, such as donor templates). Canonical HDR usually works when there is significant resection at the DSB, forming at least one single-stranded portion of DNA. In normal cells, canonical HDR usually involves a series of steps, such as recognition of breaks, stabilization of breaks, resection, stabilization of single-stranded DNA, formation of DNA crossover intermediates, decomposition of crossover intermediates, and connection. The canonical HDR process requires RAD51 and BRCA2, and the homologous nucleic acid (e.g., donor template) is typically double-stranded. In canonical HDR, a double-stranded polynucleotide (e.g., double-stranded donor template) is introduced, which contains a sequence homologous to the targeting sequence within the target endogenous locus, and will be directly integrated into the targeting sequence or will be used as a template to insert the sequence or portion of the sequence of the donor template into the target endogenous gene (e.g., B2M). Following resection at the break, repair can proceed via different pathways, for example, via the double Holliday junction model (also known as the double-strand break repair or DSBR pathway), or via the synthesis-dependent strand annealing (SDSA) pathway.

[0378] In the double Holliday junction model, a homologous sequence in a double-stranded polynucleotide (e.g., a double-stranded donor template) is strand invaded by two single-stranded overhangs of the targeting sequence, which results in the formation of an intermediate with two Holliday junctions. As new DNA is synthesized from the ends of the invading strands, the junction migrates to fill the vacancy created by the excision. The ends of the newly synthesized DNA are ligated to the excised ends, and the junction is resolved, resulting in an insertion at the targeting sequence or a portion of the targeting sequence that includes a gene variant. Crossover with a polynucleotide (e.g., a donor template) can occur when the junction is resolved.

[0379] In the SDSA approach, only one 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 vacancy created by the excision. The newly synthesized DNA is then annealed to the remaining single-stranded overhang, new DNA is synthesized to fill the vacancy, and the strands are connected to produce a modified DNA duplex.

[0380] Alternative HDR or "alternative homology directed repair" or "alternative HDR" are used interchangeably, and in some embodiments refer to the process of repairing DNA damage using homologous nucleic acids (e.g., endogenous homologous sequences, such as sister chromatids; or exogenous nucleic acids, such as donor templates). The difference between alternative HDR and standard HDR is that the process utilizes a different approach from standard HDR and can be inhibited by standard HDR mediators RAD51 and BRCA2. In addition, the difference between alternative HDR is that it also involves a single-stranded or nicked homologous nucleic acid template (e.g., a donor template), while standard HDR generally involves a double-stranded homologous template. In the alternative HDR approach, a single-stranded template polynucleotide (e.g., a donor template) is introduced. The nick, single-strand break or DSB at the cleavage site for changing the desired target site (e.g., a target endogenous gene, e.g., B2M) is mediated by a nuclease molecule (e.g., any nuclease as described herein), and excision occurs at the break to expose a single-stranded overhang. Incorporation of sequences into a template polynucleotide (eg, a donor template) to alter a target site of DNA is typically performed by the SDSA approach, as described herein.

[0381] In some embodiments, HDR is performed by introducing into the cell one or more agents capable of inducing DSB (such as any of those described herein) and a donor template (e.g., ssODN, such as any of those described herein). The introduction can be performed by any suitable delivery method (such as any of those described herein). The conditions that allow HDR to be performed can be any conditions suitable for performing HDR in a cell.

[0382] In some embodiments, HDR is performed by introducing into the cell one or more agents capable of inducing SSB in each chain (such as any of those described herein) and a donor template (e.g., ssODN, such as any of those described herein). The introduction can be performed by any suitable delivery method (such as any of those described herein). The conditions that allow HDR to be performed can be any conditions suitable for performing HDR in the cell. Donor template

[0383] In some embodiments, the method provided includes the use of a donor template, such as a donor template encoding a synthetic cytokine receptor (e.g., RACR), which is homologous to one or more portions of a targeting sequence in a target gene (e.g., B2M). In some embodiments, the targeting sequence is contained within the sense strand. In some embodiments, the targeting sequence is contained within the antisense strand. In some embodiments, a donor template for use in the method provided herein is also provided, such as a template for HDR-mediated integration of a nucleic acid sequence encoding RACR.

[0384] In some embodiments, the donor template is used in combination with one or more agents capable of inducing DNA breaks (e.g., SSB or DSB). In some embodiments, the donor template is used in combination with one or more agents capable of inducing DSB and a guide RNA (e.g., sgRNA) to knock in a nucleic acid sequence encoding a synthetic cytokine receptor (e.g., RACR) at a target endogenous locus (e.g., B2M). In some embodiments, the donor template is used in combination with one or more agents capable of inducing SSB; 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) at a target endogenous locus (e.g., B2M).

[0385] In some embodiments, the donor template comprises a nucleic acid sequence homologous to the cleavage site in the target gene (e.g., B2M). In some embodiments, the donor template comprises a nucleic acid sequence homologous to the cleavage site in the sense strand of the target gene (e.g., B2M). In some embodiments, the donor template comprises a nucleic acid sequence homologous to the cleavage site in the antisense strand of the target gene (e.g., B2M). In some embodiments, the target gene (e.g., B2M) includes a sense strand and an antisense strand, and the sense strand includes a targeting sequence. In some embodiments, the target gene (e.g., B2M) includes a sense strand and an antisense strand, and the antisense strand includes a targeting sequence.

[0386] In some embodiments, the donor template (eg, ssODN) comprises a nucleic acid sequence comprising a PAM sequence that is homologous to a PAM sequence in a targeting sequence.

[0387] 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.

[0388] In some embodiments, the ssODN comprises a 5'ssODN arm and a 3'ssODN arm. In some embodiments, the 5'ssODN arm is directly connected to the 3'ssODN arm. In some embodiments, the 5'ssODN arm is homologous to the sequence immediately upstream of the cleavage site in the target gene (e.g., B2M), and the 3'ssODN arm is homologous to the sequence immediately downstream of the cleavage site in the target gene.

[0389] In some embodiments, the 5'ssODN arm and / or 3'ssODN arm have a length between 250 and 750 nucleotides. In some embodiments, the 5'ssODN arm has a length between 250 and 750 nucleotides. In some embodiments, the 3'ssODN arm has a length between 250 and 750 nucleotides. In some embodiments, each of the 5'ssODN arm and the 3'ssODN arm has a length between 250 and 750 nucleotides. In some embodiments, the 5'ssODN arm and / or 3'ssODN arm have a length of about 500 nucleotides. In some embodiments, the 5'ssODN arm has a length of about 500 nucleotides. In some embodiments, the 3'ssODN arm has a length of about 500 nucleotides. In some embodiments, each of the 5'ssODN arm and the 3'ssODN arm has a length of about 500 nucleotides.

[0390] In some embodiments, the target gene is B2M, and the donor template comprises a nucleic acid sequence homologous to a cleavage site in the B2M gene. In some embodiments, the donor template comprises a nucleic acid sequence 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 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 immediately upstream of the cleavage site in the B2M target gene, and the 3'ssODN arm is homologous to a sequence immediately downstream of the cleavage site in the B2M target gene.

[0391] 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 shown in SEQ ID NO: 22. In some embodiments, the 5'ssODN arm comprises the nucleic acid sequence shown in SEQ ID NO: 22.

[0392] 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 shown in SEQ ID NO: 23. In some embodiments, the 3'ssODN arm comprises the nucleic acid sequence shown in SEQ ID NO: 23.

[0393] In some embodiments, the 5' ssODN arm comprises the nucleic acid sequence shown in SEQ ID NO:22, and the 3' ssODN arm comprises the nucleic acid sequence shown in SEQ ID NO:23.

[0394] Also provided herein is an isolated nucleic acid, e.g., an isolated nucleic acid used in a method of knocking a synthetic cytokine receptor (e.g., RACR) into a target gene (e.g., B2M), the isolated nucleic acid comprising any donor template described herein (e.g., ssODN) or a portion thereof (e.g., or a 5'ssODN arm or a 3'ssODN arm) of the nucleic acid sequence. In some embodiments, the 5'ssODN comprises a nucleic acid sequence as shown in SEQ ID NO: 22. In some embodiments, the 3'ssODN arm comprises a nucleic acid sequence as shown in SEQ ID NO: 23.

[0395] 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 shown 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 shown 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 shown in SEQ ID NO: 23. In some embodiments, the crRNA comprises the nucleic acid sequence shown in any one of SEQ ID NO:18; the 5'ssODN comprises the nucleic acid sequence shown in SEQ ID NO:22; and the 3'ssODN comprises the nucleic acid sequence shown in SEQ ID NO:23.

[0396] In some embodiments, the donor template (e.g., ssODN) comprises a nucleic acid sequence encoding a transgenic sequence encoding a synthetic cytokine receptor. In some embodiments, the synthetic cytokine receptor is a rapamycin-activated cytokine receptor (RACR) that responds to rapamycin or an analog (e.g., rapalog). In some embodiments, the transgenic sequence is a tandem box of two polypeptides encoding a synthetic cytokine receptor.

[0397] In some embodiments, a transgene encoding a synthetic cytokine receptor (e.g., RACR) can be inserted so that expression of the transgene is driven by an endogenous promoter at the integration site (e.g., a promoter that drives expression of the endogenous B2M gene). In some embodiments, where the polypeptide coding sequence is promoterless, expression of the integrated transgene is ensured by transcription driven by an endogenous promoter or other control elements in the region of interest. For example, a transgene encoding a partial synthetic cytokine receptor (e.g., RACR) can be inserted without a promoter, but in frame with the coding sequence of the endogenous locus (e.g., B2M locus), so that expression of the integrated transgene is controlled by transcription of an endogenous promoter and / or other regulatory elements at the integration site. In some embodiments, a polycistronic element, such as a ribosome skipping element / self-cleaving element (e.g., a 2A element or an internal ribosome entry site (IRES)), is placed upstream of the transgene such that the polycistronic element is positioned in frame with one or more exons of an endogenous open reading frame at an endogenous locus (e.g., a B2M locus) such that expression of the transgene is operably linked to an endogenous promoter.

[0398] In some embodiments, each nucleic acid encoding a polypeptide of a synthetic cytokine receptor in a "tandem" box is independently controlled by a regulatory element or all controlled as a polycistronic (e.g., bicistronic) expression system. In other embodiments, each nucleic acid encoding a polypeptide of a synthetic cytokine receptor in a "tandem" box can be operably connected to the same or different promoters. 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 nucleic acid molecules can be polycistronic (bicistronic or tricistronic, see, e.g., U.S. Patent No. 6,060,273). In some embodiments, the transcription unit can be engineered into a bicistronic unit containing an IRES (internal ribosome entry site), thereby allowing the gene product to be co-expressed by information from a single promoter. Alternatively, in some cases, a single promoter can guide the expression of an RNA that contains two polypeptides separated from each other by a sequence encoding a cleavable joint as described herein in a single open reading frame (ORF). The ORF thus encodes a single polypeptide that is processed into a separate polypeptide chain during or after translation. In some embodiments, the promoter is selected from the human elongation factor 1 alpha (EF1 alpha) promoter (as shown in SEQ ID NO: 24, 25 or 26). In some embodiments, the promoter is the MND promoter (as shown in SEQ ID NO: 27).

[0399] In some embodiments, the donor template (e.g., ssODN) includes 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) includes an EF1-α 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) includes 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 one of the foregoing, as described in Part V. In some embodiments, the nucleic acid molecule is a tandem box of a first polypeptide sequence encoding RACR and a second polypeptide sequence of RACR.

[0400] In some embodiments, the first nucleic acid sequence encoding RACR comprises a nucleic acid sequence encoding a RACR-γ chain (e.g., SEQ ID NO: 28) and a nucleic acid sequence encoding a RACR-β chain (e.g., SEQ ID NO: 33). In some embodiments, the first nucleic acid sequence encodes a RACR-γ 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 with the amino acid sequence shown in SEQ ID NO: 28. In some embodiments, the first nucleic acid sequence encodes the RACR-γ chain sequence shown in SEQ ID NO: 28. In some embodiments, the nucleic acid sequence encoding the RACR-γ chain further encodes a signal peptide at the N-terminus of the nascent protein to promote protein transport when expressed. In some embodiments, the signal peptide has the sequence shown in SEQ ID NO: 29. In some embodiments, the second nucleic acid sequence encodes a RACR-β 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 shown in SEQ ID NO: 33. In some embodiments, the second nucleic acid sequence encodes the RACR-β chain shown in SEQ ID NO: 33. In some embodiments, the nucleic acid sequence encoding the RACR-β chain further encodes a signal peptide at the N-terminus of the nascent protein to promote protein transport when expressed. In some embodiments, the signal peptide has the sequence shown in SEQ ID NO: 34.

[0401] In some embodiments, the first nucleic acid sequence encoding the RACR-γ 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 shown in SEQ ID NO: 37. In some embodiments, the first nucleic acid sequence encoding the RACR-γ chain has the sequence shown in SEQ ID NO: 37. In some embodiments, the second nucleic acid sequence encoding the RACR-β 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 shown in SEQ ID NO: 38. In some embodiments, the second nucleic acid sequence encoding the RACR-β chain is as shown in SEQ ID NO: 38.

[0402] In some embodiments, the nucleic acid sequence encoding the RACR-γ chain and the nucleic acid sequence encoding the RACR-β chain are separated by a nucleic acid sequence encoding a cleavable linker. In some embodiments, another nucleic acid sequence encoding a cleavable linker is located downstream of the nucleic acid sequence encoding the RACR-β chain.

[0403] In some embodiments, the linker is a protein quantitation reporter linker (PQR; e.g., SEQ ID NO: 42), including any of those described in Canadian Patent Application No. CA2970093, which is incorporated herein by reference in its entirety. In some embodiments, the PQR linker has the sequence shown in SEQ ID NO: 42. In some embodiments, the PQR linker is encoded by the nucleotide sequence shown in SEQ ID NO: 41.

[0404] In some embodiments, the cleavable linker is a self-cleaving peptide, such as a 2A ribosomal skipping element. In some cases, a cleavable linker (such as T2A) can cause the ribosome to skip the synthesis of the peptide bond at the C-terminus of the 2A element (ribosome skipping), resulting in a separation between the end of the 2A sequence and the next peptide downstream (see, e.g., 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 are not limited to 2A sequences from foot-and-mouth disease virus (F2A, e.g., SEQ ID NO: 43), equine rhinitis virus (E2A, e.g., SEQ ID NO: 44), Thosea asigna virus (T2A, e.g., SEQ ID NO: 45 or 46), and from porcineteschovirus-1 (P2A, e.g., SEQ ID NO: 47 or 48), as described in U.S. Patent Publication No. 20070116690.

[0405] In some embodiments, expression of a nucleic acid sequence encoding RACR produces a first peptide (ie, RACR-γ chain) and a separate second peptide (ie, RACR-β chain) due to a cleavable element located between the first and second nucleic acid sequences.

[0406] In some embodiments, the transgenic sequence can also include the sequence required for transcription termination and / or polyadenylation signal. In some aspects, exemplary polyadenylation signals are selected from SV40, hGH, BGH and rbGlob transcription termination sequences and / or polyadenylation signals. In some embodiments, the transgenic includes the SV40 polyadenylation signal. In some embodiments, if present in the transgenic, the transcription termination sequence and / or polyadenylation signal are typically the most 3' sequence in the transgenic and are connected to one of the homology arms. In some embodiments, the transgenic sequence comprises the polyadenylation sequence shown in SEQ ID NO:39.

[0407] In some embodiments, the ssODN comprises, in sequence: a 5' ssODN arm, an EF1-α promoter, a nucleic acid sequence encoding a RACR-γ chain, a nucleic acid sequence encoding a cleavable linker (eg, a PQR linker), a nucleic acid sequence encoding a RACR-β chain, a poly A sequence, and a 3' ssODN arm.

[0408] In some embodiments, the ssODN comprises the sequence shown in SEQ ID NO: 40, or a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence shown in SEQ ID NO: 40. In some embodiments, the ssODN is as shown in SEQ ID NO: 40.

[0409] In some embodiments, after ssODN is integrated into the target gene, the target gene is knocked out. In some embodiments, the target gene is human B2M, and after ssODN is integrated into B2M, B2M is knocked out. In some embodiments, the nucleic acid sequence encoding the synthetic cytokine receptor is integrated into the B2M locus. In some embodiments, the iPSC and CIL of engineering approaches have a modified B2M locus, wherein the knock-in achieved by the knockout of the B2M gene and by the targeted integration of the nucleic acid encoding the synthetic cytokine receptor carries out genetic destruction to the endogenous B2M gene. In some embodiments, the synthetic cytokine receptor is a RACR encoded by a nucleic acid sequence, and the nucleic acid sequence comprises in sequence: EF1-α promoter, a nucleic acid sequence encoding RACR-γ chain, a nucleic acid sequence encoding a cleavable joint (e.g., PQR joint), a nucleic acid sequence encoding RACR-β chain and a poly A sequence. In some embodiments, the nucleic acid sequence encoding RACR integrated into the B2M locus has the sequence shown 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 shown in SEQ ID NO: 32. In some embodiments, the nucleic acid encoding RACR integrated into the B2M locus is as shown in SEQ ID NO: 32. VII. Chimeric Antigen Receptor

[0410] In some cases, the engineered lymphocytes (such as CIL cells) of the present disclosure may contain or be transduced with a polynucleotide encoding a chimeric antigen receptor (CAR), thereby generating lymphocytes (such as CIL cells) expressing the CAR.

[0411] In some embodiments, the present disclosure contemplates a chimeric antigen receptor (CAR) system used in the treatment of a subject with cancer. In some embodiments, the engineered lymphocytes (such as CIL cells) of the present disclosure include CAR sequences (CAR-CIL cells or CAR-iCIL cells).

[0412] In some embodiments, lymphocytes (such as CIL cells) are engineered to express the CAR construct by transfecting a cell group with an expression vector encoding a CAR construct. Illustrative examples of cell groups that can be transfected include HSC, blood progenitor cells, common lymphoid progenitor cells or CIL cells. Suitable means for preparing a transduced lymphocyte (such as CIL cells) group expressing the selected CAR construct will be well known to skilled artisans, and to give a few examples, including retroviruses, slow viruses (virus-mediated CAR gene delivery systems), sleeping beauty (sleeping beauty) and piggyback (including transposon / transposase systems of non-viral mediated CAR gene delivery systems). In some embodiments, any transduction method considered in this disclosure can be used to produce CIL cells expressing CAR. A. Targeting Agents for CAR

[0413] Conventionally, CAR is produced by fusing a polynucleotide encoding VL, VH or scFv to the 5' end of a polynucleotide encoding a transmembrane and intracellular domain, and, if desired, transducing cells with the polynucleotide and with the corresponding VH or VL. Many variations of CAR are well known in the art, and the present disclosure contemplates the use of any known variation. In addition, VL / VH pairs and scFvs of numerous haptens are known in the art, or can be conventionally produced by conventional methods. Therefore, the present disclosure contemplates the use of any known hapten binding domain.

[0414] Various methods for targeting CAR and cells expressing CAR have been described in the art, including, for example, in US 2020 / 0123224, the disclosure of which is incorporated herein by reference. For example, fluorescein or fluorescein isothiocyanate (FITC) moieties can be conjugated to reagents that bind to desired target cells (such as cancer cells), thereby CAR-CIL cells expressing anti-fluorescein / FITC chimeric antigen receptors can be selectively targeted to target cells labeled by the conjugate. In a variation, other haptens recognized by CAR can be used instead of fluorescein / FITC. CAR can be produced using various scFv sequences known in the art or scFv sequences produced by conventional and routine methods. Other illustrative scFv sequences for fluorescein / FITC and for other haptens are provided in, for example, WO 2021 / 076788, the disclosure of which is incorporated herein by reference.

[0415] In some embodiments, the CAR system of the present disclosure utilizes the following CAR, the CAR targeting is not produced or expressed by the cells of the treated subject. Therefore, the CAR system allows CIL cells to be centrally targeted to target cells (such as cancer cells). By administering small conjugate molecules together with cells expressing CAR (such as CIL cells expressing CAR), the cell response of engineered cells can be targeted only to those cells expressing tumor receptors, thereby reducing off-target toxicity, and due to the rapid removal of the small conjugate molecules, the activation of engineered lymphocytes (e.g., CIL cells) can be more easily controlled. As an additional advantage, lymphocytes expressing CAR (such as CIL cells expressing CAR) can be used as "universal" cytotoxic cells to target a variety of tumors without the need to prepare a separate CAR construct. The targeted portion identified by CAR can also remain unchanged. Only the ligand portion of the small conjugate molecule needs to be changed to allow the system to target cancer cells of different identities.

[0416] In one embodiment, the present disclosure provides a description of the conjugate molecule / CAR system.

[0417] In some embodiments, the CAR system of the present disclosure utilizes a conjugate molecule as a bridge between cells expressing CAR and targeted cancer cells. The conjugate molecule is a conjugate comprising 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 small molecular weight organic molecules, such as DNP (2,4-dinitrophenol), TNP (2,4,6-trinitrophenol), biotin and digoxin, as well as fluorescein and its derivatives (including FITC (fluorescein isothiocyanate), NHS-fluorescein), and pentafluorophenyl esters (PFP) and tetrafluorophenyl esters (TFP) derivatives, knottin, centyrin and DARPin. Suitable cell targeting moieties that can themselves serve as haptens for the CAR include knottins (see Kolmar H. et al., The FEBS Journal. 2008. 275(11):26684-90), centyrins and DARPins (see Reichert, JMM Abs 2009. 1(3):190-209).

[0418] In some embodiments, the cell targeting moiety is DUPA (DUPA-(99m)Tc), a molecule that binds to PSMA-positive human prostate cancer cells with nanomolar affinity (K D=14 nM; see Kularatne, SA et al., Mol Pharm. 2009. 6(3):780-9) bound ligands. 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 WO 2015 / 057852, which is incorporated herein by reference.

[0419] In some embodiments, the cell targeting moiety is a CCK2R ligand, a ligand bound by CCK2R-positive cancer cells (e.g., cancers of the thyroid, lung, pancreas, ovary, brain, stomach, gastrointestinal stromal, and colon; see Wayua. C. et al., Molecular Pharmaceutics. 2013. ePublication).

[0420] In some embodiments, the cell targeting moiety is folate, folic acid, or an analog thereof, a ligand bound by a folate receptor on cancer cells, including cancers of the ovary, cervix, endometrium, lung, kidney, brain, breast, colon, and head and neck; see Sega, EI et al., Cancer Metastasis Rev. 2008. 27(4):655-64).

[0421] In some embodiments, the cell targeting moiety is a NK-1R ligand. Receptors for NK-1R ligands are found on cancers of, for example, the colon and pancreas. In some embodiments, the NK-1R ligand can be synthesized according to the methods disclosed in International Patent Application No. PCT / US2015 / 044229, which is incorporated herein by reference.

[0422] 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 of the NK1 receptor. In some embodiments, the small conjugate molecule ligand can be a regulatory peptide belonging to the tachykinin family that targets the tachykinin receptor. Such regulatory peptides include substance P (SP), neurokinin A (K substance) and neurokinin B (neuromediatin K) (see Hennig et al., International Journal of Cancer: 61, 786-792).

[0423] In some embodiments, the cell targeting moiety is a CAIX ligand. Receptors for the CAIX ligand are found, for example, on renal cancer, ovarian cancer, vulvar cancer, and breast cancer. The CAIX ligand may also be referred to herein as CA9.

[0424] In some embodiments, the cell targeting moiety is a ligand for gamma-glutamyl transpeptidase, which is overexpressed in, for example, ovarian cancer, colon cancer, liver cancer, astrocytic glioma, melanoma, and leukemia.

[0425] In some embodiments, the cell targeting moiety is a CCK2R ligand. Receptors for the CCK2R ligand are found, inter alia, on cancers of the thyroid, lung, pancreas, ovary, brain, stomach, gastrointestinal stroma, and colon.

[0426] In one embodiment, the cell targeting moiety can 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.

[0427] In an illustrative embodiment, the connection in the conjugate described herein can be a direct connection (e.g., a reaction between the isothiocyanate group of FITC and the free amine group of the small molecule ligand), or the connection can be achieved through an intermediate linker. In one embodiment, if present, the intermediate linker can be any biocompatible linker known in the art, such as a divalent linker. In an illustrative embodiment, the divalent linker can include about 1 to about 30 carbon atoms. In another illustrative embodiment, the divalent linker can include about 2 to about 20 carbon atoms. In other embodiments, a divalent linker with a lower molecular weight (i.e., those with an approximate molecular weight of about 30 to about 300 Da) is 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.

[0428] In some embodiments, the hapten and the 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., folic acid, DUPA, and CCK2R ligand). However, the use of a linker domain to connect the two molecules can be helpful because it can provide flexibility and stability. Examples of suitable linker domains include: 1) polyethylene glycol (PEG); 2) polyproline; 3) hydrophilic amino acids; 4) sugars; 5) non-natural peptidoglycans; 6) polyvinyl pyrrolidone; 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.

[0429] In some embodiments, the linker can be a bivalent linker that can include one or more spacers.

[0430] An illustrative conjugate of the present disclosure is FITC-folic acid An illustrative conjugate of the present disclosure is FITC-CA9

[0431] Illustrative conjugates of the present disclosure include the following molecules: FITC-(PEG)12 -Folic acid, FITC-(PEG) 20 -Folic acid, FITC-(PEG) 108 -Folic acid, FITC-DUPA, FITC-(PEG) 12 -DUPA, FITC-CCK2R ligand, FITC-(PEG) 12 -CCK2R ligand, FITC-(PEG) 11 -NK1R ligand and FITC-(PEG)2-CA9.

[0432] Although the affinity with which a ligand binds to a cancer cell receptor can vary, and in some cases low affinity binding may be preferred (e.g., about 1 μM), the binding affinity of a ligand to a cancer cell receptor will typically be at least about 100 μM, 1 nM, 10 nM, or 100 nM, preferably at least about 1 pM or 10 pM, and even more preferably at least about 100 pM.

[0433] Examples of conjugates and methods for making them are provided in U.S. patent applications US2017 / 0290900, US 2019 / 0091308, and US2020 / 0023009, all of which are incorporated herein by reference. B. CAR Constructs

[0434] In some embodiments, the CAR construct comprises an extracellular binding portion, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a costimulatory signaling domain and / or an activation signaling domain. In some embodiments, the CAR construct comprises an extracellular binding portion, a transmembrane domain, and an intracellular signaling domain containing a costimulatory signaling domain. In some embodiments, the CAR construct comprises an extracellular binding portion, a transmembrane domain, and an intracellular signaling domain containing an activation signaling domain. In some embodiments, the CAR construct comprises an extracellular binding portion, a transmembrane domain, and an intracellular signaling domain containing a costimulatory signaling domain and an activation signaling domain.

[0435] 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 unmodified (ie, 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.

[0436] In some embodiments, the costimulatory domain is used to enhance the proliferation and survival of lymphocytes after CAR is combined with the targeted portion. The identity of the costimulatory domain is limited only in that it has the ability to enhance cell proliferation and survival activation after CAR is combined with the targeted portion. Suitable costimulatory domains include, but are not limited to: CD28 (see, for example, 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, for example, Imai, C. et al., Leukemia. 2004. 18: 676-84); and CD134 (OX40), a member of the TNFR receptor superfamily (see, for example, Latza, U. et al., Eur. J. Immunol. 1994. 24: 677). It will be appreciated by a skilled person that sequence variants of these costimulatory domains can be used, wherein the variant has the same or similar activity to the domain modeled therewith. 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.

[0437] In some embodiments of the present invention, the CAR construct comprises two costimulatory domains. Although a specific combination includes all possible variants of the four described domains, specific examples include: 1) CD28+CD137 (4-1BB) and 2) CD28+CD134 (OX40).

[0438] In some embodiments, the activation signaling domain is used to activate cells after CAR binds to the targeted portion. The identity of the activation signaling domain is limited only in that it has the ability to induce activation of selected cells after CAR binds to the targeted portion. Suitable activation signaling domains include CD3 ζ chain and Fc receptor γ. Skilled technicians will understand that sequence variants of these activation signaling domains can be used without adversely affecting the present invention, wherein the variants have the same or similar activity as the domains modeled by them. 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 with the amino acid sequence of the domain from which they are derived.

[0439] In some embodiments, the CAR may include additional elements, such as a signal peptide to ensure proper export 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 to confer flexibility to the recognition region and allow strong binding to the targeted moiety.

[0440] Illustrative CAR constructs suitable for CAR-engineered lymphocytes (such as CAR-CIL cells) are provided below: (1) scFv-CD8 TM -4-1BB IC -CD3ζs (see, e.g., Liu E, Tong Y, Dotti G, et al., Leukemia. 2018; 32: 520-531); (2) scFv-CD28 TM+IC -CD3ζs (see, 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 -CD3ζs (see, e.g., Xu Y, Liu Q, Zhong M et al., J Hematol Oncol. 2019; 12:49); (5) scFv-2B4 TM+IC -CD3ζs (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 -CD3ζs (see, e.g., Kloss S, Oberschmidt O, Morgan M et al., Hum Gene Ther. 2017; 28: 897-913); (7) scFv-CD16 TM -2B4 IC-CD3ζs (see, e.g., Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192); (8)scFv-NKp44 TM -DAP10 IC -CD3ζs (see, e.g., Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192); (9)scFv-NKp46 TM -2B4 IC -CD3ζs (see, e.g., Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192); (10)scFv-NKG2D TM -2B4 IC -CD3ζs (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 -CD3ζs (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 -CD3ζs (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 -CD3ζs (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).

[0441] In some embodiments, the binding portion of the CAR can be directed to any antigen that is desired to be targeted, such as due to its overexpression on a cell or association with a disease or disorder (such as cancer).

[0442] In some embodiments, the binding portion of the CAR is specific for a tumor antigen. The choice of antigen binding domain will depend on the specific 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, polysialic acid, PLAC1, RU1, RU2 (AS), intestinal carboxylesterase, lewisY, sLe, LY6K, mut hsp70-2, M-CSF, MYCN, RhoC, TRP-2, CYPIBI, BORIS, prostate enzymes, prostate-specific antigen (PSA), PAX3, PAP, NY-ESO-1, LAGE-la, LMP2, NCAM, p53, p53 mutants, Ras mutants, gplOO, prostate-specific protein (prostein), OR51E2, PANX3, PSMA, PSCA, Her2 / neu, hTERT, HMWMAA, HAVCR1, VEGFR2, PDGFR-β, survivin and telomerase, pod protein, 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 (TMPRSS2 ETS 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 the following: differentiation antigens, such as tyrosinase, TRP-1, TRP-2, and tumor-specific multi-lineage 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 generated by 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-based 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, β-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, β-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, SDCCAG1 6, TA-90\Mac-2 binding protein / cyclophilin C-associated protein, TAAL6, TAG72, TLP, TPS, GPC3, MUC16, LMP1, EBMA-1, BARF-1, CS1, CD319, HER1, B7H6, L1CAM, IL6 and MET.

[0443] Skilled technicians are easily familiar with CARs for different tumor antigens. Any of such CARs can be used as CAR. Many CARs have been approved by the FDA, and include but are not limited to anti-CD19 and anti-BCMACAR T cells such as Kymriah, Yescarta, Tecartus, Breyanzi or Abecma. Skilled technicians can produce similar constructs for specific targeting of desired tumor antigens.

[0444] In some embodiments, the binding portion of CAR can be directed to a universal antigen to target a variety of tumors without the need to prepare a separate CAR construct. The targeted portion recognized by CAR can also remain unchanged. In some embodiments, a ligand can be administered to a subject to allow interaction with target cells and interaction with the binding portion of 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. An exemplary universal CAR system is described in the above section.

[0445] In some embodiments, the CAR is an anti-FITC CAR, and the ligand is composed of a fluorescein or fluorescein isothiocyanate (FITC) portion conjugated to an agent, which is combined with a desired target cell (such as a cancer cell). Exemplary ligands are described in the above section. In some embodiments, the ligand is FITC-folic acid.

[0446] An illustrative CAR of the present disclosure is shown in Figure 8 wherein the fusion protein is encoded by a lentiviral expression vector and wherein "SP" is a signal peptide, CAR is an anti-FITC CAR, the CD8α hinge is present, the transmembrane domain is present ("TM"), the co-stimulatory domain is 4-1BB, and the activation signaling domain is CD3ζ.

[0447] An illustrative nucleotide sequence encoding a CAR may comprise SEQ ID NO: 13:

[0448] The exemplary CAR amino acid sequence can include SEQ ID NO:14: MALPVTALLLPLALLLHAARPDVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRVSGVPDRFSGSGSGTDFTLKINRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIKSSADDAKKDAAKKDDAKKDDAKKDGGVKLDETGGGLVQPGGAMKLSCVTSGFTFGHYWMNWVRQSPEKGLEWVAQFRNKPYNYETYYSDSVKGRFTISRDDSKSSVYLQMNNLRVEDTGIYYCTGASYGMEYLGQGTSVTVSFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:14)

[0449] The exemplary nucleotide insert can include SEQ ID NO:15: GCCACCATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGATGTCGTGATGACCCAGACCCCCCTCAGCCTCCCAGTGTCCCTCGGTGACCAGGCTTCTATTAGTTGCAGATCCAGCCAGTCCCTCGTGCACTCTAACGGTAATACCTACCTGAGATGGTATCTCCAGAAGCCCGGACAGAGCCCTAAGGTGCTGATCTACAAAGTCTCCAACCGGGTGTCTGGAGTCCCTGACCGCTTCTCAGGGAGCGGTTCCGGCACCGACTTCACCCTGAAGATCAACCGGGTGGAGGCCGAAGACCTCG GCGTCTATTTCTGCTCTCAGAGTACACATGTGCCCTGGACCTTCGGCGGAGGGACC AAGCTGGAGATCAAAAGCTCCGCAGACGATGCCAAGAAAGATGCCGCTAAGAAA GACGATGCTAAGAAAGACGATGCAAAGAAAGACGGTGGCGTGAAGCTGGATGAA ACCGGAGGAGGTCTCGTCCAGCCAGGAGGAGCCATGAAGCTGAGTTGCGTGACCA GCGGATTCACCTTTGGGCACTACTGGATGAACTGGGTGCGACAGTCCCCAGAGAA GGGGCTCGAATGGGTCGCTCAGTTCAGGAACAAACCCTACAATTATGAGACATAC TATTCAGACAGCGTGAAGGGCAGGTTTACTATCAGTAGAGACGATTCCAAATCTA GCGTGTACCTGCAGATGAACAATCTCAGGGTCGAAGATACAGGCATCTACTATTG CACAGGGGCATCCTATGGTATGGAGTATCTCGGTCAGGGGACAAGCGTCACAGTC AGTTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAGCGCCGCGACC ACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCG TGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTG ATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTG GTTATCACCCTTTACTGCAACCACAGGAACCGTTTCTCTGTTGTTAAACGGGGCAG AAAGAAACTCCGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTC AAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTG AACTGAGAGTGAAGTTCAGCAGGAGCCGCAGACGCCCCCGCGTACCAGCAGGGCCA GAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTG GACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAAC CCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACA GTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTT ACCAGGGTCCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA (SEQ ID NO: 15)

[0450] In some embodiments, the CAR can be encoded by a nucleic acid sequence encoding a signal peptide, which is used for the signal transduction of CAR in cells. It should be understood that the signal peptide is typically removed from the protein.

[0451] An illustrative CAR amino acid sequence without a signal peptide can comprise SEQ ID NO: 16: DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRVSGVPDRFSGSGSGTDFTLKINRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIKSSADDAKKDAAKKDDAKKDDAKKDGGVKLDETGGGLVQPGGAMKLSCVTSGFTFGHYWMNWVRQSPEKGLEWVAQFRNKPYNYETYYSDSVKGRFTISRDDSKSSVYLQMNNLRVEDTGIYYCTGASYGMEYLGQ GTSVTVSFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCR FPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:16)

[0452] An illustrative CAR amino acid sequence signal peptide may comprise SEQ ID NO: 17: MALPVTALLLPLALLLHAARP (SEQ ID NO: 17)

[0453] In various embodiments, a CAR-expressing cell comprising a nucleic acid of SEQ ID NO: 13 or 15 is provided. 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.

[0454] In some embodiments, variant nucleic acid sequences 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.

[0455] Although the affinity with which a CAR expressed by a lymphocyte binds to a targeted moiety can vary, and in some cases low affinity binding may be preferred (e.g., about 50 nM), the binding affinity of the CAR to the targeted ligand will generally be at least about 100 nM, 1 pM, or 10 pM, more preferably at least about 100 pM, 1 fM, or 10 fM, and even more preferably at least about 100 fM. VII. Characterization of iCIL cells expressing synthetic cytokine receptors

[0456] In some embodiments, the CIL cells are CD3-CD5-, CD16+, CD56+, CD57+, NKp30+, NKp46+, NKG2A+ and / or NKG2D+.

[0457] In some embodiments, the engineered cell population is 40% to 60% CD16+, 50% to 70% CD16+, 60% to 80% CD16+, 70% to 90% CD16+, 80% to 100% CD16+, or any percentage within a range bounded by any two of the foregoing values.

[0458] In some embodiments, the engineered cell population is 60% to 80% CD56+, 65% to 85% CD56+, 70% to 90% CD56+, 75% to 95% CD56+, 80% to 99% CD56+, or any percentage within a range bounded by any two of the foregoing values.

[0459] In some embodiments, the engineered CIL cell population is CD56lo.In some embodiments, the engineered CIL cell population is CD56high.

[0460] In some embodiments, the engineered CIL cell population is 60% to 80% CD16+CD56+, 65% to 85% CD16+CD56+, 70% to 90% CD16+CD56+, 75% to 95% CD16+CD56+, 80% to 99% CD16+CD56+, or any percentage within the range defined by any two of the foregoing values.

[0461] In some embodiments, the engineered CIL cell population is at least 40% CD16+, at least 50% CD16+, at least 60% CD16+, at least 70% CD16+, at least 80% CD16+, at least 90% CD16+, or 100% CD16+.

[0462] In some embodiments, the engineered CIL cell population is at least 80% CD56+, at least 85% CD56+, at least 90% CD56+, at least 95% CD56+, or 100% CD56+.

[0463] In some embodiments, the engineered CIL cell population is at least 40% CD16+CD56+, at least 50% CD16+CD56+, at least 60% CD16+CD56+, at least 70% CD16+CD56+, at least 80% CD16+CD56+, at least 90% CD16+CD56+, or 100% CD16+CD56+.

[0464] In some embodiments, the CIL cells are characterized as being CD45+CD56+.

[0465] In some embodiments, the engineered CIL cell population is 40% to 60% CD45+, 50% to 70% CD45+, 60% to 80% CD45+, 70% to 90% CD45+, 80% to 100% CD45+, or any percentage within a range bounded by any two of the foregoing values.

[0466] In some embodiments, the engineered CIL cell population is 60% to 80% CD45+CD56+, 65% to 85% CD45+CD56+, 70% to 90% CD45+CD56+, 75% to 95% CD45+CD56+, 80% to 99% CD45+CD56+, or any percentage within the range defined by any two of the foregoing values.

[0467] In some embodiments, the engineered CIL cell population is at least 40% CD45+, at least 50% CD45+, at least 60% CD45+, at least 70% CD45+, at least 80% CD45+, at least 90% CD45+, or 100% CD45+.

[0468] In some embodiments, the engineered CIL cell population is at least 40% CD45+CD56+, at least 50% CD45+CD56+, at least 60% CD45+CD56+, at least 70% CD45+CD56+, at least 80% CD45+CD56+, at least 90% CD45+CD56+, or 100% CD45+CD56+.

[0469] K562 cells are highly sensitive targets for in vitro cytotoxic innate lymphoid cell cytotoxic activity assays. In the assay, CIL cells are co-incubated with K562 target tumor cells known to be sensitive to CIL cell-mediated cytotoxicity at different ratios. Target cells (K562) are pre-labeled with fluorescent dyes so that they can be distinguished from effector cells (CIL cells). After the incubation period, the target cells killed are identified by nucleic acid stains that specifically penetrate dead cells. Death % is calculated by comparing the total number of live cells in each experimental assay well with those in the non-effector control well. As used herein, the term "activity" refers to the measurement of the cytotoxic ability of CIL cells against target cells.

[0470] In some embodiments, the engineered CIL cells secrete CD107a in response to an antigen recognized by the engineered CIL cells.

[0471] Innate CIL cell receptor stimulation causes CIL cells to secrete CD107a. CD107a expression is associated with cytokine secretion and CIL cell-mediated target cell lysis, and therefore, as used herein, CD107a secretion is a marker of CIL cell functional activity.

[0472] 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.

[0473] CIL cell activation leads to the secretion of IFNγ and TNF-α, which synergistically enhance the cytotoxicity of CIL cells. In engineered CIL cells, the expression of IFNγ and / or TNF-α is a marker of CIL cell activity.

[0474] As described above, provided herein are hematopoietic stem cells or progenitor cells that can be differentiated into lymphoid cells using synthetic cytokine receptor complexes activated by non-physiological ligands, and differentiated cells produced by those stem cells or progenitor cells used in medical treatment. The differentiated cells can be, but are not limited to, iCIL cells. The advantages of the embodiments may include the ability to produce abundant cell sources (e.g., induced pluripotent stem cells) of effector cells expressing synthetic cytokine receptor complexes activated by non-physiological ligands, so that the proliferation of effector cells in patients can be controlled by administering or stopping the administration of the non-physiological ligands. Other advantages of the embodiments include, but are not limited to, the ability to produce effector cells from source cells in a culture medium that is substantially free of cytokines (such as IL-2, IL-7, and / or IL-15) that are routinely used for differentiation of iCIL cells.

[0475] CIL cells can be generated from multiple sources, illustrative examples include: iPSC, PBMC or UCB. In some embodiments, the CIL source cells are autologous cells. In some embodiments, the CIL source cells are allogeneic cells. In some embodiments, the CIL source cells are heterologous cells. For example, when the subject treated with the composition of the present disclosure has received high-dose chemotherapy or radiation therapy to destroy the subject's immune system, allogeneic cells can be used.

[0476] In some embodiments, hematopoietic stem cells can be engineered to express synthetic cytokine receptors. In some embodiments, blood progenitor cells (leukocytes) 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.

[0477] In some embodiments, in addition to or in place of exogenous cytokines, non-physiological ligands can induce differentiation. In some embodiments, the non-physiological ligands can induce differentiation during one or more of mesoderm formation, hematopoietic specialization, lymphoid progenitor differentiation, and CIL cell differentiation.

[0478] In some embodiments, the non-physiological ligand may be contacted with cells at a stage of differentiation requiring IL-7 and / or IL-15 signaling.

[0479] In some embodiments, the non-physiological ligand may be contacted with cells that are in an expansion phase requiring an IL-2 signal.

[0480] 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 differentiate and / or expand without the use of exogenous factors.

[0481] Because it is well known in the relevant art that the differentiation and / or expansion of CIL cells depends on the presence of various exogenous stimuli, as described herein, the differentiation and / or expansion of engineered CIL cells in the absence of one, two or all of IL-2, IL-15 and IL-7 is surprising and unexpected.

[0482] In some embodiments, the engineered common lymphoid progenitor cells are differentiated into CIL cells in the absence of IL-15 in the differentiation medium.

[0483] In some embodiments, the engineered common lymphoid progenitor cells are differentiated into CIL cells in the absence of IL-7 in the differentiation medium.

[0484] In some embodiments, the engineered common lymphoid progenitor cells are differentiated into CIL cells in the absence of IL-15 or IL-7 in the differentiation medium.

[0485] In some embodiments, the common lymphoid progenitor cells are engineered to express a synthetic cytokine receptor (eg, RACR) and are differentiated into CIL cells in the presence of a rapalog in the differentiation medium and in the absence of IL-15 or IL-7 in the differentiation medium.

[0486] In some embodiments, the engineered CIL cells are expanded in the absence of IL-2 in the expansion medium.

[0487] In some embodiments, CIL cells are engineered to express a synthetic cytokine receptor (eg, RACR) and are expanded in the presence of a rapalog in the expansion medium and in the absence of a recombinant cytokine in the expansion medium.

[0488] In some embodiments, the CIL cell expansion step is performed in a cell culture medium that is substantially free of recombinant cytokines.

[0489] In some embodiments, the expanding step is performed in feeder-free cell culture.

[0490] In some embodiments, the CIL cell expansion step is performed in a culture vessel that is not coated with a recombinant ligand used for CIL cell expansion.

[0491] In some embodiments, a method of administering an effective amount of a non-physiological ligand to a subject is provided, wherein the non-physiological ligand causes common lymphoid progenitor cells to differentiate into CIL cells according to any of the preceding embodiments.

[0492] In some embodiments, a method of administering an effective amount of a non-physiological ligand to a subject is provided, wherein according to any of the preceding embodiments, the non-physiological ligand causes cell proliferation.

[0493] In some embodiments, the non-physiological ligand is present or provided in an amount effective to cause ex vivo expansion of the CIL cells.

[0494] In some embodiments, the non-physiological ligand is present or provided in an amount effective to induce cytokine secretion by CIL cells ex vivo.

[0495] In some embodiments, the non-physiological ligand is present or provided in an amount effective to cause CIL cells to secrete CD107a, interferon gamma (IFNγ) and / or tumor necrosis factor alpha (TNF-α) ex vivo.

[0496] In some embodiments, the non-physiological ligand is present or provided in an amount effective to cause tumor cell killing.

[0497] In some embodiments, the non-physiological ligand is present or provided in a therapeutically effective amount. IX. Methods of treating a subject using the disclosed compositions

[0498] The present disclosure provides methods for treating subjects in need with compositions, therapeutic compositions, cells, vectors and polynucleotides disclosed herein. In some embodiments, the present disclosure provides a method for treating cancer and / or killing cancer cells in a subject, the method comprising administering a therapeutically effective amount of the disclosed cells to the subject. Also provided is a method for treating a tumor and / or killing tumor cells in a subject, the method comprising administering an effective amount of a non-physiological ligand to the subject, wherein according to any of the foregoing embodiments, the non-physiological ligand causes cell proliferation.

[0499] In some embodiments, the malignancy is one or more of 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 myeloid 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 myeloid leukemia (CML), B cell prolymphocytic leukemia, blastic plasma cell The invention relates to a malignant lymphoma, a leukemia, a small cell-like dendritic cell neoplasm, a Burkitt lymphoma, a diffuse large B-cell lymphoma, a follicular lymphoma, a hairy cell leukemia, a small cell or large cell follicular lymphoma, a malignant lymphoproliferative disorder, a MALT lymphoma, a mantle cell lymphoma, a marginal zone lymphoma, a myelodysplastic and myelodysplastic syndrome, a plasmablastic lymphoma, a plasmacytoid dendritic cell neoplasm, a Waldenstrom macroglobulinemia, a plasma cell proliferative disorder (e.g., asymptomatic myeloma (smoldering multiple myeloma or indolent myeloma)), a monoclonal gammopathy of undetermined significance (MGUS), a plasma cell neoplasm (e.g., a plasma cell disorder, a solitary myeloma, a solitary plasmacytoma, an extramedullary plasmacytoma, and multiple plasmacytoma), a systemic amyloid light chain amyloidosis, a POEMS syndrome (also known as Kroll-Fukase syndrome, Takatsuki disease, and PEP syndrome), or a combination thereof.

[0500] In some embodiments, the methods disclosed herein can 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.

[0501] The present disclosure also provides methods of treating cancer and / or killing cancer cells in a subject, the method comprising administering to the subject a system of any of the foregoing embodiments.

[0502] In some embodiments, the present disclosure provides methods for treating cancer with any composition provided herein. When read according to the specification, "cancer" has its obvious and common meaning, and may include, but is not limited to, for example, a group of diseases involving abnormal cell growth, which may invade or spread to other parts of the body. Subjects that can be treated using the methods described herein include subjects identified or selected as having cancer, including but not limited to colon, lung, liver, breast, kidney, prostate, ovary, skin (including melanoma), bone and brain cancer, etc. This identification and / or selection can be performed by clinical or diagnostic evaluation. In some embodiments, tumor-associated antigens or 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, one or more oncogenic polypeptides are associated with kidney, uterus, colon, lung, liver, breast, kidney, prostate, ovary, skin (including melanoma), bone, brain cancer, adenocarcinoma, pancreatic cancer, chronic myeloid leukemia or leukemia. In some embodiments, a method of treating, ameliorating or inhibiting a subject's cancer is provided. In some embodiments, the cancer is breast cancer, ovarian cancer, lung cancer, pancreatic cancer, prostate cancer, melanoma, kidney cancer, pancreatic cancer, glioblastoma, neuroblastoma, medulloblastoma, sarcoma, liver cancer, colon cancer, skin cancer (including melanoma), bone cancer or brain cancer.

[0503] In some embodiments, the target cell is a tumor cell. In some embodiments, the target cell is present in a tumor microenvironment.

[0504] In some embodiments, the present disclosure provides a method for treating cancer with any composition provided herein without preconditioning the subject. In some embodiments, the subject of the present disclosure does not need to receive lymphocyte clearance therapy. Those skilled in the art will understand the available common lymphocyte clearance therapy, such as chemotherapy. In some embodiments, the subject of the present disclosure does not receive lymphocyte clearance therapy. In some embodiments, differentiated cells are provided to subjects who have not received lymphocyte clearance therapy. In some embodiments, the subject does not receive lymphocyte clearance therapy in 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 days before the administration of differentiated cells.

[0505] In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 24, 36, 48, 60 or 72 hours after applying the ligand composition or at any time within the range limited by any two aforementioned values, the subject is provided with differentiated cells. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 24, 36 or 48 hours before applying the ligand composition or at any time within the range limited by any two aforementioned values, the subject is provided with the cell. In some embodiments, within a few seconds or a few minutes (such as less than one hour) of providing the composition to the subject, the subject is provided with the cell. In some embodiments, the subject is provided with the strengthening of the cell and / or the composition.

[0506] In some aspects, the present disclosure provides methods of treating cancer as described in WO 2019 / 144095, which is incorporated herein by reference in its entirety.

[0507] In some embodiments, the disclosure provides a method for treating cancer, the method comprising administering a chimeric antigen receptor cell (e.g., NK cell), wherein the CAR comprises an E2 anti-fluorescein antibody fragment. In some embodiments, the method for treating cancer further comprises administering a small molecule connected to a targeting moiety via a linker.

[0508] In some embodiments, the targeting moiety depends on the type of cancer treated in the subject. As an example, folate receptors are highly expressed on the surface of various solid tumor cells, and the solid tumors include breast cancer (for example, triple-negative breast cancer), ovarian cancer, endometrial cancer, kidney 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 includes folic acid, and the folic acid will be combined with the folate receptor expressed on cancer cells or tumor cells. In other embodiments, the folic acid can be folic acid, folic acid analogs or any folate receptor binding molecules. In some embodiments, the small molecule includes fluorescein, fluorescein isothiocyanate (FITC), NHS-fluorescein or any other fluorophore. In some embodiments, the small molecule connected to the targeting moiety is FITC-folate.

[0509] In some embodiments, the present disclosure contemplates the transplantation of engineered stem cells used in the treatment of subjects with cancer. In some embodiments, engineered cytotoxic innate lymphoid cells derived from iPSC are stably transplanted in subjects. In some embodiments, engineered cytotoxic innate lymphoid cells derived from iPSC show long-term transplantation in subjects. In some embodiments, the administration of engineered cytotoxic innate lymphoid cells derived from iPSC allows for transplantation and further differentiation in subjects. In some embodiments, engineered cytotoxic innate lymphoid cells derived from iPSC are natural killer cells.

[0510] In some embodiments, additional cancer therapies are provided, such as small molecules (eg, compounds), antibody therapies (eg, humanized monoclonal antibodies conjugated with or without radionuclides, toxins, or drugs), surgery, and / or radiation.

[0511] In some embodiments, the subject is selected to receive an additional cancer therapy, which may include a cancer therapeutic agent, radiation, chemotherapy, or a drug for cancer treatment. In some embodiments, the drug includes abiraterone, alentuzumab, anastrozole, aprepitant, arsenic trioxide, atelizumab, azacitidine, bevacizumab, bleomycin, bortezomib, cabazitaxel, capecitabine, carboplatin, cetuximab, chemotherapy drug combination, cisplatin, crizotinib, cyclophosphamide, cytarabine, denosumab, docetaxel, doxorubicin, eribulin, erlotinib, etoposide, everolimus, exemestane, filgrastim, fluorouracil , fulvestrant, gemcitabine, imatinib, imiquimod, ipilimumab, ixabepilone, lapatinib, lenalidomide, letrozole, leuprolide, mesna, methotrexate, nivolumab, oxaliplatin, paclitaxel, palonosetron, pembrolizumab, pemetrexed, prednisone, radium-223, rituximab, siprucept-T, sorafenib, sunitinib, talc (intrapleural), tamoxifen, temozolomide, temsirolimus, thalidomide, trastuzumab, vinorelbine, or zoledronic acid. Administration and dosing

[0512] In some embodiments, the transduced lymphocytes (such as CIL cells) engineered with synthetic cytokine receptors as described herein can be grown under conditions suitable for a cell population to be introduced into a subject such as a human. Special considerations include the use of a culture medium lacking any animal product (such as bovine serum). Other considerations include aseptic conditions to avoid contamination with bacteria, fungi, and mycoplasmas. In some embodiments, any cell culture method contemplated in this disclosure can be used to grow lymphocytes (such as CIL cells expressing CAR) expressing CAR.

[0513] In some embodiments, after transfection, cells can be immediately administered to patients or cells can be cultured for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11.12, 13, 14, 15, 16, 17, 18 or more days, or 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, for example, to allow time for cells to recover from transfection. Suitable culture conditions can be similar to containing or not containing agents for promoting activation to culture cells for activation. In some embodiments, any method of applying the engineered lymphocytes (such as CIL cells) considered in the present disclosure can be used to apply lymphocytes (such as CIL cells expressing CAR) expressing CAR to patients.

[0514] The disclosed cells can be administered in a variety of ways, depending on whether local or systemic treatment is desired.

[0515] In the case of adoptive cell therapy, methods of administering cells for adoptive cell therapy are known and can be used in conjunction with the provided methods and compositions.

[0516] 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 includes any route of administration, and administration is characterized by physical destruction of the tissue of the subject and administration of the pharmaceutical composition through a laceration in the tissue, thus generally resulting in direct administration into the bloodstream, into the muscle, or into an internal organ. Therefore, parenteral administration includes, but is not limited to, administration of the pharmaceutical composition by injection of the composition, administration of the composition through a surgical incision, administration of the composition through a non-surgical wound penetrating the tissue, etc. In particular, it is contemplated that parenteral administration includes, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal, intravenous, intraarterial, intrathecal, intraventricular, intraurethral, ​​intracranial, intratumoral, intrasynovial injection or infusion; and renal dialysis infusion techniques. In an embodiment, the parenteral administration of the compositions of the present disclosure includes intravenous administration.

[0517] The formulation of a pharmaceutical composition suitable for parenteral administration generally comprises a combination of an active ingredient and a pharmaceutically acceptable carrier (such as sterile water or sterile isotonic saline). Such formulations can be prepared, packaged or sold in a form suitable for push injection or continuous administration. Injectable formulations can be prepared, packaged or sold in unit dosage forms (such as ampoules or multi-dose containers containing preservatives). Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, etc. Such formulations may also include one or more additional ingredients, including but not limited to suspending agents, stabilizers or dispersants. In one embodiment 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) before parenteral administration of the reconstituted composition. Parenteral formulations also include aqueous solutions, which may contain excipients such as salts, carbohydrates, and buffers (preferably to a pH of 3 to 9), but for some applications, they may be more suitably formulated as sterile non-aqueous solutions or dry forms for use in combination with suitable vehicles (such as sterile pyrogen-free water). Illustrative parenteral administration forms include solutions or suspensions in sterile aqueous solutions (e.g., aqueous propylene glycol solutions or dextrose solutions). Such dosage forms may be appropriately buffered if desired. Available other parenterally administrable formulations include those comprising active ingredients in microcrystalline forms or liposome formulations. Formulations for parenteral administration may be formulated to release immediately and / or regulate release. Regulated release formulations include delayed release, sustained release, pulsed release, controlled release, targeted release, and programmed release.

[0518] Composition of the present invention can contain other auxiliary components that are conventionally present in the pharmaceutical composition in addition.Therefore, for example, described composition can contain other compatible pharmaceutically active materials, for example antipruritic, astringent, local anesthetic or anti-inflammatory agent, or can contain other materials that can be used for the various dosage forms of physical preparation of the present composition, such as dye, flavoring, preservative, antioxidant, emulsifier, thickener and stabilizer.However, when adding such materials, the biological activity of the component of the present composition should not be disturbed inappropriately.Preparation can be sterilized, and if necessary, mixed with auxiliary agent, described auxiliary agent for example lubricant, preservative, stabilizer, wetting agent, emulsifying agent, salt, buffer, coloring agent, flavoring and / or aromatic substance that affect osmotic pressure and similar substances that do not interact harmfully with one or more nucleic acids of preparation.

[0519] The compositions of the viral particles, adapter molecules and / or immune cells of the present invention can be administered in an amount effective to treat or prevent a disease or condition (e.g., a therapeutically effective amount or a prophylactically effective amount). In some embodiments, the therapeutic or prophylactic efficacy is monitored by regularly evaluating the treated subject. For repeated administration over several days or longer, depending on the condition, the treatment is repeated until the desired suppression of disease symptoms occurs. However, other dosage regimens may be useful and can be determined. The desired dose can be delivered by administering the composition as a single bolus, by administering the composition as multiple boluses, or by administering the composition as a continuous infusion.

[0520] In certain embodiments, in the context of an infusion of differentiated cells or transgenic differentiated cells according to the present disclosure, cells are administered to a subject in a range of about 1 million to about 100 billion cells per kilogram of subject body weight, such as, for example, 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 bounded by any two of the foregoing values), such as 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, or a range bounded by any two of the foregoing values). In some embodiments, the administration of a cell or cell population may include administering 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 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), and in some cases 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 90 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells) or any value between these ranges, and / or such numbers of cells. For example, in some embodiments, administration of a cell or cell population may include administering about 10 3 to about 10 9 cells, including all integer values ​​of the number of cells within those ranges. X. Exemplary Embodiments

[0521] The provided implementations include: 1. A cell population comprising engineered cytotoxic innate lymphoid cells engineered for controlled expansion and / or activity, wherein the engineered cytotoxic innate lymphoid cells comprise synthetic cytokine receptors for non-physiological ligands, Wherein the cytokine receptor comprises: A synthetic gamma chain polypeptide comprising a first dimerization domain, a first transmembrane domain and an interleukin-2 receptor subunit gamma (IL-2RG) intracellular domain, and A synthetic β chain polypeptide comprising a second dimerization domain, a second transmembrane domain and an intracellular domain selected from the group consisting of an interleukin-2 receptor subunit β (IL-2RB) intracellular domain, an interleukin-7 receptor subunit β (IL-7RB) intracellular domain and / or an interleukin-21 receptor subunit β (IL-21RB) intracellular domain; Wherein the non-physiological ligand activates a synthetic cytokine receptor in the cytotoxic innate lymphoid cells to induce expansion and / or activation of the engineered cytotoxic innate lymphoid cells. 2. A cell population according to embodiment 1, wherein the β chain intracellular domain includes the IL-2RB intracellular domain. 3. The cell population of embodiment 2, wherein the IL-2RB intracellular domain comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO: 2, or a polypeptide sequence as shown in SEQ ID NO: 2. 4. A cell population according to embodiment 1, wherein the β chain intracellular domain includes the IL-7RB intracellular domain. 5. The cell population of embodiment 4, wherein the IL-7RB intracellular domain comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO: 3, or a polypeptide sequence as shown in SEQ ID NO: 3. 6. The cell population according to embodiment 1, wherein the β chain intracellular domain includes 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 SEQ ID NO: 4, or a polypeptide sequence as shown in SEQ ID NO: 4. 8. The cell population according to any one of embodiments 1 to 7, wherein the first dimerization domain and the second dimerization domain are extracellular domains; The synthetic γ chain polypeptide comprises the first dimerization domain, the first transmembrane domain and the interleukin-2 receptor subunit γ (IL-2RG) intracellular domain in the order from N to C terminus, and The synthetic β chain polypeptide comprises the second dimerization domain, the second transmembrane domain and the intracellular domain in order from N-terminus to C-terminus. 9. A cell population according to any one of embodiments 1 to 8, wherein the first dimerization domain and the second dimerization domain are heterodimerization domains selected from the following: FK506 binding protein (FKBP) with a size of 12kD and FKBP12-rapamycin binding (FRB) domain; and / or The non-physiological ligand is rapamycin or rapalog. 10. The cell population of embodiment 9, wherein the FRB domain comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:6 or SEQ ID NO:7. 11. A cell population according to embodiment 9, wherein the FRB domain comprises the polypeptide sequence shown in SEQ ID NO:6 or SEQ ID NO:7. 12. A cell population according to any one of embodiments 1 to 8, wherein the first dimerization domain and the second dimerization domain are heterodimerization domains selected from the following: a FK506 binding protein (FKBP) of 12 kD in size and a calcineurin domain; and / or The non-physiological ligand is FK506 or an analog thereof. 13. A cell population according to any one of embodiments 1 to 12, wherein the FKBP domain comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:5. 14. A cell population according to any one of embodiments 1 to 12, wherein the FKBP domain comprises the polypeptide sequence shown in SEQ ID NO:5. 15. The cell population according to any one of embodiments 1 to 8, wherein the first dimerization domain and the second dimerization domain are homodimerization domains selected from the following: i) FK506 binding protein (FKBP) with a size of 12 kD; ii) Cyclophilin A (CypA); or iii) gyrase B (CyrB); And the non-physiological ligands are: i) FK1012, AP1510, AP1903 or AP20187 or their analogs; ii) cyclosporine A (CsA) or an analogue thereof; or iii) coumermycin or its analogs. 16. The cell population of any one of embodiments 1 to 15, wherein the cytotoxic innate lymphoid cells express a cytoplasmic polypeptide that binds to the non-physiological ligand. 17. The cell population of any one of embodiments 1 to 15, wherein the non-physiological ligand is rapamycin or rapalog and the cytotoxic innate lymphoid cells express a cytoplasmic FRB domain or a variant thereof. 18. A cell population according to embodiment 17, wherein the cytoplasmic FRB domain comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:6 or SEQ ID NO:7. 19. A cell population according to embodiment 17, wherein the cytoplasmic FRB domain comprises a polypeptide sequence that is at least 98% identical to SEQ ID NO:6 or SEQ ID NO:7. 20. The cell population of any one of embodiments 1 to 19, wherein the cytotoxic innate lymphoid cells are primary cytotoxic innate lymphoid cells. 21. The cell population of any one of embodiments 1 to 19, wherein the cytotoxic innate lymphoid cells are blood-derived cytotoxic innate lymphoid cells. 22. The cell population of any one of embodiments 1 to 19, wherein the cytotoxic innate lymphoid cells are induced cytotoxic innate lymphoid (iCIL) cells. 23. A cell population according to any one of embodiments 1 to 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 embodiments 1 to 23, wherein the engineered cytotoxic innate lymphoid cell population is at least 40% CD16+. 25. The cell population of any one of embodiments 1 to 24, wherein the engineered cytotoxic innate lymphoid cell population is or is at least 40% CD56+. 26. A cell population according to any one of embodiments 1 to 23, wherein the engineered cytotoxic innate lymphoid cell population is at least 40% CD16+CD56+. 27. A cell population according to any one of embodiments 1 to 26, wherein the engineered cytotoxic innate lymphoid cells express CD16, CD56, CD57, NKG2A, NKG2D and / or CD57 at a level at least equivalent to that of control cytotoxic innate lymphoid cells expanded with IL-2. 28. A cell population according to any one of embodiments 1 to 27, wherein the engineered cytotoxic innate lymphoid cells express NKp30 and / or NKp46 at a level higher than that of control cytotoxic innate lymphoid cells expanded with IL-2. 29. A cell population according to any one of embodiments 1 to 28, wherein the engineered cytotoxic innate lymphoid cells secrete CD107a in response to an antigen recognized by the engineered cytotoxic innate lymphoid cells. 30. A cell population according to any one of embodiments 1 to 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. A method of preparing engineered cytotoxic innate lymphoid cells for controlled expansion and / or activity, the method comprising: contacting the source cell with a vector comprising a polynucleotide encoding a synthetic cytokine receptor for a non-physiological ligand, thereby transducing the source cells to produce a cell population comprising transduced cells; Wherein the cytokine receptor comprises: A synthetic gamma chain polypeptide comprising a first dimerization domain, a first transmembrane domain and an interleukin-2 receptor subunit gamma (IL-2RG) intracellular domain, and A synthetic β chain polypeptide comprising a second dimerization domain, a second transmembrane domain and an intracellular domain selected from the group consisting of an interleukin-2 receptor subunit β (IL-2RB) intracellular domain, an interleukin-7 receptor subunit β (IL-7RB) intracellular domain and / or an interleukin-21 receptor subunit β (IL-21RB) an intracellular domain; and expanding the engineered cytotoxic innate lymphoid cells in the cell population by contacting the cell population with the non-physiological ligand to activate the synthetic cytokine receptor, This results in the expansion of the engineered cytotoxic innate lymphoid cells within the cell population. 32. A method according to embodiment 31, wherein the source cell is a primary cytotoxic innate lymphoid cell. 33. A method according to 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. A method according to embodiment 31, wherein the source cells are human pluripotent stem cells. 35. The method of embodiment 33 or embodiment 34, wherein the method comprises contacting the cell population with the non-physiological ligand during differentiation of the transduced cells into engineered cytotoxic innate lymphoid cells. 36. A method according to any one of embodiments 31 to 35, wherein the expanding step is performed in a cell culture medium that is substantially free of recombinant cytokines; wherein the expanding step is performed in a feeder-free cell culture; and / or wherein the expanding 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 according to any one of embodiments 31 to 36. 38. A pharmaceutical composition comprising the cell population according to any one of embodiments 1 to 30 or embodiment 37. 39. A method for immunotherapy of a subject in need thereof, the method comprising administering to the subject an effective amount of the cell population according to any one of Embodiments 1 to 30 or Embodiment 37 or the pharmaceutical composition according to Embodiment 38. 40. The method of embodiment 39, comprising administering to the subject the non-physiological ligand in an amount effective to cause expansion of the engineered cytotoxic innate lymphoid cells in the subject. 41. The method of embodiment 39 or embodiment 40, comprising administering to the subject the 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 embodiments 39 to 41, comprising administering to the subject the non-physiological ligand in an amount effective to cause the engineered cytotoxic innate lymphoid cells to kill tumor cells. 43. A kit comprising a cell population according to any one of embodiments 1 to 30 or embodiment 37 or a pharmaceutical composition according to 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, Wherein the cytokine receptor comprises: A synthetic gamma chain polypeptide comprising a first dimerization domain, a first transmembrane domain and an interleukin-2 receptor subunit gamma (IL-2RG) intracellular domain, and A synthetic β chain polypeptide comprising a second dimerization domain, a second transmembrane domain and an intracellular domain selected from the group consisting of an interleukin-2 receptor subunit β (IL-2RB) intracellular domain, an interleukin-7 receptor subunit β (IL-7RB) intracellular domain and / or an interleukin-21 receptor subunit β (IL-21RB) intracellular domain; The non-physiological ligand activates the synthetic cytokine receptor in the engineered cells to induce expansion and / or activation of the engineered cells. 45. A cell population according to embodiment 44, wherein the engineered cells are human pluripotent stem cells. 46. ​​A cell population according to embodiment 44, wherein the engineered cells are hematopoietic stem cells or hematopoietic progenitor cells. 47. A cell population according to embodiment 44, wherein the engineered cells are common lymphoid progenitor cells. 48. A cell population according to any one of embodiments 44 to 47, wherein the cell population is a cell population according to any one of embodiments 2 to 30 or embodiment 37. 49. A pharmaceutical composition or kit comprising the cell population according to any one of embodiments 44 to 48. 50. A method for preparing the cell population according to embodiment 1 according to any one of embodiments 31 to 37. 51. Use of the cell population according to embodiment 50 in a method according to any one of embodiments 38 to 42. 52. A method for preparing and / or expanding an engineered cell population comprising a synthetic cytokine receptor for a non-physiological ligand, the method comprising: a) providing an engineered cell comprising a synthetic cytokine receptor, optionally by introducing into a source cell a polynucleotide encoding a synthetic cytokine receptor, and b) incubating the engineered source cells in a culture medium comprising a non-physiological ligand, Wherein the cytokine receptor comprises: A synthetic gamma chain polypeptide comprising a first dimerization domain, a first transmembrane domain and an interleukin-2 receptor subunit gamma (IL-2RG) intracellular domain, and A synthetic β chain polypeptide comprising a second dimerization domain, a second transmembrane domain and an intracellular domain selected from the group consisting of an interleukin-2 receptor subunit β (IL-2RB) intracellular domain, an interleukin-7 receptor subunit β (IL-7RB) intracellular domain and / or an interleukin-21 receptor subunit β (IL-21RB) intracellular domain; The non-physiological ligand activates the synthetic cytokine receptor in the engineered cells to induce expansion and / or activation of the engineered cells. 53. A method according to embodiment 52, wherein the source cells are induced pluripotent stem cells. 54. A method according to embodiment 52, wherein the source cells are hematopoietic stem cells or hematopoietic progenitor cells. 55. A method according to embodiment 52, wherein the source cell is a common lymphoid progenitor cell. 56. A method according to embodiment 52, wherein the source cell is a cytotoxic innate lymphoid cell. 57. A method according to embodiment 52, wherein the vector is a lentiviral vector. 58. The method of embodiment 52, wherein the non-physiological ligand is 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, Wherein the cytokine receptor comprises: A synthetic gamma chain polypeptide comprising a first dimerization domain, a first transmembrane domain and an interleukin-2 receptor subunit gamma (IL-2RG) intracellular domain, and A synthetic β chain polypeptide comprising a second dimerization domain, a second transmembrane domain and an intracellular domain selected from the group consisting of an interleukin-2 receptor subunit β (IL-2RB) intracellular domain, an interleukin-7 receptor subunit β (IL-7RB) intracellular domain and / or an interleukin-21 receptor subunit β (IL-21RB) intracellular domain; The binding of the non-physiological ligand to the synthetic cytokine receptor activates the synthetic cytokine receptor in the engineered CIL cells to induce expansion and / or activation of the engineered CIL cells in 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. An engineered CIL cell according to embodiment 59 or embodiment 60, wherein the synthetic γ chain polypeptide comprises the first dimerization domain, the first transmembrane domain and the interleukin-2 receptor subunit γ (IL-2RG) intracellular domain in order from N to C terminus, and the synthetic β chain polypeptide comprises the second dimerization domain, the second transmembrane domain and the intracellular domain in order from N to C terminus. 62. The engineered CIL cell according to any one of embodiments 59-61, wherein the IL-2RG intracellular domain comprises a polypeptide sequence at least 95% identical to SEQ ID NO: 1, or a polypeptide sequence as shown in SEQ ID NO: 1. 63. An engineered CIL cell according to any one of embodiments 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 SEQ ID NO: 8 or 31, or a polypeptide sequence as shown in SEQ ID NO: 8 or 31. 65. An engineered CIL cell according to any one of embodiments 59-64, wherein the β chain intracellular domain includes the IL-2RB intracellular domain. 66. The CIL cell of embodiment 65, wherein the IL-2RB intracellular doma...

Claims

1. An engineered cytotoxic innate lymphoid (CIL) cell engineered for controlled expansion and / or activity, said engineered lymphocyte or progenitor cell comprising a synthetic cytokine receptor for a non-physiological ligand, Wherein the cytokine receptor comprises: A synthetic gamma chain polypeptide comprising a first dimerization domain, a first transmembrane domain and an interleukin-2 receptor subunit gamma (IL-2RG) intracellular domain, and A synthetic α or β chain polypeptide comprising a second dimerization domain, a second transmembrane domain, and an intracellular domain selected from the group consisting of an interleukin-2 receptor subunit β (IL-2RB) intracellular domain, an interleukin-7 receptor subunit β (IL-7RB) intracellular domain, an interleukin-21 receptor subunit β (IL-21RB) intracellular domain, or an interleukin-9 receptor subunit α (IL-9RA) intracellular domain; The binding of the non-physiological ligand to the synthetic cytokine receptor activates the synthetic cytokine receptor in the engineered CIL cells to induce expansion and / or activation of the engineered CIL cells in the cell population.

2. The engineered CIL cell according to claim 1, Wherein the cytokine receptor comprises: A synthetic gamma chain polypeptide comprising a first dimerization domain, a first transmembrane domain and an interleukin-2 receptor subunit gamma (IL-2RG) intracellular domain, and A synthetic β chain polypeptide comprising a second dimerization domain, a second transmembrane domain and an intracellular domain selected from the following: an interleukin-2 receptor subunit β (IL-2RB) intracellular domain, an interleukin-7 receptor subunit β (IL-7RB) intracellular domain and / or an interleukin-21 receptor subunit β (IL-21RB) intracellular domain.

3. The engineered CIL cell according to claim 1 or claim 2, wherein the first dimerization domain and the second dimerization domain are extracellular domains.

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

5. The engineered CIL cell according to any one of claims 1 to 4, wherein the IL-2RG intracellular domain comprises a polypeptide sequence at least 95% identical to SEQ ID NO: 1, or a polypeptide sequence as shown in SEQ ID NO:

1.

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

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

8. The engineered CIL cell according to any one of claims 1-7, wherein the β chain intracellular domain includes the IL-2RB intracellular domain.

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

2.

10. The engineered CIL cell according to any one of claims 1-7, wherein the β chain intracellular domain comprises the IL-7RB intracellular domain.

11. The engineered CIL cell according to any one of claims 1-7, wherein the IL-7RB intracellular domain comprises a polypeptide sequence at least 95% identical to SEQ ID NO: 3, or a polypeptide sequence as shown in SEQ ID NO:

3.

12. The engineered CIL cell according to any one of claims 1-7, wherein the β chain intracellular domain comprises the IL-21RB intracellular domain.

13. The engineered CIL cell according to claim 12, wherein the IL-21RB intracellular domain comprises a polypeptide sequence at least 95% identical to SEQ ID NO:4, or a polypeptide sequence as shown in SEQ ID NO:

4.

14. The engineered CIL cell of any one of claims 2-7, wherein the synthetic α chain polypeptide comprises the IL-9RA intracellular domain.

15. The engineered CIL cell of claim 14, wherein the IL-9RA intracellular domain comprises a polypeptide sequence at least 95% identical to SEQ ID NO: 51, or a polypeptide sequence as shown in SEQ ID NO:

51.

16. The engineered CIL cell of any one of claims 1-13, wherein the second transmembrane domain comprises a transmembrane domain from the same polypeptide as the intracellular domain.

17. The engineered CIL cell according to claims 1-9 and 16, wherein the second transmembrane domain is a transmembrane domain of IL-2RB comprising a polypeptide sequence at least 95% identical to SEQ ID NO: 35 or 36, or a polypeptide sequence as shown in SEQ ID NO: 35 or 36.

18. The engineered CIL cell according to any one of claims 1-9, 16 and 17, wherein: The synthetic gamma chain polypeptide comprises an IL-2RG TM domain comprising the sequence shown in SEQ ID NO: 8 or 31 and an IL-2RG intracellular domain comprising the sequence shown in SEQ ID NO: 1; and The synthetic β chain polypeptide comprises an IL-2RB TM domain comprising the sequence shown in SEQ ID NO:35 or 36 and an IL-2RB intracellular domain comprising the sequence shown in SEQ ID NO:

2.

19. The engineered CIL cell according to any one of claims 1-18, wherein the first dimerization domain and the second dimerization domain are heterodimerization domains selected from the following: FK506 binding protein (FKBP) of 12 kD in size and FKBP12-rapamycin binding (FRB) domain; and / or The non-physiological ligand is rapamycin or rapalog.

20. The engineered CIL cell of claim 19, wherein the first dimerization domain is FKBP and the second dimerization domain is FRB.

21. The engineered CIL cell of claim 19, wherein the first dimerization domain is FRB and the second dimerization domain is FKBP.

22. The engineered CIL cell of any one of claims 19-21, wherein the FRB domain comprises a polypeptide sequence at least 95% identical to SEQ ID NO: 6 or SEQ ID NO:

7.

23. The engineered CIL cell according to any one of claims 19-21, wherein the FRB domain comprises the polypeptide sequence shown in SEQ ID NO: 6 or SEQ ID NO:

7.

24. The engineered CIL cell according to any one of claims 1-18, wherein the first dimerization domain and the second dimerization domain are heterodimerization domains selected from the group consisting of: a 12 kD FK506 binding protein (FKBP) and a calcineurin domain; and / or The non-physiological ligand is FK506 or an analog thereof.

25. The engineered CIL cell of any one of claims 1-19, wherein the FKBP domain comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:5 or SEQ ID NO:

30.

26. The engineered CIL cell according to any one of claims 1-19, wherein the FKBP domain comprises the polypeptide sequence shown in SEQ ID NO: 5 or SEQ ID NO:

30.

27. The engineered CIL cell according to any one of claims 1-9 and 16-26, wherein the synthetic γ chain polypeptide has the amino acid sequence shown in SEQ ID NO:28, or an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:28, and the synthetic β chain polypeptide has the amino acid sequence shown in SEQ ID NO:33, or an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:

33.

28. The engineered CIL cell according to any one of claims 1-9 and 16-27, wherein the synthetic γ chain polypeptide has the amino acid sequence shown in SEQ ID NO:28, and the synthetic β chain polypeptide has the amino acid sequence shown in SEQ ID NO:

33.

29. The engineered CIL cell according to any one of claims 1-18, wherein the first dimerization domain and the second dimerization domain are homodimerization domains selected from the following: i) FK506 binding protein (FKBP) with a size of 12 kD; ii) Cyclophilin A (CypA); or iii) gyrase B (CyrB); And the non-physiological ligands are: i) FK1012, AP1510, AP1903 or AP20187 or their analogs; ii) cyclosporine A (CsA) or an analogue thereof; or iii) coumermycin or its analogs.

30. The engineered CIL cell of any one of claims 1-29, wherein the CIL cell is resistant to rapamycin-mediated inhibition of mTOR.

31. The engineered CIL cell of any one of claims 1-30, wherein the cytotoxic innate lymphoid cell expresses a cytoplasmic polypeptide that binds to the non-physiological ligand, optionally wherein the cytoplasmic polypeptide is a cytoplasmic FRB domain.

32. The engineered CIL cell of any one of claims 1-31, wherein the non-physiological ligand is rapamycin or rapalog, and the cytotoxic innate lymphoid cell expresses a cytoplasmic FRB domain or a variant thereof.

33. The engineered CIL cell of claim 26 or claim 27, wherein the cytoplasmic FRB domain comprises a polypeptide sequence at least 95% identical to SEQ ID NO: 6 or SEQ ID NO:

7.

34. The engineered CIL cell of claim 26 or claim 27, wherein the cytoplasmic FRB domain comprises a polypeptide sequence at least 98% identical to SEQ ID NO: 6 or SEQ ID NO:

7.

35. The engineered CIL cell of any one of claims 1-34, wherein the CIL cell comprises a disrupted FKBP12 gene that reduces FKBP12 expression.

36. The engineered CIL cell according to any one of claims 1-35, wherein the CIL cell comprises a knockout of the FKBP12 gene.

37. The engineered CIL cell according to any one of claims 1-36, wherein the CIL cell comprises a nucleotide sequence encoding the synthetic cytokine receptor inserted into the genome of the CIL cell.

38. The engineered CIL cell of claim 37, wherein the nucleotide sequence encoding the synthetic cytokine receptor is inserted into a non-target locus in the genome of the CIL cell.

39. The engineered CIL cell of claim 37, wherein the nucleotide sequence encoding the synthetic cytokine receptor is inserted into an endogenous gene of the CIL cell.

40. The engineered CIL cell of claim 39, wherein the insertion reduces expression of an endogenous gene in the locus.

41. The engineered CIL cell of claim 39 or claim 40, wherein the insertion knocks out the endogenous gene in the locus.

42. The engineered CIL cell according to any one of claims 39-41, wherein the insertion is performed by homology directed repair.

43. The engineered CIL cell according to any one of claims 39-42, wherein the endogenous gene is a housekeeping gene, a blood lineage-specific locus, or an immune-related gene.

44. The engineered CIL cell of claim 43, wherein the endogenous gene is a housekeeping gene, and the housekeeping gene is selected from eukaryotic translation elongation factor 1 alpha (EEF1A), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ubiquitin C (UBC), and actin beta (ACTB).

45. The engineered CIL cell of claim 43, wherein the endogenous gene is a blood lineage-specific locus, and the blood lineage-specific locus is selected from the group consisting of protein tyrosine phosphatase receptor type C (PTPRC), IL2RG, and IL2RB.

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

47. The engineered CIL cell of any one of claims 1-46, wherein the CIL cell comprises a B2M knockout.

48. The engineered CIL cell according to any one of claims 1-47, wherein the CIL cell comprises a B2M knockout and a FKBP12 knockout.

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

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

51. A cell population comprising the engineered CIL cells according to any one of claims 1-50.

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

53. The method of claim 52, wherein the nucleotide sequence is inserted via homology directed repair (HDR).

54. The method of claim 53, wherein the vector comprises a nucleic acid comprising, from 5' to 3', (a) a nucleotide sequence homologous to a region located upstream of the target site, (b) the nucleotide sequence encoding a synthetic cytokine receptor for a non-physiological ligand, and (c) a nucleotide sequence homologous to a region located downstream.

55. The method of claim 52, wherein the nucleotide sequence is inserted via non-homologous end joining (NHEJ).

56. The method of any one of claims 52-55, wherein the RNA-guided endonuclease is selected from a Cas endonuclease, a Mad endonuclease, and a Cpf1 endonuclease.

57. The method of any one of claims 52-56, wherein the RNA-guided endonuclease is Cas9 or Mad7.

58. The method of any one of claims 52-57, wherein the endogenous gene is selected from B2M, TRAC and SIRPA.

59. The method of any one of claims 52-58, wherein the endogenous gene is B2M.

60. The method of any one of claims 52-59, wherein the gRNA comprises the sequence shown in SEQ ID NO:

18.

61. The method of any one of claims 54-60, wherein the nucleotide sequence homologous to the region located upstream of the target site comprises a nucleic acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the nucleic acid sequence shown in SEQ ID NO:22; and the nucleotide sequence homologous to the region located downstream comprises a nucleic acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the nucleic acid sequence shown in SEQ ID NO:

23.

62. The method of any one of claims 52-61, wherein the nucleotide sequence encoding the synthetic cytokine receptor comprises a first nucleic acid sequence encoding a γ chain and a second nucleic acid sequence encoding a β chain, wherein the first nucleic acid sequence has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the nucleic acid sequence shown in SEQ ID NO:37, and the second nucleic acid sequence has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the nucleic acid sequence shown in SEQ ID NO:

38.

63. The method of claim 62, wherein the first nucleic acid sequence and the second nucleic acid sequence are separated by a cleavable linker or an IRES.

64. The method of claim 63, wherein the cleavable linker is a protein quantitation reporter (PQR) linker, optionally as shown in SEQ ID NO:

42.

65. The method of any one of claims 52-64, wherein the nucleotide sequence encoding a synthetic cytokine receptor for a non-physiological ligand is under the operable control of a heterologous promoter.

66. The method of claim 65, wherein the heterologous promoter is the EF1α promoter or the MND promoter.

67. The method of any one of claims 52-65, wherein the nucleotide sequence encoding the synthetic cytokine receptor comprises a polyadenylation sequence.

68. The method of any one of claims 52-67, wherein the recombinant vector comprises the sequence shown in SEQ ID NO:40 or a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the nucleic acid sequence shown in SEQ ID NO:

40.

69. The method of any one of claims 52-68, comprising engineering the CIL cell population to be resistant to rapamycin-mediated inhibition of mTOR.

70. The method of claim 69, wherein engineering the CIL cell population to be resistant to rapamycin comprises knocking out the FKBP12 gene.

71. The method of claim 69, wherein the method further comprises contacting the CIL cell population with a guide RNA (gRNA) targeted to a target site in the FKBP12 gene.

72. The method of claim 69, wherein the gRNA comprises one or more gRNAs selected from the group consisting of gRNAs containing the sequence shown in SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO:

21.

73. The method of claim 72, wherein the gRNA is a gRNA library comprising 2 or 3 gRNAs.

74. The method of any one of claims 52-73, further comprising introducing a chimeric antigen receptor (CAR) into the CIL cell population.

75. The method of claim 74, wherein the CAR is an anti-FITC CAR.

76. A cell population produced by the method of any one of claims 52-75.

77. A pharmaceutical composition comprising a cell population according to any one of claims 1-37 and 76.

78. A method of expanding engineered cytotoxic innate lymphoid (CIL) cells, the method comprising contacting the CIL of any one of claims 1-50 with a non-physiological ligand for the synthetic cytokine receptor.

79. A method of treating cancer in a subject, the method comprising administering to the subject an effective amount of the engineered CIL of any one of claims 1-50, the cell population of claim 76, or the pharmaceutical composition of claim 77.

80. A method of treating cancer in a subject, the method comprising administering to the subject an effective amount of the engineered CIL of any one of claims 1-50, the cell population of claim 76, or the pharmaceutical composition of claim 77.

81. The method of claim 80, wherein the subject has not been administered lymphodepleting therapy prior to administration of the CIL, the population of CIL, or the pharmaceutical composition.

82. The method of claim 80 or claim 81, wherein the CIL expresses a CAR that targets cancer cells in the subject.

83. The method of claim 82, wherein the CAR is an anti-FITC CAR and the subject has been administered a FITC-ligand to mark cancer cells in the subject, wherein the ligand specifically binds to a molecule expressed on the tumor.

84. The method of claim 83, wherein the FITC-ligand is FITC-folate.

85. The method of claim 84, wherein the non-physiological ligand is rapamycin or a rapamycin analog, optionally wherein the rapamycin analog is a rapalog.

86. The method of any one of claims 78-85, wherein the non-physiological ligand is contacted at a concentration of between 5 nM and 200 nM, between 5 nM and 150 nM, between 5 nM and 100 nM, between 5 nM and 50 nM, between 5 nM and 20 nM, between 5 nM and 10 nM, between 10 nM and 200 nM, between 10 nM and 150 nM, between 10 nM and 100 nM, between 10 ... Between 50 nM and 100 nM, between 20 nM and 200 nM, between 20 nM and 150 nM, between 20 nM and 100 nM, between 20 nM and 50 nM, between 50 nM and 200 nM, between 50 nM and 150 nM, between 50 nM and 100 nM, between 100 nM and 200 nM, between 100 nM and 150 nM, and between 150 nM and 200 nM.

87. The method of any one of claims 78-85, wherein the non-physiological ligand is contacted at a concentration of at or about 10 nM.

88. The method of any one of claims 78-85, wherein the non-physiological ligand is contacted at a concentration of at or about 100 nM.

89. A method for killing cancer cells or inhibiting the proliferation of cancer cells, the method comprising contacting the cancer cells with the engineered CIL according to any one of claims 1-50, the cell population according to claim 76, or the pharmaceutical composition according to claim 77.

90. The method for killing cancer cells or inhibiting the proliferation of cancer cells according to claim 89, wherein the method is performed in a subject.

91. A kit comprising the engineered CIL of any one of claims 1-50, the cell population of claim 76, or the pharmaceutical composition of claim 77 and instructions for administration to a subject in need thereof.

92. The kit of claim 91, further comprising a container comprising the non-physiological ligand and instructions for administering the non-physiological ligand to the subject following administration of the cell population.

93. The kit of claim 91 or 92, wherein the subject has cancer.

94. The method of claim 89 or claim 90 or the kit of any one of claims 91-93, wherein the non-physiological ligand is administered at a dose of 1 mg to 100 mg, optionally between 10-100 mg, optionally at or about 10 mg, 20 mg, 25 mg, 30 mg, 40 mg, 50 mg, or any value between any of the foregoing values.

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