Signaling-enhanced engineered cells and methods of use thereof

Abstract

The present disclosure provides cytokine signaling receptors to provide STAT3 and STATS signaling in cells such as iPSC-derived CD8+ T cells. The cytokine signaling receptors may be under the control of endogenous or exogenous promoters to regulate their expression in a cell. The present disclosure also provides engineered cells comprising the cytokine signaling receptors, or a combination of a cytokine signaling receptor and a secreted cytokine, as well as methods of use of the engineered cells.

Description

Signaling-Enhanced Engineered Cells and Methods of Use ThereofFIELD

[0001] The present invention relates generally to methods of engineering cells to modulate cytokine signaling.BACKGROUND OF THE DISCLOSURE

[0002] Enhancement of signal transducer and activator of transcription 3 (STAT3) and / or STAT5, such as through supplementation with exogenous cytokines, has been shown to enhance T cell survival, persistence and function. Primary T cells have been engineered with a retroviral vector to constitutively express an IL2, IL7, IL15 or IL21 cytokine (Markley and Sadelain, 2010). Primary T cells have also been engineered with a constitutively active IL7 receptor variant (Shum et al., 2017). The engineering of donor T cells with an IL7 receptor, IL21 receptor or a hybrid IL7 and IL21 receptor, using non-site-specific insertion and a constitutive promoter, has also been shown to enhance function in vitro (Saxby and Jensen, WO 2023192948). In addition to primary T cells, induced pluripotent stem cell (iPSC)-derived T cells have been engineered to express STAT5-dominated signaling constructs, such as an I L15 / IL15 receptor construct at the TRAC locus (Lu et al., US 20240033355), an IL7 / IL7 receptor construct at the TRAC locus (Valamehr et al., US 20230390337) or I L15 at the CIITA locus (Naso et al., US 11661459). These approaches in primary T cells or iPSC-derived T cells provided uncontrolled STAT3 and / or STAT5 signalling, which may be unsuitable for safe and efficacious clinical cellular therapies. The need remains for an approach to provide defined, rigorous control of STAT3 and STAT5 signals in an engineered cell product.

[0003] It is desirable to obviate or mitigate one or more of the above deficiencies.SUMMARY OF THE DISCLOSURE

[0004] In a first aspect of the disclosure, provided is an engineered lymphoid cell comprising a STAT3 and STAT5 hybrid cytokine signaling receptor expressed at the cell surface that induces both STAT3 and STAT5 signaling in the engineered cell. The cell, in the absence of the hybrid cytokine signaling receptor, has attenuated STAT5 signaling in comparison to a primary CD8+ T cell.

[0005] In an embodiment of the first aspect of the disclosure, the engineered lymphoid cell is a CD8+ T cell.

[0006] In an embodiment of the first aspect of the disclosure, the hybrid cytokine signaling receptor is constitutively active.

[0007] In an embodiment of the first aspect of the disclosure, the hybrid cytokine signaling receptor is under the control of a promoter associated with T cell activation or late-stage T cell differentiation.

[0008] In an embodiment of the first aspect of the disclosure, the promoter is an endogenous promoter.

[0009] In an embodiment of the first aspect of the disclosure, the promoter is a granzyme A promoter, a granzyme B promoter, a granulocyte-macrophage colony-stimulating factor (GM-CSF) promoter, a CD25 promoter, a CD86 promoter or a CD70 promoter.

[0010] In an embodiment of the first aspect of the disclosure, the hybrid cytokine signaling receptor is under the control of an inducible promoter.

[0011] In an embodiment of the first aspect of the disclosure, the inducible promoter comprises an antigen-responsive element activated by T cell antigen recognition.

[0012] In an embodiment of the first aspect of the disclosure, the promoter is a NFKB promoter, a NFAT promoter or a granulocyte-macrophage colony-stimulating factor (GM-CSF) promoter.

[0013] In an embodiment of the first aspect of the disclosure, the STAT3 and STAT5 hybrid cytokine signaling receptor comprises an extracellular domain, a transmembrane domain coupled to the extracellular domain, a STAT5 signaling domain coupled to the transmembrane domain and a STAT3 signaling domain attached to the STAT5 signaling domain via a linker.

[0014] In an embodiment of the first aspect of the disclosure, the STAT5 signaling domain comprises an IL7 receptor alpha (IL7Ra) signaling domain.

[0015] In an embodiment of the first aspect of the disclosure, the STAT3 signaling domain comprises an IL21 receptor signaling domain.

[0016] In an embodiment of the first aspect of the disclosure, the IL21 receptor signaling domain comprises a tyrosine-containing motif.

[0017] In an embodiment of the first aspect of the disclosure, the transmembrane domain comprises an IL7 receptor alpha (IL7Ra) transmembrane domain.

[0018] In an embodiment of the first aspect of the disclosure, the IL7 receptor alpha (IL7Ra) transmembrane domain is constitutively active.

[0019] In an embodiment of the first aspect of the disclosure, the hybrid signaling receptor comprises a polypeptide encoded by SEQ ID NO: 2 or SEQ ID NO:4.

[0020] In an embodiment of the first aspect of the disclosure, the STAT3 and STAT5 hybrid cytokine signaling receptor comprises an extracellular domain, a transmembrane domain coupled to theextracellular domain, a STAT3 signaling domain coupled to the transmembrane domain and a STAT5 signaling domain attached to the STAT3 signaling domain via a linker.

[0021] In an embodiment of the first aspect of the disclosure, the engineered cell is derived from a pluripotent stem cell.

[0022] In an embodiment of the first aspect of the disclosure, the engineered lymphoid cell further comprises a nucleic acid sequence encoding a chimeric antigen receptor (CAR) and / or a nucleic acid sequence encoding an exogenous T cell receptor (TCR).

[0023] In a second aspect of the disclosure, provided is an engineered lymphoid cell comprising an exogenous STAT5 signaling receptor and an exogenous STAT3 signaling cytokine. The STAT5 signaling receptor is expressed on the cell surface and the STAT3 signaling cytokine is secreted from the cell, and the engineered cell, in the absence of the signaling receptor and the cytokine, has attenuated STAT5 signaling in comparison to a primary CD8+ T cell.

[0024] In an embodiment of the second aspect of the disclosure, the STAT5 signaling receptor and the STAT3 signaling cytokine are inserted into the same insertion site of the engineered cell.

[0025] In an embodiment of the second aspect of the disclosure, the STAT5 signaling receptor is constitutively active.

[0026] In an embodiment of the second aspect of the disclosure, the STAT5 signaling receptor and the STAT3 signaling cytokine are under the control of an endogenous or exogenous promoter associated with T cell activation or late-stage T cell differentiation.

[0027] In an embodiment of the second aspect of the disclosure, the STAT5 signaling receptor and the STAT3 signaling cytokine are under the control of an inducible promoter.

[0028] In an embodiment of the second aspect of the disclosure, the STAT5 signaling receptor and the STAT3 signaling cytokine are expressed from a single construct, and the construct comprises the nucleic acid sequence of SEQ ID NO: 5.

[0029] In an embodiment of the second aspect of the disclosure, the engineered lymphoid cell further comprises a nucleic acid sequence encoding a chimeric antigen receptor (CAR) and / or a nucleic acid sequence encoding an exogenous T cell receptor (TCR).

[0030] In a third aspect of the disclosure, provided is a STAT3 and STAT5 hybrid cytokine signaling receptor construct comprising a promoter, an extracellular domain, a transmembrane domain, a STAT3 signaling domain and a STAT5 signaling domain. The promoter is a synthetic promoter associated with T cell activation or late-stage T cell differentiation.

[0031] In an embodiment of the third aspect of the disclosure, the promoter is a NFKB promoter or a granulocyte-macrophage colony-stimulating factor (GM-CSF) promoter.

[0032] In a fourth aspect of the disclosure, provided is an engineered CD8+ T cell population comprising a STAT3 and STAT5 hybrid cytokine signaling receptor. The hybrid cytokine signaling receptor is expressed at the cell surface and induces both STAT3 and STAT5 signaling in the engineered cell, and the engineered CD8+ T cell population is derived in vitro from a pluripotent stem cell.

[0033] In an embodiment of the fourth aspect of the disclosure, the STAT3 and STAT5 cytokine signaling receptor comprises an extracellular domain, a transmembrane domain coupled to the extracellular domain, a STAT5 signaling domain coupled to the transmembrane domain and a STAT3 signaling domain attached to the STAT5 signaling domain via a linker.

[0034] In an embodiment of the fourth aspect of the disclosure, the STAT3 and STAT5 cytokine signaling receptor is constitutively active.

[0035] In an embodiment of the fourth aspect of the disclosure, the STAT3 and STAT5 hybrid cytokine signaling receptor is under the control of a promoter associated with T cell activation, or late-stage T cell differentiation, or under the control of an inducible promoter.

[0036] In an embodiment of the fourth aspect of the disclosure, the STAT3 and STAT5 hybrid cytokine signaling receptor is a polypeptide encoded by SEQ ID NO: SEQ ID NO: 2 or SEQ ID NO:4.

[0037] In an embodiment of the fourth aspect of the disclosure, the engineered CD8+ T cell population further comprises a nucleic acid sequence encoding a chimeric antigen receptor (CAR) and / or a nucleic acid encoding an exogenous T cell receptor (TCR).

[0038] In a fifth aspect of the disclosure, provided is a use of an engineered CD8+ T cell population comprising a STAT3 and STAT5 hybrid cytokine signaling receptor in the treatment of a disease or a condition in a subject. The hybrid cytokine signaling receptor is expressed at the cell surface and induces both STAT3 and STAT5 signaling in the engineered cell, and the engineered CD8+ T cell population is derived in vitro from a pluripotent stem cell.

[0039] In a sixth aspect of the disclosure, provided is an engineered CD8+ T cell population comprising an exogenous STAT5 signaling receptor and an exogenous STAT3 signaling cytokine. The STAT5 signaling receptor is expressed on the cell surface and the STAT3 signaling cytokine is secreted from the cell, and the engineered CD8+ T cell population is derived in vitro from a pluripotent stem cell.

[0040] In an embodiment of the sixth aspect of the disclosure, the STAT5 signaling receptor and the STAT3 signaling cytokine are inserted into the same insertion site of the engineered cell population.

[0041] In an embodiment of the sixth aspect of the disclosure, the STAT5 signaling receptor is constitutively active.

[0042] In an embodiment of the sixth aspect of the disclosure, the STAT5 signaling receptor and the STAT3 signaling cytokine are under the control of a promoter associated with T cell activation, or latestage T cell differentiation, or under the control of an inducible promoter.

[0043] In an embodiment of the sixth aspect of the disclosure, the STAT5 signaling receptor comprises an extracellular domain, a transmembrane domain and a STAT5 signaling domain.

[0044] In an embodiment of the sixth aspect of the disclosure, the STAT5 signaling domain comprises an IL7 receptor alpha (IL7Ra) signaling domain.

[0045] In an embodiment of the sixth aspect of the disclosure, the STAT3 signaling cytokine is IL21.

[0046] In an embodiment of the sixth aspect of the disclosure, the STAT5 signaling receptor and the STAT3 signaling cytokine are expressed from a single construct, and the construct comprises the nucleic acid sequence of SEQ ID NO: 5.

[0047] In an embodiment of the sixth aspect of the disclosure, the engineered CD8+ T cell population further comprises a nucleic acid sequence encoding a chimeric antigen receptor (CAR) and / or a nucleic acid encoding an exogenous T cell receptor (TCR).

[0048] In a seventh aspect of the disclosure, provided is a use of an engineered CD8+ T cell population comprising an exogenous STAT5 signaling receptor and an exogenous STAT3 signaling cytokine in the treatment of a disease or a condition in a subject. The STAT5 signaling receptor is expressed on the cell surface and the STAT3 signaling cytokine is secreted from the cell, and the engineered CD8+ T cell population is derived in vitro from a pluripotent stem cell.

[0049] In an eight aspect of the disclosure, provided is an induced pluripotent stem cell (iPSC) comprising a STAT3 and STAT5 hybrid cytokine signaling receptor construct, the construct comprising a promoter, an extracellular domain, a transmembrane domain, a STAT5 signaling domain and a STAT3 signaling domain coupled to the STAT5 signaling domain via a linker.

[0050] In an embodiment of the eighth aspect of the disclosure, the promoter is associated with T cell activation, or late-stage T cell differentiation, or is an inducible promoter.

[0051] In an embodiment of the eighth aspect of the disclosure, the STAT5 signaling domain is an IL7 receptor alpha (IL7Ra) signaling domain and the STAT3 signaling domain is an IL21 receptor signaling domain.

[0052] In a ninth aspect of the disclosure, provided is an induced pluripotent stem cell (iPSC) comprising a STAT3 and STAT5 hybrid cytokine signaling receptor construct, the construct comprising a promoter, a STAT5 signaling receptor and a STAT3 signaling cytokine.

[0053] In an embodiment of the ninth aspect of the disclosure, the promoter is associated with T cell activation, or late-stage T cell differentiation, or is an inducible promoter.

[0054] In an embodiment of the ninth aspect of the disclosure, the STAT5 signaling receptor comprises an IL7 receptor alpha (IL7Ra) signaling domain, and the STAT3 signaling cytokine is IL21.BRIEF DESCRIPTION OF THE DRAWINGS

[0055] In order that the subject matter may be readily understood, embodiments are illustrated by way of non-limiting examples in the accompanying drawings.

[0056] FIG. 1A is a schematic of STAT3 and STAT5 engineered cells according to an embodiment of the invention, depicting a cell containing a STAT3 / STAT5 hybrid cytokine signaling receptor.

[0057] FIG. IB is a schematic of STAT3 and STAT5 engineered cells according to an alternate embodiment of the invention, depicting a cell containing a combination of a STAT3 or STAT5 cytokine signaling receptor and engineered cytokine support.

[0058] FIG. 2A is a schematic of regulation strategies for the cytokine signaling receptor constructs: capture of an endogenous promoter at a late-stage site (top) or insertion of a synthetic inducible promoter (bottom).

[0059] FIG. 2B is a schematic of the desired expression profiles associated with each of the regulation strategies of Fig. 27A over the course of T cell differentiation, CD3 stimulation ("CD3 stim") and target antigen exposure ("+ / - Ag exposure").

[0060] FIG. 3A is a graph of phosphorylated signal transducer and activator of transcription 3 (pSTAT3) and phosphorylated STAT5 (pSTAT5) expression in primary and iPSC-derived T cells cultured with and without IL2, as detected by flow cytometry. gMFI, geometric median fluorescence intensity.

[0061] FIG. 3B is a graph of pSTAT3 and pSTAT5 expression in primary and iPSC-derived T cells cultured with and without IL7, as detected by flow cytometry.

[0062] FIG. 3C is a graph of pSTAT3 and pSTAT5 expression in primary and iPSC-derived T cells cultured with and without IL15, as detected by flow cytometry.

[0063] FIG. 3D is a graph of pSTAT3 and pSTAT5 expression in primary and iPSC-derived T cells cultured with and without IL21, as detected by flow cytometry.

[0064] FIG. 4A is a graph of pSTAT3 and pSTAT5 expression in iPSC-derived T cells cultured with and without IL2 at baseline and following four rounds of antigen stimulation ("post-stim 4"), as detected by flow cytometry.

[0065] FIG. 4B is a graph of pSTAT3 and pSTAT5 expression in iPSC-derived T cells cultured with and without IL15 at baseline and following four rounds of antigen stimulation ("post-stim 4"), as detected by flow cytometry.

[0066] FIG. 4C is a graph of pSTAT3 and pSTAT5 expression in iPSC-derived T cells cultured with and without IL7 at baseline and following four rounds of antigen stimulation ("post-stim 4"), as detected by flow cytometry.

[0067] FIG. 4D is a graph of pSTAT3 and pSTAT5 expression in iPSC-derived T cells cultured with and without IL21 at baseline and following four rounds of antigen stimulation ("post-stim 4"), as detected by flow cytometry.

[0068] FIG. 5A is a graph of iPSC-derived T cell proliferation following a serial antigen restimulation assay supplemented with individual or combinations of cytokines providing STAT3 and / or STAT5 signals.

[0069] FIG. 5B is a graph of iPSC-derived T cell proliferation during a serial antigen restimulation assay supplemented with combinations of interleukins providing STAT3 and / or STAT5 signals.

[0070] FIG. 6A is a graph of primary T cell or iPSC-derived T cell proliferation during a serial antigen restimulation assay with and without cytokine supplementation.

[0071] FIG. 6B is a graph of iPSC-derived T cell proliferation following a serial antigen restimulation assay with and without cytokine supplementation.

[0072] FIG. 7A is a schematic of cytokine signaling receptor constructs providing STAT3 and / or STAT5 signals; all constructs included the constitutive EFla promoter.

[0073] FIG. 7B is a schematic of four of the cytokine signaling receptor constructs in Fig. 7A providing STAT3 and / or STAT5 signals (pNT466, pNT549, pNT581 and pNT729). ECD, extracellular domain; TMD, transmembrane domain; ICD, intracellular domain; aa, amino acid.

[0074] FIG. 8A is a graph of fold change in cell numbers over time in culture for one primary T cell line ("Donor 31") untransduced ("UTD") and transduced with various cytokine signaling receptor constructs as shown in FIG. 7.

[0075] FIG. 8B is a graph of viability over time in culture for one primary T cell line ("Donor 31") untransduced and transduced with various cytokine signaling receptor constructs as shown in FIG. 7.

[0076] FIG. 8C is a graph of fold change in cell numbers over time in culture for a second primary T cell line ("Donor 33") untransduced and transduced with various cytokine signaling receptor constructs as shown in FIG. 67.

[0077] FIG. 8D is a graph of viability over time in culture for one T cell line ("Donor 33") untransduced and transduced with various cytokine signaling receptor constructs as shown in FIG. 7.

[0078] FIG. 9A is a graph of expression of cytokine signaling receptor constructs, as determined by flow cytometry analysis of CD34 extracellular domain expression, in two independent transduced primary T cell lines ("Donor 33" and "Donor 35") in comparison with untransduced control ("UTD").

[0079] FIG. 9B is a graph of vector copy number (VCN) of cytokine signaling receptor constructs in two independent transduced primary T cell lines ("Donor 33" and "Donor 35") in comparison with untransduced control ("UTD").

[0080] FIG. 9C is a graph of pSTAT3 expression, as determined by flow cytometry, for two independent primary T cell lines ("Donor 33" and "Donor 35") transduced with cytokine signaling receptor constructs in comparison with untransduced control ("UTD").

[0081] FIG. 10A is a graph of cytokine signaling receptor construct expression, as determined by flow cytometry analysis of CD34 ECD expression, for iPSC-derived CD8+ CAR-TCR T cells transduced with the pNT466 construct in comparison to untransduced control ("UTD").

[0082] FIG. 10B is a graph of pSTAT5 expression, as determined by flow cytometry, for iPSC-derived CD8+ CAR-TCR T cells transduced with the pNT466 construct in comparison to untransduced control ("UTD").

[0083] FIG. 10C is a graph of proliferation during a serial antigen restimulation assay for iPSC-derived CD8+ CAR-TCR T cells transduced with the pNT466 construct and supplemented with exogenous IL21 in comparison to untransduced control ("UTD") supplemented with exogenous IL21 or IL2, IL7 and IL21.

[0084] FIG. 11A is a graph of fold change in proliferation of iPSC-derived CD8+ CAR-TCR T cells transduced with pNT466 cytokine signaling receptor construct, with and without exogenous IL21, or untransduced iPSC-derived CD8+ CAR-TCR T cells ("UTD") with exogenous IL21, in a serial antigen restimulation assay.

[0085] FIG. 11B is a graph of cytotoxicity of iPSC-derived CD8+ CAR-TCR T cells transduced with pNT466 cytokine signaling receptor construct, with and without exogenous IL21, or untransduced iPSC-derived CD8+ CAR-TCR T cells ("UTD") with exogenous IL21, co-cultured with target cells.

[0086] FIG. 12A is a graph of fold change in proliferation of iPSC-derived CD8+ CAR-TCR T cells transduced with various cytokine signaling receptor constructs in a serial antigen restimulation assay.

[0087] FIG. 12B is a graph of fold change in proliferation of iPSC-derived CD8+ CAR-TCR T cells transduced with various cytokine signaling receptor constructs after one round of antigen stimulation ("Stim 1").

[0088] FIG. 12C is a graph of fold change in proliferation of iPSC-derived CD8+ CAR-TCR T cells transduced with various cytokine signaling receptor constructs after three rounds of antigen stimulation ("Stim 3").

[0089] FIG. 12D is a graph of cytotoxicity of iPSC-derived CD8+ CAR-TCR T cells transduced with various cytokine signaling receptor constructs in a serial antigen restimulation assay.

[0090] FIG. 13A is a graph of fold change in proliferation of untransduced or cytokine signaling receptor construct-transduced iPSC-derived CD8+ CAR-TCR T cells in a serial antigen restimulation assay, without cytokine support or supplemented with exogenous I L2, I L7, and IL21.

[0091] FIG. 13B is a graph of fold change in proliferation of untransduced or cytokine signaling receptor construct-transduced iPSC-derived CD8+ CAR-TCR T cells, without cytokine support or supplemented with exogenous IL2, IL7 and IL21, after one round of antigen stimulation ("Stim 1").

[0092] FIG. 13C is a graph of fold change in proliferation of untransduced or cytokine signaling receptor construct-transduced iPSC-derived CD8+ CAR-TCR T cells, without cytokine support or supplemented with exogenous IL2, IL7, and IL21, after three rounds of antigen stimulation ("Stim 3").

[0093] FIG. 14A is a graph of cytotoxicity, as assessed by tumor cell growth inhibition (%TGI) of untransduced or cytokine signaling receptor construct-transduced iPSC-derived CD8+ CAR-TCR T cells in comparison with primary CD3+ T cells, supplemented with exogenous IL2, IL7 and IL21. E:T, effector to target cell ratio.

[0094] FIG. 14B is a graph of cytotoxicity, as assessed by tumor cell growth inhibition (%TGI) of untransduced or cytokine signaling receptor construct-transduced iPSC-derived CD8+ CAR-TCR T cells in comparison with primary CD3+ T cells, without exogenous cytokine supplementation. E:T, effector to target cell ratio.

[0095] FIG. 15A is a graph of granzyme A secretion in iPSC-derived CD8+ TCR-T cells transduced with pNT549 cytokine signaling receptor construct or pNT581 cytokine signaling receptor construct and cocultured with target cells with and without exogenous cytokine supplementation, in comparison with untransduced iPSC-derived CD8+ TCR-T cells, primary CD3+ T cells or target cell controls.

[0096] FIG. 15B is a graph of perforin secretion in iPSC-derived CD8+ TCR-T cells transduced with pNT549 cytokine signaling receptor construct or pNT581 cytokine signaling receptor construct and co-cultured with target cells with and without exogenous cytokine supplementation, in comparison with untransduced iPSC-derived CD8+ TCR-T cells, primary CD3+ T cells or target cell controls.

[0097] FIG. 15C is a graph of granzyme B secretion in iPSC-derived CD8+ TCR-T cells transduced with pNT549 cytokine signaling receptor construct or pNT581 cytokine signaling receptor construct and cocultured with target cells with and without exogenous cytokine supplementation, in comparison with untransduced iPSC-derived CD8+ TCR-T cells, primary CD3+ T cells or target cell controls.

[0098] FIG. 15D is a graph of GM-CSF secretion in iPSC-derived CD8+ TCR-T cells transduced with pNT549 cytokine signaling receptor construct or pNT581 cytokine signaling receptor construct and cocultured with target cells with and without exogenous cytokine supplementation, in comparison with untransduced iPSC-derived CD8+ TCR-T cells, primary CD3+ T cells or target cell controls.

[0099] FIG. 15E is a graph of interferon-y (IFNy) secretion in iPSC-derived CD8+ TCR-T cells transduced with pNT549 cytokine signaling receptor construct or pNT581 cytokine signaling receptor construct and co-cultured with target cells with and without exogenous cytokine supplementation, in comparison with untransduced iPSC-derived CD8+ TCR-T cells, primary CD3+ T cells or target cell controls.

[0100] FIG. 15F is a graph of tumor necrosis factor a (TNFa) secretion in iPSC-derived CD8+ TCR-T cells transduced with pNT549 cytokine signaling receptor construct or pNT581 cytokine signaling receptor construct and co-cultured with target cells with and without exogenous cytokine supplementation, in comparison with untransduced iPSC-derived CD8+ TCR-T cells, primary CD3+ T cells or target cell controls.

[0101] FIG. 16A is a graph of fold change proliferation for iPSC-derived CD8+ T cells transduced with cytokine signaling receptor constructs in a serial antigen restimulation assay with and without exogenous cytokines, in comparison to untransduced iPSC-derived CD8+ T cells and primary CD3+ T cells.

[0102] FIG. 16B is a graph of cell viability for iPSC-derived CD8+ T cells transduced with cytokine signaling receptor constructs following a serial antigen restimulation assay with and without exogenous cytokines, in comparison to untransduced iPSC-derived CD8+ T cells and primary CD3+ T cells.

[0103] FIG. 17A is a graph of tumor growth inhibition of iPSC-derived CD8+ T cells transduced with cytokine signaling receptor constructs following a serial antigen restimulation assay with and without exogenous cytokines, in comparison to untransduced iPSC-derived CD8+ T cells and primary CD3+ T cells.

[0104] FIG. 17B is a graph of cytokine signaling receptor construct expression for iPSC-derived CD8+ T cells transduced with cytokine signaling receptor constructs before ("baseline"), during (post-stimulation1, "PSI") or following (post-stimulation 3, "PS3") a serial antigen restimulation assay. Expression was determined by % of CD34 extracellular domain-positive cells by flow cytometry, gated on live cells.

[0105] FIG. 17C is a graph of cytokine signaling receptor construct expression for iPSC-derived CD8+ CAR-T cells transduced with cytokine signaling receptor constructs before ("baseline"), during (poststimulation 1, "PSI") or following (post-stimulation 3, "PS3") a serial antigen restimulation assay. Construct expression was determined by the geometric mean fluorescence intensity (gMFI) of the CD34 extracellular domain, as analyzed by flow cytometry and gated on CAR+ cells.

[0106] FIG. 18A is a graph of proliferation of iPSC-derived CD8+ TCR-T cells transduced with cytokine signaling receptor constructs in a serial antigen restimulation assay, with and without exogenous cytokine supplementation, in comparison to untransduced control.

[0107] FIG. 18B is a graph of proliferation of iPSC-derived CD8+ TCR-T cells transduced with cytokine signaling receptor constructs in a separate serial antigen restimulation assay, with and without exogenous cytokine supplementation, in comparison to untransduced control.

[0108] FIG. 18C is a graph of proliferation of iPSC-derived CD8+ CAR-T cells transduced with cytokine signaling receptor constructs in a separate serial antigen restimulation assay, with and without exogenous cytokine supplementation, in comparison to untransduced control.

[0109] FIG. 19A is a graph of granzyme A secretion in iPSC-derived CD8+ CAR-T cells transduced with pNT549 cytokine signaling receptor construct or pNT581 cytokine signaling receptor construct and cocultured with target cells with and without exogenous cytokine supplementation, in comparison with untransduced iPSC-derived CD8+ CAR-T cells, primary CD3+ T cells or target cell controls.

[0110] FIG. 19B is a graph of granzyme B secretion in iPSC-derived CD8+ CAR-T cells transduced with pNT549 cytokine signaling receptor construct or pNT581 cytokine signaling receptor construct and cocultured with target cells with and without exogenous cytokine supplementation, in comparison with untransduced iPSC-derived CD8+ CAR-T cells, primary CD3+ T cells or target cell controls.

[0111] FIG. 19C is a graph of perforin secretion in iPSC-derived CD8+ CAR-T cells transduced with pNT549 cytokine signaling receptor construct or pNT581 cytokine signaling receptor construct and cocultured with target cells with and without exogenous cytokine supplementation, in comparison with untransduced iPSC-derived CD8+ CAR-T cells, primary CD3+ T cells or target cell controls.

[0112] FIG. 19D is a graph of GM-CSF secretion in iPSC-derived CD8+ CAR-T cells transduced with pNT549 cytokine signaling receptor construct or pNT581 cytokine signaling receptor construct and cocultured with target cells with and without exogenous cytokine supplementation, in comparison with untransduced iPSC-derived CD8+ CAR-T cells, primary CD3+ T cells or target cell controls.

[0113] FIG. 19E is a graph of I FNy secretion in iPSC-derived CD8+ CAR-T cells transduced with pNT549 cytokine signaling receptor construct or pNT581 cytokine signaling receptor construct and co-cultured with target cells with and without exogenous cytokine supplementation, in comparison with untransduced iPSC-derived CD8+ CAR-T cells, primary CD3+ T cells or target cell controls.

[0114] FIG. 19F is a graph of TNFa secretion in iPSC-derived CD8+ CAR-T cells transduced with pNT549 cytokine signaling receptor construct or pNT581 cytokine signaling receptor construct and co-cultured with target cells with and without exogenous cytokine supplementation, in comparison with untransduced iPSC-derived CD8+ CAR-T cells, primary CD3+ T cells or target cell controls.

[0115] FIG. 20A is a graph of proliferation of iPSC-derived CD8+ CAR-T cells transduced with pNT549 cytokine signaling receptor construct or pNT581 cytokine signaling receptor construct in comparison with CAR-only cells ("CAR").

[0116] FIG. 20B is a graph of viability of iPSC-derived CD8+ CAR-T cells transduced with pNT549 cytokine signaling receptor construct or pNT581 cytokine signaling receptor construct in comparison with CAR-only cells ("CAR").

[0117] FIG. 20C is a graph of pre-enrichment percent transduction of iPSC-derived CD8+ CAR-T cells transduced with pNT549 cytokine signaling receptor construct or pNT581 cytokine signaling receptor construct in comparison with CAR-only cells ("CAR").

[0118] FIG. 20D a graph of post -enrichment percent transduction of iPSC-derived CD8+ CAR-T cells transduced with pNT549 cytokine signaling receptor construct or pNT581 cytokine signaling receptor construct in comparison with CAR-only cells ("CAR").

[0119] FIG. 21A is a graph of cumulative proliferation during a serial antigen restimulation assay of iPSC-derived CD8+ CAR-T cells transduced with pNT549 cytokine signaling receptor construct or pNT581 cytokine signaling receptor construct in comparison with CAR-only cells, with and without exogenous cytokine supplementation.

[0120] FIG. 21B is a graph of fold change in proliferation at the end of a serial antigen restimulation assay of iPSC-derived CD8+ CAR-T cells transduced with pNT549 cytokine signaling receptor construct or pNT581 cytokine signaling receptor construct in comparison with CAR-only cells, with and without exogenous cytokine supplementation.

[0121] FIG. 21C is a graph of IFN-y secretion following a serial antigen restimulation assay in iPSC- derived CD8+ CAR-T cells transduced with pNT549 cytokine signaling receptor construct or pNT581 cytokine signaling receptor construct in comparison with CAR-only cells or a target cell control, with and without exogenous cytokine supplementation.

[0122] FIG. 21D is a graph of TNFa secretion following a serial antigen restimulation assay in iPSC- derived CD8+ CAR-T cells transduced with pNT549 cytokine signaling receptor construct or pNT581 cytokine signaling receptor construct in comparison with CAR-only cells or a target cell control, with and without exogenous cytokine supplementation.

[0123] FIG. 22A is a graph of proliferation during a serial antigen restimulation assay for iPSC-derived CD8+ CAR-T cells transduced with pNT549 cytokine signaling receptor construct or pNT581 cytokine signaling receptor construct in comparison with CAR-only cells with cytokine supplementation or primary T cells without cytokine supplementation.

[0124] FIG. 22B is a graph of tumor cell growth inhibition during a serial antigen restimulation assay for of iPSC-derived CD8+ CAR-T cells transduced with pNT549 cytokine signaling receptor construct or pNT581 cytokine signaling receptor construct in comparison with CAR-only cells with cytokine supplementation or primary T cells without cytokine supplementation.

[0125] FIG. 23A is a graph of tumor growth inhibition (% TGI) by iPSC-derived CD8+ CAR-T cells transduced with pNT581 cytokine signaling receptor construct in comparison with CAR-only cells ("UTD"), with and without cytokine supplementation.

[0126] FIG. 23B is a graph of tumor growth inhibition (% TGI) by cryopreserved iPSC-derived CD8+ CAR- T cells transduced with pNT581 cytokine signaling receptor construct in comparison with CAR-only cells ("UTD"), with and without cytokine supplementation.

[0127] FIG. 23C is a graph of viability post -thaw for cryopreserved iPSC-derived CD8+ CAR-T cells transduced with pNT581 cytokine signaling receptor construct in comparison with CAR-only cells ("UTD").

[0128] FIG. 23D is a graph of IFN-y secretion by cryopreserved or fresh iPSC-derived CD8+ CAR-T cells transduced with pNT581 cytokine signaling receptor construct in comparison with CAR-only cells ("UTD"), cultured with and without cytokine supplementation.

[0129] FIG. 24 is a graph of proliferation during a serial antigen restimulation assay for iPSC-derived cells transduced with cytokine signaling receptor constructs in comparison with untransduced ("UTD") cells, with and without exogenous cytokine supplementation.

[0130] FIG. 25A is a graph of expression of cytokine signaling receptor constructs in primary T cells, as determined by CD34 extracellular domain expression.

[0131] FIG. 25B is a graph of vector copy number in primary T cells transduced with cytokine signaling receptor constructs in comparison with untransduced ("UTD") cells.

[0132] FIG. 25C is a graph of pSTAT3 expression in primary T cells transduced with cytokine signaling receptor constructs in comparison with untransduced ("UTD") cells.

[0133] FIG. 25D is a graph of pSTAT5 expression in primary T cells transduced with cytokine signaling receptor constructs in comparison with untransduced ("UTD") cells.

[0134] FIG. 26A is a graph of proliferation of iPSC-derived CD8+ T cells cultured with exogenous IL21 at indicated concentrations.

[0135] FIG. 26B is a graph of pSTAT3 expression in iPSC-derived CD8+ T cells cultured with exogenous IL21 at indicated concentrations.

[0136] FIG. 26C is a graph of pSTAT5 expression in iPSC-derived CD8+ T cells cultured with exogenous IL21 at indicated concentrations.

[0137] FIG. 26D is a graph of IL21 secretion in iPSC-derived CD8+ T cells cultured with exogenous IL21 at indicated concentrations, in comparison to primary T cells, untransduced or transduced with cytokine signaling receptor constructs.

[0138] FIG. 27A is a graph of proliferation following the first round of antigen stimulation for iPSC- derived CD8+ T cells transduced with a cytokine signaling receptor construct, as indicated. Untransduced control cells were supplemented with exogenous IL2, IL7 and IL21 ("UTD + IL2 / 7 / 21") and cells transduced with the pNT466 STAT5 construct were supplemented with exogenous IL21 ("pNT466 + IL21").

[0139] FIG. 27B is a graph of pSTAT3 MFI following the first round of antigen stimulation in a persistence assay for iPSC-derived CD8+ cells transduced with a cytokine signaling receptor construct, as indicated. Untransduced control cells were supplemented with exogenous IL2, IL7 and IL21 ("UTD + IL2 / 7 / 21") and cells transduced with the pNT466 STAT5 construct were supplemented with exogenous IL21 ("pNT466 + IL21").

[0140] FIG. 27C is a graph of pSTAT5 MFI following the first round of antigen stimulation in a persistence assay for iPSC-derived CD8+ T cells transduced with a cytokine signaling receptor construct, as indicated. Untransduced control cells were supplemented with exogenous IL2, IL7 and IL21 ("UTD + IL2 / 7 / 21") and cells transduced with the pNT466 STAT5 construct were supplemented with exogenous IL21 ("pNT466 + IL21").

[0141] FIG. 27D is a graph of proliferation over the course of a persistence assay for iPSC-derived CD8+ T cells transduced with a cytokine signaling receptor construct, as indicated. Cells were co-cultured with A549-CD19+ target cells at a 2:1 E:T ratio. Untransduced control cells were supplemented with exogenous IL2, IL7 and IL21 ("UTD + IL2 / 7 / 21") and cells transduced with the pNT466 STAT5 constructwere supplemented with exogenous IL21 ("pNT466 + IL21"). The cumulative fold proliferation for each group at the end of the assay is noted at the right.

[0142] FIG. 27E is a graph of proliferation over the course of a persistence assay for iPSC-derived CD8+ T cells transduced with a cytokine signaling receptor construct, as indicated. Cells were co-cultured with A549-CD19+ target cells at a 1:1 E:T ratio. Untransduced control cells were supplemented with exogenous I L2, IL7 and IL21 ("UTD + IL2 / 7 / 21") and cells transduced with the pNT466 STAT5 construct were supplemented with exogenous IL21 ("pNT466 + IL21"). The cumulative fold proliferation for each group at the end of the assay is noted at the right.

[0143] FIG. 27F is a graph of construct expression, as assessed by the percentage of CD34 ECD-positive cells, over the course of a persistence assay for iPSC-derived CD8+ T cells transduced with the cytokine signaling receptor construct pNT466 or pNT729. Cells were co-cultured with A549-CD19+ target cells at a 2:1 E:T ratio. Cells transduced with the pNT466 STAT5 construct were supplemented with exogenous IL21 ("pNT466 + IL21").

[0144] FIG. 28 is a schematic of cytokine signaling receptor constructs and target insertion sites using endogenous promoters (FIG. 28A) or exogenous synthetic promoters (FIG. 28B).

[0145] FIG. 29A is a graph of granzyme A secretion by iPSC-derived cell lines during CD8+ T cell differentiation.

[0146] FIG. 29B is a graph of granzyme B secretion by iPSC-derived cell lines during CD8+ T cell differentiation.

[0147] FIG. 29C is a graph of GM-CSF secretion by iPSC-derived cell lines during CD8+ T cell differentiation.

[0148] FIG. 30A is a graph of CD5 and CD7 expression in iPSC-derived cells transduced with cytokine signaling receptor construct in comparison with untransduced ("UTD") or TCR-transduced cells ("pNT150 TD"), and cells cultured with exogenous I L15 during differentiation.

[0149] FIG. 30B is a graph of CD16 and CD56 expression in iPSC-derived cells transduced with cytokine signaling receptor construct in comparison with untransduced ("UTD") or TCR-transduced cells ("pNT150 TD"), and cells cultured with exogenous I L15 during differentiation.

[0150] FIG. 30C is a graph of CD5 and CD7 expression in iPSC-derived cells cultured with exogenous IL21 during differentiation.

[0151] FIG. 30D is a graph of CD16 and CD56 expression in iPSC-derived cells cultured with exogenous IL21 during differentiation.

[0152] FIG. 31 is a gene expression plot of iPSC-derived cells during differentiation or post-antigen stimulation in comparison to primary CAR-T cells.

[0153] FIG. 32 is a schematic of insertion site and templates for cytokine signaling receptor constructs at an endogenous promoter site.

[0154] FIG. 33A is a graph of expression of endogenous promoter genes in iPSC-derived cells during differentiation. ITGB7 was included as a positive control.

[0155] FIG. 33B is a graph of mRNA expression of endogenous promoter genes in iPSC-derived cells during differentiation. ITGB7 was included as a positive control.

[0156] FIG. 33C is a graph of expression of endogenous promoter genes in iPSC-derived cells during differentiation.

[0157] FIG. 33D is a graph of mRNA expression of endogenous promoter genes in iPSC-derived cells during differentiation. ITGB7 was included as a positive control.

[0158] FIG. 34A is a graph of expression of endogenous promoter genes in iPSC-derived CD8+ T cells before expansion ("Unexp."), post-expansion ("Exp.") and after one round of antigen stimulation ("Post- Stiml").

[0159] FIG. 34B is a graph of expression of endogenous promoter genes in iPSC-derived CD8+ T cells before expansion ("Unexp."), post-expansion and cryopreservation ("D7 exp. cryo"), post-expansion ("D7 exp. fresh"), after one round of antigen stimulation ("Post-Stiml") and after three rounds of antigen stimulation ("Post-Stim3").

[0160] FIG. 35A is a graph of granzyme A, granzyme B and GM-CSF secretion in iPSC-derived CD8+ cells during differentiation. WP, well-plate differentiation; STR, bioreactor differentiation.

[0161] FIG. 35B is a graph of granzyme A, granzyme B and GM-CSF secretion in iPSC-derived CD8+ cells during co-culture with A549-CD19+ target cells or wild-type (WT) control cells.

[0162] FIG. 36A is a graph of EBV TCR expression, quantified by percent positive cells or gMFI as assessed by flow cytometry, for iPSC-derived cells modified with EBV TCR at the CIITA or P2M loci under a CAG promoter. Cells were assessed during differentiation or post-activation following differentiation.

[0163] FIG. 36B is a graph of mRNA expression of the indicated promoter site genes during iPSC-derived cell differentiation.

[0164] FIG. 36C is a graph of gene expression for iPSC-derived CD8+ T cells before expansion ("Unexp."), after expansion ("Exp."), after one round of antigen stimulation ("Post-Stiml") and after three rounds of antigen stimulation ("Post-Stim3"). N.D., not detected.

[0165] FIG. 37 is a schematic of inducible synthetic promoters evaluated with the pNT581 cytokine signaling receptor construct, as well as the EFla constitutive promoter used as a control.

[0166] FIG. 38A is a graph of cytokine signaling receptor construct expression, as determined by CD34 extracellular domain expression, for constructs with varying synthetic promoters as labelled, in iPSC- derived CD8+ T cells before ("Unstim") and after antigen stimulation ("Stim").

[0167] FIG. 38B is a graph of cytokine signaling receptor construct expression, as determined by gMFI of CD34 extracellular domain expression, for constructs with varying synthetic promoters as labelled, in iPSC-derived CD8+ T cells before ("Unstim") and after antigen stimulation ("Stim").

[0168] FIG. 39 is a graph of cytokine signaling receptor construct expression under the EFla promoter, NFKB promoter, the synthetic antigen-responsive promoter ("ARP") or the GM-CSF promoter as determined by CD34 extracellular domain expression. Cells were cultured without target cells ("Effector Only") or with A549-CD19+ target cells, A549 WT cells or Dynabead™ stimulation, in the presence (left, "+ Cytokine") or absence of exogenous cytokines (right, "- Cytokine").

[0169] FIG. 40A is a graph of cytokine signaling receptor construct expression under the EFla promoter, as determined by CD34 extracellular domain expression following culture with target cells ("On Target") or 48h after co-culture ("Off Target (48h)").

[0170] FIG. 40B is a graph of cytokine signaling receptor construct expression under the NFKB promoter, as determined by CD34 extracellular domain expression following culture with target cells ("On Target") or 48h after co-culture ("Off Target (48h)").

[0171] FIG. 40C is a graph of cytokine signaling receptor construct expression under the synthetic antigen-responsive promoter ("ARP"), as determined by CD34 extracellular domain expression following culture with target cells ("On Target") or 48h after co-culture ("Off Target (48h)").

[0172] FIG. 40D is a graph of cytokine signaling receptor construct expression under the GM-CSF promoter, as determined by CD34 extracellular domain expression following culture with target cells ("On Target") or 48h after co-culture ("Off Target (48h)").

[0173] FIG. 40E is a graph of cytokine signaling receptor construct expression under the EFla promoter, as determined by CD34 extracellular domain expression after 36 or 48 hours of stimulation with Dynabeads™ and anti-41BB antibody.

[0174] FIG. 40F is a graph of cytokine signaling receptor construct expression under the NFKB promoter, as determined by CD34 extracellular domain expression after 36 or 48 hours of stimulation with Dynabeads™ and anti-41BB antibody.

[0175] FIG. 40G is a graph of cytokine signaling receptor construct expression under the synthetic antigen-responsive promoter ("ARP"), as determined by CD34 extracellular domain expression after 36 or 48 hours of stimulation with Dynabeads™ and anti-41BB antibody.

[0176] FIG. 40H is a graph of cytokine signaling receptor construct expression under the GM-CSF promoter, as determined by CD34 extracellular domain expression after 36 or 48 hours of stimulation with Dynabeads™ and anti-41BB antibody.

[0177] FIG. 41A is a graph of in vitro cytotoxicity of iPSC-derived CD8+ cells transduced with a STAT3 / STAT5 cytokine signaling receptor construct (pNT581) under the control of a constitutive EFla promoter, or the inducible promoters NFKB, synthetic antigen-responsive promoter ("ARP") or GM-CSF. Cells were co-cultured with A549-CD19+ target cells in a persistence assay for a total of 18 days.

[0178] FIG. 41B is a graph of cell number at the end of the persistence assay depicted in FIG. 41A.

[0179] FIG. 41C is a graph of the percentage of transduced cells, as determined by CD34 extracellular domain expression, after cell expansion and before the persistence assay ("Post-Expansion").

[0180] FIG. 41D is a graph of the percentage of transduced cells, as determined by CD34 extracellular domain expression, following 5 rounds of target cell stimulation ("5x Post -Stimulation") with antigenpositive or antigen-negative cells.

[0181] FIG. 42A is a graph of in vitro cytotoxicity of iPSC-derived CD8+ T cells transduced with a STAT3 / STAT5 cytokine signaling receptor construct (pNT581) under the control of a constitutive EFla promoter, or the inducible promoters NFKB, synthetic antigen-responsive promoter ("ARP") or GM-CSF, in comparison to untransduced cells cultured with exogenous cytokines ("UTD + IL2 / 7 / 21"). Cells were co-cultured with A549-CD19+ target cells in a persistence assay for a total of 14 days.

[0182] FIG. 42B is a graph of proliferation of iPSC-derived CD8+ T cells transduced with a STAT3 / STAT5 cytokine signaling receptor construct (pNT581) under the control of a constitutive EFla promoter, or the inducible promoters NFKB, the synthetic antigen-responsive promoter ("ARP") or GM-CSF, in comparison to untransduced cells cultured with exogenous cytokines ("UTD + IL2,7,21").

[0183] FIG. 43A is a graph of fold expansion of untransduced or cytokine receptor-transduced iPSC- derived CD8+ T cells in a serial restimulation assay with A549-CD19+ target cells. Cells were transduced with a STAT3 / STAT5 cytokine signaling receptor construct (pNT581) under the control of a constitutive EFla promoter, or the inducible promoters NFKB, synthetic antigen-responsive promoter ("ARP") or GM-CSF. Total fold expansion at the end of the assay is noted on the right.

[0184] FIG. 43B is a graph of the percentage of transduced cells, as determined by CD34 extracellular domain expression, over the course of cell expansion ("Exp.") and the serial restimulation assay.

[0185] FIG. 43C is a graph of the specificity of tumor growth inhibition for untransduced and transduced cells. The growth inhibition of antigen-positive target cells (solid lines, "+Ag") and antigennegative target cells (dotted lines, "-Ag") is shown.

[0186] FIG. 44A is a graph of vector copy number (VCN) of STAT3 / STAT5 cytokine signaling receptor construct (pNT581) inserted at the indicated loci of iPS cells using MAD7 or Cas9 nuclease.

[0187] FIG. 44B is a graph of vector copy number (VCN) of STAT3 / STAT5 cytokine signaling receptor construct (pNT581) including indicated exogenous promoters inserted at CIITA or AAVS1 loci of iPS cells using MAD7 or Cas9 nuclease.

[0188] FIG. 44C is a graph of vector copy number (VCN) of STAT3 / STAT5 cytokine signaling receptor construct (pNT581) including indicated exogenous promoters integrated into iPS cells.

[0189] FIG. 45A is a graph of the percentage of clones identified without extra copies of STAT3 / STAT5 cytokine signaling receptor construct (pNT581) inserted at the indicated loci of iPS cells using MAD7 or Cas9 nuclease.

[0190] FIG. 45B is a graph of the number of clones generated for insertion of a STAT3 / STAT5 cytokine signaling receptor construct (pNT581) at the indicated loci of iPS cells using MAD7 or Cas9 nuclease.

[0191] FIG. 45C is a graph of the percentage of clones identified without extra copies of STAT3 / STAT5 cytokine signaling receptor construct (pNT581) including indicated exogenous promoters integrated into iPS cells.

[0192] FIG. 45D is a graph of the number of clones generated for insertion of a STAT3 / STAT5 cytokine signaling receptor construct (pNT581) including indicated exogenous promoters into iPS cells.

[0193] FIG. 45E is a graph of vector copy number (VCN) of cytokine signaling receptor construct pNT729 inserted at the indicated loci of iPS cells.

[0194] FIG. 45F is a graph of vector copy number (VCN) of cytokine signaling receptor construct pNT729 including indicated exogenous promoters integrated into iPS cells.

[0195] FIG. 46A is a graph of CD5 and / or CD7 expression in iPSC-derived cells during differentiation, either without a cytokine signaling receptor ("No CyR") or with a cytokine signaling receptor inserted at the GZMA locus ("GZMA CyR").

[0196] FIG. 46B is a graph of CD56 and / or CD7 expression in iPSC-derived cells during differentiation, either without a cytokine signaling receptor ("No CyR") or with a cytokine signaling receptor inserted at the GZMA locus ("GZMA CyR").

[0197] FIG. 46C is a graph of CD4 and / or CD8a expression in iPSC-derived cells during differentiation, either without a cytokine signaling receptor ("No CyR") or with a cytokine signaling receptor inserted at the GZMA locus ("GZMA CyR").

[0198] FIG. 46D is a graph of CD8a and / or CD8P expression in iPSC-derived cells during differentiation, either without a cytokine signaling receptor ("No CyR") or with a cytokine signaling receptor inserted at the GZMA locus ("GZMA CyR").

[0199] FIG. 46E is a graph of CD56, CD16, NKp46, or CD94 expression in iPSC-derived cells during differentiation, either without a cytokine signaling receptor ("No CyR") or with a cytokine signaling receptor inserted at the GZMA locus ("GZMA CyR").

[0200] FIG. 46F is a graph of cumulative fold expansion and viability of iPSC-derived cells during differentiation, either without a cytokine signaling receptor ("No CyR") or with a cytokine signaling receptor inserted at the GZMA locus ("GZMA CyR").

[0201] FIG. 47A is a graph of membrane-bound TCRP (mTCRP) and CD3 expression and mTCRP and CD3 MFI in iPSC-derived cells during differentiation, either without a cytokine signaling receptor ("No CyR") or with a cytokine signaling receptor inserted at the GZMA locus ("GZMA CyR").

[0202] FIG. 47B is a graph of CAR expression and MFI in iPSC-derived cells during differentiation, either without a cytokine signaling receptor ("No CyR") or with a cytokine signaling receptor inserted at the GZMA locus ("GZMA CyR").

[0203] FIG. 47C is a graph of intracellular granzyme A expression in iPSC-derived cells during differentiation, either without a cytokine signaling receptor ("No CyR") or with a cytokine signaling receptor inserted at the GZMA locus ("GZMA CyR").

[0204] FIG. 47D is a graph of construct expression, as determined by CD34 extracellular domain expression, in iPSC-derived cells during differentiation, either without a cytokine signaling receptor ("No CyR") or with a cytokine signaling receptor inserted at the GZMA locus ("GZMA CyR").

[0205] FIG. 48A is a graph of granzyme A and / or construct expression, as determined by CD34 extracellular domain expression, of iPSC-derived CD8+ T cells, either without a cytokine signaling receptor ("No CyR") or with a cytokine signaling receptor inserted at the GZMA locus ("GZMA CyR"), before expansion or after expansion with and without exogenous cytokines.

[0206] FIG. 48B is a graph of construct expression, as determined by CD34 extracellular domain expression, of iPSC-derived CD8+ T cells, either without a cytokine signaling receptor ("No CyR") or with a cytokine signaling receptor inserted at the GZMA locus ("GZMA CyR"), after expansion with and without exogenous cytokines.

[0207] FIG. 48C is a graph of pSTAT3 expression of iPSC-derived CD8+ T cells, either without a cytokine signaling receptor ("No CyR") or with a cytokine signaling receptor inserted at the GZMA locus ("GZMA CyR"), after expansion with and without exogenous cytokines.

[0208] FIG. 48D is a graph of pSTAT5 expression of iPSC-derived CD8+ T cells, either without a cytokine signaling receptor ("No CyR") or with a cytokine signaling receptor inserted at the GZMA locus ("GZMA CyR"), after expansion with and without exogenous cytokines.

[0209] FIG. 49A is a graph of tumor growth inhibition at varying effector to target cell (E:T) ratios of iPSC-derived CD8+ T cells, either without a cytokine signaling receptor ("No CyR") or with a cytokine signaling receptor inserted at the GZMA locus ("GZMA CyR"), and with or without exogenous cytokine supplementation.

[0210] FIG. 49B is a graph of fold change in proliferation of iPSC-derived CD8+ T cells, either without a cytokine signaling receptor ("No CyR") or with a cytokine signaling receptor inserted at the GZMA locus ("GZMA CyR"), following target antigen stimulation with or without exogenous cytokine supplementation.

[0211] FIG. 49C is a graph of fold change in proliferation of iPSC-derived CD8+ T cells after 5 days, either without a cytokine signaling receptor ("No CyR") or with a cytokine signaling receptor inserted at the GZMA locus ("GZMA CyR"), following target antigen stimulation with exogenous cytokine supplementation.

[0212] FIG. 49D is a graph of tumor growth inhibition of iPSC-derived CD8+ T cells, either without a cytokine signaling receptor ("No CyR") or with a cytokine signaling receptor inserted at the GZMA locus ("GZMA CyR"), and with or without exogenous cytokine supplementation.

[0213] FIG. 49E is a graph of tumor growth inhibition in a serial antigen restimulation assay for iPSC- derived CD8+ T cells, either without a cytokine signaling receptor ("No CyR") or with a cytokine signaling receptor inserted at the GZMA locus ("GZMA CyR"), and with or without exogenous cytokine supplementation.

[0214] FIG. 50A is a graph of fold change in proliferation of iPSC-derived CD8+ T cells, either without a cytokine signaling receptor ("No CyR") or with a cytokine signaling receptor inserted at the GZMA locus ("GZMA CyR"), expanded under indicated cytokine supplementation, following target antigen stimulation with exogenous cytokine supplementation.

[0215] FIG. 50B is a graph of tumor growth inhibition of iPSC-derived CD8+ T cells, either without a cytokine signaling receptor ("No CyR") or with a cytokine signaling receptor inserted at the GZMA locus("GZMA CyR"), expanded under indicated cytokine supplementation, following target antigen stimulation exogenous cytokine supplementation.

[0216] FIG. 50C is a graph of fold change in proliferation of iPSC-derived CD8+ T cells, either without a cytokine signaling receptor ("No CyR") or with a cytokine signaling receptor inserted at the GZMA locus ("GZMA CyR"), expanded under indicated cytokine supplementation, following target antigen stimulation without exogenous cytokine supplementation.

[0217] FIG. 50D is a graph of tumor growth inhibition of iPSC-derived CD8+ T cells, either without a cytokine signaling receptor ("No CyR") or with a cytokine signaling receptor inserted at the GZMA locus ("GZMA CyR"), expanded under indicated cytokine supplementation, following target antigen stimulation without exogenous cytokine supplementation.

[0218] FIG. 51 is a graph of cumulative fold expansion during in vitro T cell differentiation of iPSC- derived cells without a cytokine receptor ("PCE309 (Parental Ctrl)"), or with a cytokine signaling receptor under the control of an exogenous NFKB promoter ("PCE434 (NFKB)", PCE436 (NFKB)"), an exogenous GM-CSF promoter ("PCE438 (GM-CSFp)") or a synthetic antigen-responsive promoter ("PCE481 (ARP)").

[0219] FIG. 52A is a graph of CD5 and CD7 expression at day 21 of in vitro T cell differentiation of iPSC- derived cells without a cytokine signaling receptor ("PCE309 (Parental Ctrl)"), or with a cytokine signaling receptor under the control of an exogenous NFKB promoter ("PCE434 (NFKB)", "PCE436 (NFKB)"), an exogenous GM-CSF promoter ("PCE438, (GM-CSFp)") or a synthetic antigen-responsive promoter ("PCE481 (ARP)").

[0220] FIG. 52B is a graph of CD56 and CD7 expression at day 21 of in vitro T cell differentiation of iPSC- derived cells without a cytokine signaling receptor ("PCE309 (Parental Ctrl)"), or with a cytokine signaling receptor under the control of an exogenous NFKB promoter ("PCE434 (NFKB)", "PCE436 (NFKB)"), an exogenous GM-CSF promoter ("PCE438 (GM-CSFp)") or a synthetic antigen-responsive promoter ("PCE481 (ARP)").

[0221] FIG. 52C is a graph of CD4 and CD8a expression at day 21 of in vitro T cell differentiation of iPSC- derived cells without a cytokine signaling receptor ("PCE309 (Parental Ctrl)"), or with a cytokine signaling receptor under the control of an exogenous NFKB promoter ("PCE434 (NFKB)", "PCE436 (NFKB)"), an exogenous GM-CSF promoter ("PCE438 (GM-CSFp)") or a synthetic antigen-responsive promoter ("PCE481 (ARP)").

[0222] FIG. 52D is a graph of CD8a and CD8P expression at day 21 of in vitro T cell differentiation of iPSC-derived cells without a cytokine signaling receptor ("PCE309 (Parental Ctrl)"), or with a cytokine signaling receptor under the control of an exogenous NFKB promoter ("PCE434 (NFKB)", "PCE436(NFKB)"), an exogenous GM-CSF promoter ("PCE438 (GM-CSFp)") or a synthetic antigen-responsive promoter ("PCE481 (ARP)").

[0223] FIG. 53 is a heat map of NK lineage marker expression ("CD94", "CD16", "NKp44" and "NKp46") in iPSC-derived CD8+ T cells without a cytokine receptor ("PCE309 (Parental Ctrl)"), or with a cytokine signaling receptor under the control of an exogenous NFKB promoter ("PCE434 (NFKB)", "PCE436 (NFKB)"), an exogenous GM-CSF promoter ("PCE438 (GM-CSFp)") or a synthetic antigen-responsive promoter ("PCE481 (ARP)").

[0224] FIG. 54A is a graph of cumulative fold expansion and viability during in vitro activation of iPSC- derived CD8+ T cells without a cytokine receptor ("PCE309 (Parental Ctrl)"), or with a cytokine signaling receptor under the control of an exogenous NFKB promoter ("PCE434 (NFKB)", "PCE436 (NFKB)"), an exogenous GM-CSF promoter ("PCE438 (GM-CSFp)") or a synthetic antigen-responsive promoter ("PCE481 (ARP)").

[0225] FIG. 54B is a graph of CD25 expression intensity (gMFI) during in vitro activation of iPSC-derived CD8+ T cells without a cytokine signaling receptor ("PCE309 (Parental Ctrl)"), or with a cytokine signaling receptor under the control of an exogenous NFKB promoter ("PCE434 (NFKB)", "PCE436 (NFKB)"), an exogenous GM-CSF promoter ("PCE438 (GM-CSFp)") or a synthetic antigen-responsive promoter ("PCE481 (ARP)").

[0226] FIG. 55A is a graph of cytokine signaling receptor surface expression (as indicated by CD34 expression) during differentiation ("D17", "D21") and in vitro activation ("D1X"-"D7X") of iPSC-derived CD8+ T cells with a cytokine signaling receptor under the control of an exogenous NFKB promoter ("PCE434 (NFKB)", "PCE436 (NFKB)") or an exogenous GM-CSF promoter ("PCE438 (GM-CSFp)").

[0227] FIG. 55B is a graph of cytokine signaling receptor surface expression (as indicated by CD34 expression) during differentiation ("D17", "D21") and in vitro activation ("D1X"-"D7X") of iPSC-derived CD8+ T cells with a cytokine signaling receptor under the control of a synthetic antigen-responsive promoter ("PCE481 (ARP)") or without a cytokine receptor ("PCE309 (Parental Ctrl)").

[0228] FIG. 56A is a graph of proliferation following one round of antigen stimulation ("Post-Stim 1") at varying E:T ratios of iPSC-derived CD8+ T cells without a cytokine signaling receptor ("Parental" ) in the presence or absence of exogenous cytokines, or iPSC-derived CD8+ T cells with a cytokine signaling receptor under the control of an exogenous NFKB promoter ("NFKB (PCE434)", "NFKB (PCE436)"), an exogenous engineered GM-CSF promoter ("GM-CSFp") or a synthetic antigen-responsive promoter ("ARP").

[0229] FIG. 56B is a graph of proliferation following three rounds of antigen stimulation of iPSC-derived CD8+ T cells without a cytokine receptor ("Parental") in the presence or absence of exogenous cytokines, or iPSC-derived CD8+ T cells with a cytokine signaling receptor under the control of an exogenous NFKB promoter ("NFKB (PCE434)", "NFKB (PCE436)"), an exogenous engineered GM-CSF promoter ("GM-CSFp") or an synthetic antigen-responsive promoter ("ARP").

[0230] FIG. 57A is a graph of tumor growth inhibition (% TGI) at varying E:T ratios for iPSC-derived CD8+ T cells without a cytokine receptor ("Parental") in the presence or absence of exogenous cytokines, or iPSC-derived CD8+ T cells with a cytokine signaling receptor under the control of an exogenous NFKB promoter ("NFKB (PCE434)", "NFKB (PCE436)"), an exogenous engineered GM-CSF promoter ("GM- CSFp") or a synthetic antigen-responsive promoter ("ARP").

[0231] FIG. 57B is a graph of tumor growth inhibition for iPSC-derived CD8+ T cells without a cytokine receptor ("Parental") in the presence or absence of exogenous cytokines, or iPSC-derived CD8+ T cells with a cytokine signaling receptor under the control of an exogenous NFKB promoter ("NFKB (PCE434)", "NFKB (PCE436)"), an exogenous engineered GM-CSF promoter ("GM-CSFp") or a synthetic antigen- responsive promoter ("ARP").

[0232] FIG. 58 is a graph of in vivo tumor growth inhibition, as assessed by bioluminescence imaging (region of interest, "ROI flux"), for iPSC-derived CD8+ CAR-T cells with and without transduced cytokine signaling receptor ("iPSC-CD8+ pNT549", "iPSC-CD8+ UTD") and primary CD4+ and CD8+ CAR-T cell control ("Primary CD4 / CD8 T").

[0233] FIG. 59A is a graph of CAR-T cell concentration in the lungs, as assessed by hCD45 expression, at the end of an in vivo tumor implant study for iPSC-derived CD8+ CAR-T cells with and without transduced cytokine signaling receptor ("iPSC-CD8+ pNT549", "iPSC-CD8+ UTD") and primary CD4+ and CD8+ CAR-T cell control ("Primary CD4 / CD8 T").

[0234] FIG. 59B is a graph of the percentage of CAR+ cells, gated on the cells assessed in Fig. 59A, for iPSC-derived CD8+ CAR-T cells with transduced cytokine signaling receptor ("iPSC-CD8+ pNT549) and primary CD4+ and CD8+ CAR-T cell control ("Primary CD4 / CD8 T").

[0235] FIG. 59C is a graph of the percentage of CD3+ cells, gated on the cells assessed in Fig. 59A, for iPSC-derived CD8+ CAR-T cells with transduced cytokine signaling receptor ("iPSC-CD8+ pNT549") and primary CD4+ and CD8+ CAR-T cell control ("Primary CD4 / CD8 T").

[0236] FIG. 60A is a graph of CD34 (transduced cytokine signaling receptor extracellular domain) and CD3 expression for cells harvested from the lungs at the end of an in vivo tumor implant study. Groupsanalyzed were iPSC-derived CD8+ CAR-T cells with transduced cytokine signaling receptor ("iPSC-CD8+ pNT549") and primary CD4+ and CD8+ CAR-T cell control ("Primary CD4 / CD8 T").

[0237] FIG. 60B is a graph of CD8a and CD8P expression, gated on the cells assessed in Fig. 59A, for iPSC-derived CD8+ CAR-T cells with transduced cytokine signaling receptor ("iPSC-CD8+ pNT549") and primary CD4+ and CD8+ CAR-T cell control ("Primary CD4 / CD8 T").

[0238] FIG. 60C is a graph of CD8a and CD4 expression, gated on the cells assessed in Fig. 59A, for iPSC- derived CD8+ CAR-T cells with transduced cytokine signaling receptor ("iPSC-CD8+ pNT549") and primary CD4+ and CD8+ CAR-T cell control ("Primary CD4 / CD8 T").

[0239] FIG. 61A is a graph of biodistribution of CAR+ cells at the end of an in vivo tumor implant study. Groups analyzed were iPSC-derived CD8+ CAR-T cells with and without transduced cytokine signaling receptor ("iPSC-CD8+ pNT549", "iPSC-CD8+ UTD") and primary CD4+ and CD8+ CAR-T cell control ("Primary CD4 / CD8 T").

[0240] FIG. 61B a graph of biodistribution of CAR+ cells at the end of an in vivo tumor implant study, normalized to the detection in the lung tissue. Groups analyzed were iPSC-derived CD8+ CAR-T cells with and without transduced cytokine signaling receptor ("iPSC-CD8+ pNT549", "iPSC-CD8+ UTD") and primary CD4+ and CD8+ CAR-T cell control ("Primary CD4 / CD8 T").

[0241] FIG. 62A is a graph of fold proliferation of iPSC-derived CD8+ T cells cultured with exogenous STAT3 cytokine support (IL21), STAT5 cytokine support (IL2 and IL7) or STAT3 and STAT5 cytokine support (IL2, IL7 and IL21).

[0242] FIG. 62B is a graph of cytotoxicity of iPSC-derived CD8+ T cells against A549 CD19+ tumor cells without cytokine support or with exogenous STAT3 and STAT5 cytokine support (IL2, IL7 and IL21).

[0243] FIG. 62C is a graph of proliferation of iPSC-derived CD8+ T cells in the cytotoxicity assay of Fig. 62B, without cytokine support or with exogenous STAT3 and STAT5 cytokine support (IL2, IL7 and IL21).

[0244] FIG. 63A is a graph of phosphorylated STAT3 (pSTAT3) expression, as reported by gMFI, for untransduced primary T cells ("UTD") primary T cells transduced with a STAT5 cytokine receptor construct (pNT466, "CytR (STAT5)") or a STAT3 / STAT5 cytokine receptor construct (pNT581, "CytR (STAT3 / 5)").

[0245] FIG. 63B is a graph of phosphorylated STAT5 (pSTAT5) expression, as reported by gMFI, for untransduced primary T cells ("UTD") or primary T cells transduced with a STAT5 cytokine receptor construct (pNT466, "CytR (STAT5)") or a STAT3 / STAT5 cytokine receptor construct (pNT581, "CytR (STAT3 / 5)").

[0246] FIG. 63C is a graph of cytotoxicity of untransduced iPSC-derived CD8+ T cells ("UTD") or iPSC- derived CD8+ T cells transduced with a STAT5 cytokine receptor construct (pNT466, "CytR (STAT5)") or a STAT3 / STAT5 cytokine receptor construct (pNT581, "CytR (STAT3 / 5)") in a serial restimulation assay with A549 CD19+ tumor target cells.

[0247] FIG. 63D is a graph of proliferation of iPSC-derived CD8+ T cells in the cytotoxicity assay of Fig. 63C, for untransduced iPSC-derived CD8+ T cells ("UTD"), untransduced iPSC-derived CD8+ T cells with exogenous IL2, IL7 and IL21 ("UTD +STAT5 + STAT3") or iPSC-derived CD8+ T cells transduced with a STAT5 cytokine receptor construct (pNT466, "CytR (STAT5)") or a STAT3 / STAT5 cytokine receptor construct (pNT581, "CytR (STAT3 / 5)").

[0248] FIG. 64A is a graph of cytokine receptor expression ("% CytR expression") during iPSC-derived CD8+ T cell differentiation for three different cytokine receptor expression profiles numbered 1-3.

[0249] FIG. 64B is flow cytometry plots of CDllb and CD7 expression for iPSC-derived cells with ("(+) CytR") and without ("(-) CytR") a cytokine receptor with the first profile (1) of Fig. 64A.

[0250] FIG. 64C is a graph of CD5 and CD7 expression for iPSC-derived cells with ("(+) CytR") and without ("(-) CytR") a cytokine receptor with the first profile (1) of Fig. 64A.

[0251] FIG. 64D is flow cytometry plots of CD5 and CD7 expression for iPSC-derived cells with ("(+) CytR") and without ("(-) CytR") a cytokine receptor with the second profile (2) of Fig. 64A.

[0252] FIG. 64E is a heat map of CD56 MFI, NKp44 MFI and CD16 MFI for iPSC-derived cells with ("(+) CytR") and without ("(-) CytR") a cytokine receptor with the second profile (2) of Fig. 64A.

[0253] FIG. 64F is flow cytometry plots of CD5 and CD7 expression for iPSC-derived cells with ("(+) CytR") and without ("(-) CytR") a cytokine receptor with the third profile (3) of Fig. 64A.

[0254] FIG. 64G is a heat map of CD56 MFI, NKp44 MFI and CD16 MFI for iPSC-derived cells with ("(+) CytR") and without ("(-) CytR") a cytokine receptor with the third profile (3) of Fig. 64A.

[0255] FIG. 65A is a graph of CD5 and CD7 expression during early-, mid- and late-T cell differentiation for iPSC-derived cells engineered with a cytokine receptor.

[0256] FIG. 65B is a graph of CD8a and CD8P expression during early-, mid- and late-T cell differentiation for iPSC-derived cells engineered with a cytokine receptor.

[0257] FIG. 65C is a graph of CD56 and CD7 expression during early-, mid- and late-T cell differentiation for iPSC-derived cells engineered with a cytokine receptor.

[0258] FIG. 65D is a graph of percent CAR expression and gMFI during early-, mid- and late-T cell differentiation for iPSC-derived cells engineered with a cytokine receptor.

[0259] FIG. 65E is representative flow cytometry plots of CD5 and CD7 expression, CD8a and CD8P expression, CD56 and CD7 expression and CD3 and CAR expression for iPSC-derived CD8+ T cells engineered with a cytokine receptor.

[0260] FIG. 65F is a graph of cytokine receptor ("CytR") expression during early-, mid- and late-T cell differentiation, following CD8+ T cell activation ("Post-Act.") and during or after CD8+ T cell expansion ("Exp. (Mid)", "Exp. (End)") for iPSC-derived cells engineered with a cytokine receptor.

[0261] FIG. 66A is a heatmap of tumor growth inhibition (TGI) for iPSC-derived T cells with exogenous cytokine support (IL2, IL7 and IL21 "Exogenous Support") or an engineered cytokine receptor ("Engineered CytR") co-cultured with A549 CD19+ tumor target cells at varying effector to target cell (E:T) ratios as indicated.

[0262] FIG. 66B is a heatmap of tumor growth inhibition (TGI) for iPSC-derived CD19 CAR-T cells with exogenous cytokine support (IL2, IL7 and IL21 "Exogenous Support") or an engineered cytokine receptor ("Engineered CytR") co-cultured with Raji CD19+ tumor target cells at varying E:T ratios as indicated.

[0263] FIG. 66C is a graph of secretion of I FNy, TNFa, perforin, granzyme B ("GzmB") and granzyme A ("GzmA") by iPSC-derived CD19 CAR-T cells with exogenous cytokine support (IL2, IL7 and IL21 "Exogenous Support") or an engineered cytokine receptor ("Engineered CytR"), as measured by a Meso Scale Discovery (MSD®, CellCarta) panel at 48 hours after CD19 antigen stimulation via co-culture with A549 CD19+ target cells.

[0264] FIG. 66D is a graph of pSTAT3 and pSTAT5 expression for iPSC-derived CD19 CAR-T cells with exogenous cytokine support (IL2, IL7 and IL21 "Exogenous Support") or an engineered cytokine receptor ("Engineered CytR") following CD19 antigen stimulation via co-culture with A549 CD19+ target cells.

[0265] FIG. 67A is a graph of tumor cell growth in a co-culture assay of A549 CD19+ tumor target cells and iPSC-derived CD19 CAR-T cells without cytokine support ("No Support") with exogenous cytokine support (IL2, IL7 and IL21, "Exogenous Support") or an engineered cytokine receptor ("Engineered CytR"), in comparison to co-culture with primary CD3+ CD19 CAR-T cells ("Primary CD3 CAR-T") or A549 CD19+ target cells alone.

[0266] FIG. 67B is a graph of tumor cell growth in a co-culture assay of Raji CD19+ tumor target cells and iPSC-derived CD19 CAR-T cells without cytokine support ("No Support") with exogenous cytokine support (IL2, IL7 and IL21, "Exogenous Support") or an engineered cytokine receptor ("Engineered CytR"), in comparison to co-culture with primary CD3+ CD19 CAR-T cells ("Primary CD3 CAR-T") or Raji CD19+ target cells alone.

[0267] FIG. 67C is a graph of proliferation of iPSC-derived CD19 CAR-T cells with exogenous cytokine support (IL2, IL7 and IL21, "Exogenous Support") or an engineered cytokine receptor ("Engineered CytR") in comparison to primary CD3+ CD19 CAR-T cells ("Primary CD3 CAR-T") in the A549 cell coculture assay of Fig. 67A.

[0268] FIG. 67D is a graph of proliferation of iPSC-derived CD19 CAR-T cells with exogenous cytokine support (IL2, IL7 and IL21, "Exogenous Support") or an engineered cytokine receptor ("Engineered CytR") in comparison to primary CD3+ CD19 CAR-T cells ("Primary CD3 CAR-T") in the Raji cell co-culture assay of Fig. 67B.

[0269] FIG. 67E is a graph of cytokine receptor expression ("STAT3 / 5 Signal") in iPSC-derived CD19 CAR- T cells with an engineered cytokine receptor ("Engineered CytR") in response to co-culture with CD19+ target cells or CD19- control cells.

[0270] FIG. 67F is a graph of graph of tumor cell growth in a co-culture assay of A549 CD19+ tumor target cells or A549 CD19- control cells and iPSC-derived CD19 CAR-T cells with an engineered cytokine receptor ("Engineered CytR"), in comparison with CD19+ tumor target cells or CD19- control target cells alone.

[0271] FIG. 68A is a graph of tumor burden, as determined by bioluminescence imaging (region of interest, "ROI") in NSG mice bearing disseminated A549-CD19 tumors, untreated ("Tumor only" or treated with iPSC-derived CD8+ CD19 CAR-T cells with an engineered cytokine receptor ("Engineered CytR"), unmodified iPSC-derived CD8+ CD19 CAR-T cells ("No Cytokine Support") or primary CD3+ CD19 CAR-T cells ("Primary CD3 CAR-T"). On Day 14, all treatment groups were rechallenged with additional A549-CD19 cells ("Tumor rechallenge").

[0272] FIG. 68B is a percent change in bodyweight, an indicator of acute toxicity, in NSG mice bearing disseminated A549-CD19 tumors, untreated ("Tumor only" or treated with iPSC-derived CD8+ CD19 CAR-T cells with an engineered cytokine receptor ("Engineered CytR"), unmodified iPSC-derived CD8+ CD19 CAR-T cells ("No Cytokine Support") or primary CD3+ CD19 CAR-T cells ("Primary CD3 CAR-T"). On Day 14, all treatment groups were rechallenged with additional A549-CD19 cells ("Tumor rechallenge").DETAILED DESCRIPTION OF THE DISCLOSURE

[0273] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0274] Generally, the present disclosure provides methods of engineering T cells, iPSCs and iPSC- derived T lineage cells to express constructs to induce STAT3 and / or STAT5 signaling.Definitions

[0275] As used herein, the term "attenuated STAT5 signaling" refers to a lymphoid cell with a reduced level of STAT5 signaling in comparison to a primary CD8+ T cell. The cell with attenuated STAT5 signaling may have, for example, reduced response to exogenous STAT5-signaling cytokines, reduced secretion of STAT5-signaling cytokines and / or reduced expression of STAT5-signaling receptor(s) in comparison to a primary CD8+ T cell.

[0276] As used herein, the term "hybrid cytokine signaling receptor", "CyR" or "CytR" refers to an engineered cell surface receptor that provides one or more cytokine signals when active in a cell, such as, for example, a STAT5 and a STAT3 signal. The hybrid cytokine signaling receptor may include an extracellular domain, a transmembrane domain, and one or more intracellular domains. The hybrid cytokine signaling receptor may be constitutively active, for example by modification of the transmembrane domain.

[0277] As used herein, the term "cytokine signaling receptor construct" refers to a nucleic acid sequence for expression in a cell that includes an engineered cytokine signaling receptor providing, for example, one or more of a STAT3 and a STAT5 signal when expressed and active in a cell. The cytokine signaling receptor construct may further include an exogenous promoter sequence to control expression of the cytokine signaling receptor independent of the insertion site of the construct in the cell genome, or the cytokine signaling receptor construct may be expressed under the control of an endogenous promoter at the integration site.

[0278] As used herein, the term "promoter associated with late-stage T cell differentiation" refers to an endogenous or exogenous promoter that is not active in undifferentiated iPSCs, hematopoietic stem / progenitor cells or progenitor T cells, but becomes activated during late-stage T cell commitment and differentiation, such as, for example, emergence of CD8 single-positive T cells and / or expansion of CD8+ T cells.

[0279] As used herein, the term "promoter associated with T cell activation" refers to an endogenous or exogenous promoter that is induced by CD8+ T cell activation, such as, for example, antigen stimulation and / or CD3 stimulation. Methods to activate CD8+ T cells in vitro and in vivo are known in the art and include, for example, exposure to antigen-positive cells, co-culture with antigen-presentingcells or artificial antigen-presenting cells and stimulation with CD3 / CD28 antibody-coated beads or tetramers.

[0280] As used herein, the term "inducible promoter" refers to a regulatory promoter that is activated in a cell in response to a cell-extrinsic stimulus such as, for example, antigen stimulation.

[0281] As used herein, the term "antigen-responsive element" refers to a component of an inducible promoter that is responsive to antigen stimulation, thereby inducing activation of the promoter.

[0282] As used herein, the term "stem cell" refers to a cell that can differentiate into more specialized cells and has the capacity for self-renewal. Stem cells include pluripotent stem cells (PSCs), such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), and multipotent stem cells, such as mobilized peripheral blood-derived CD34+ stem cells, umbilical cord blood stem cells and adult stem cells, which are found in various tissues. Methods for obtaining, deriving or producing stem cells are known in the art.

[0283] As used herein, the term "progenitor cell" refers to a cell that can differentiate into one or more types of cells, but typically has a limited capacity for self-renewal. Progenitor cells are derivatives of stem cells and have more limited potency relative to their corresponding source stem cells. For example, hematopoietic stem cells (HSCs), found in adult bone marrow, peripheral blood (in smaller numbers) and in umbilical cord blood, have the capacity to give rise to all other blood cells. Hematopoietic progenitor cells are multipotent or lineage-committed cells derived from HSCs that have the capacity to give rise to a more limited or specific type of blood cell. Hematopoietic stem and progenitor cells (HSPCs) typically exist as a heterogeneous population in vivo and have use as a heterogeneous population as described herein. Hematopoietic stem and progenitor cells may be characterized, for example, by surface CD34 (CD34+).

[0284] As used herein, the terms "progenitor T cell" and "proT cell" refer to a cell that is derived from a pluripotent stem cell or a CD34+ hematopoietic stem and / or progenitor cell and expresses at least CD7+, and has the capacity to differentiate into one or more types of immature and mature T cells. Examples of progenitor T cells include, but are not limited to, CD7+ cells, CD7+CD5+ cells, CD7+CD5+CD34+ cells, CD7+CD5+CD45RA+ cells and / or CD7+CD5+CDla+ cells.

[0285] As used herein, an "immature T cell" or mature T cell is a T lineage cell derived from a progenitor T cell. T cell development may be characterized by the progressive expression of cell surface receptors, particularly CD4 and CD8. In vivo, T lineage cells progress from progenitor T cells through CD4-CD8- (double-negative, DN), CD4+CD8- (CD4 immature single-positive, CD4ISP), CD4+CD8+ (doublepositive, DP), and CD4-CD8+ (CD8 single-positive, CD8SP) and CD4 single-positive (CD4SP) stages. CD8may be expressed as a heterodimer of CD8a and CD8P, resulting in CD8aP+ cells, or as CD8aa homodimer, resulting in CD8aa+ cells. CD4-CD8+ cells may also be characterized by cell-surface expression of CD3 and one of TCRyS (y8 T cells) or TCRaP (aP T cells).

[0286] As used herein, the term "subject" refers to a vertebrate, preferably a mammal (e.g., a nonhuman mammal), more preferably a primate and still more preferably a human. Mammals include, but are not limited to, primates, humans, farm animals, sport animals, and pets.

[0287] As used herein, the term "treatment", "treat" or "treating" is an approach for obtaining beneficial or desired clinical results. For purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, one or more of the following: increased immune response, increased T cell response, decreased extent of damage from a disease, condition, or disorder, decreased duration of a disease, condition, or disorder, and / or reduction in the number, extent, or duration of symptoms related to a disease, condition, or disorder. The term includes the administration of the compounds, agents, drugs or pharmaceutical compositions of the present disclosure to prevent or delay the onset of one or more symptoms, complications, or biochemical indicia of a disease or condition; lessening or improving one or more symptoms; shortening or reduction in duration of a symptom; or arresting or inhibiting further development of a disease, condition, or disorder. Treatment may be prophylactic (to prevent or delay the onset of a disease, condition, or disorder, or to prevent the manifestation of clinical or subclinical symptoms thereof) or therapeutic suppression or alleviation of symptoms after the manifestation of the disease, condition, or disorder. The beneficial or desired clinical result may be an increase or decrease (as appropriate) of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% relative to an appropriate control, for example, a subject that did not receive the therapy.

[0288] The term "administering" or "administration" as used herein refers to the placement of an agent, a drug, a compound, or a pharmaceutical composition as disclosed herein into a subject by a method or route which results in at least partial delivery of the composition to a desired site. The compounds and pharmaceutical compositions disclosed herein can be administered by any appropriate route which results in an effective treatment in the subject. Routes of administration of the compounds and pharmaceutical compositions disclosed herein include, but are not limited to, intravenous or intraperitoneal routes of administration, or a combination thereof.

[0289] The term "effective amount" or "therapeutically effective amount", for example an effective amount or therapeutically effective amount of an engineered lymphoid cell population or iPSC-derived T cell population as used herein is an amount sufficient to bring about any one or more beneficial ordesired results. In more specific aspects, an effective amount may alleviate or ameliorate one or more symptoms of a disease; decrease the duration of time that one or more symptoms of a disease, are present in a subject; increase the survival rate of a subject having a disease. For prophylactic use, beneficial or desired results may include eliminating or reducing the risk, lessening the severity, or delaying the onset of a disease, including biochemical and / or histological symptoms of the infection, its complications and intermediate pathological phenotypes presenting during development of the disease. For therapeutic use, beneficial or desired results may include clinical results such as reducing one or more symptoms of a disease; decreasing the dose or length of administration of other medications required to treat the disease; enhancing the effect and / or reducing the toxicity of another medication; delaying the progression of the disease in a subject, decreasing the duration of time that one or more symptoms of a disease, are present in a subject, and / or increasing the overall survival rate of a subject having a disease. An effective amount can be administered in one or more than one dose, round of administration, or course of treatment.

[0290] For purposes of this disclosure, an effective dosage of a cell population or a pharmaceutical composition is an amount sufficient to accomplish prophylactic or therapeutic treatment either directly or indirectly. As is understood in the clinical context, an effective dosage of a compound, or a pharmaceutical composition may or may not be achieved in conjunction with another agent, drug, compound, or pharmaceutical composition. Thus, an "effective dosage" may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desirable result may be or is achieved. The amount may vary from one subject to another and may depend upon one or more factors, such as, for example, subject gender, age, body weight, subject's health history, and / or the underlying cause of the disease, condition, or disorder to be prevented, inhibited and / or treated.

[0291] The term "pharmaceutically acceptable carrier, diluent, or excipient" as used herein includes any material which, when combined with an active ingredient, allows the ingredient to retain biological activity and is non-reactive with the subject's immune system. Examples include, but are not limited to, any of the standard pharmaceutical carriers such as a phosphate buffered saline solution, water, emulsions such as oil / water emulsion, and various types of wetting agents. In some embodiments, diluents for aerosol or parenteral administration are phosphate buffered saline (PBS) or normal (0.9%) saline. Compositions comprising such carriers are formulated by well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18thedition, A. Gennaro, ed., Mack Publishing Co., Easton, PA, 1990; and Remington, The Science and Practice of Pharmacy 20thEd. Mack Publishing, 2000).

[0292] As used herein, the singular forms "a," "an," and "the," are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0293] The phrase "and / or" should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Thus, as a non-limiting example, a reference to "A and / or B", when used in conjunction with open-ended language such as "comprising" can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0294] As used herein, the phrase "one or more," in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "one or more" refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, "one or more of A and B" (or, equivalently, "one or more of A or B," or, equivalently "one or more of A and / or B") can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0295] When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below those numerical values. In general, the term "about" is used herein to modify a numerical value above and below the stated value by a variance of 20%, 10%, 5%, or 1%. In certain embodiments, the term "about" is used to modify a numerical value above and below the stated value by a variance of 10%. In certain embodiments, the term "about" is used to modify a numerical value above and below the stated value by a variance of 5%. In certain embodiments, the term "about" is used to modify a numerical value above and below the stated value by a variance of 1%.

[0296] When a range of values is listed herein, it is intended to encompass each value and sub-range within that range. For example, "1-5 mL" is intended to encompass 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 1-2 mL, 1-3 mL, 1-4 mL, 1-5 mL, 2-3 mL, 2-4 mL, 2-5 mL, 3-4 mL, 3-5 mL, and 4-5 mL.

[0297] It will be further understood that the terms "comprises," "comprising," "includes," and / or "including," when used herein, specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0298] The term "consisting of" and its derivatives, as used herein, are intended to be closed terms that specify the presence of stated features, integers, steps, operations, elements, and / or components, and exclude the presence or addition of one or more other features, integers, steps, operations, elements and / or components.General techniques

[0299] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art.

[0300] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (MJ. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R.l. Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J.B. Griffiths, and D.G. Newell, eds., 1993-1998) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D.M. Weir and C.C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Cales, eds., 1987); Current Protocols in Molecular Biology (F.M. Ausubel eta / ., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis eta / ., eds., 1994); Current Protocols in Immunology (J.E. Coligan et al., eds., 1991); Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rded., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, NY (2002); Harlow and Lane Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1998); Coligan et al., Short Protocols in Protein Science, John Wiley & Sons, NY (2003); Short Protocols in Molecular Biology (Wiley and Sons, 1999); and Immunobiology (C.A. Janeway and P. Travers, 1997).Gene ues

[0301] Various techniques are available to express an exogenous factor in a cell or to suppress the activity of an endogenous factor, including viral vector-mediated or nuclease-mediated gene editing. For example, transfection of a cell with a lentiviral vector (LVV) enables non-specific insertion of a construct into the genome (Ellis et al., 2021). Site-specific insertion (SSI) may be achieved with nuclease editing systems such as MAD7 or CRISPR-Cas (including Cas9 nuclease) using, for example, co-transfection of a guide RNA (gRNA) and a non-viral DNA template, or ribonucleic protein (RNP) complex (Ellis et al., 2021).

[0302] In an embodiment, SSI of a cytokine signaling receptor construct is performed by transfection of a non-viral plasmid DNA template, MAD7 nuclease and gRNA. In an embodiment, the SSI is performed in an iPSC. In an alternate embodiment, non-specific insertion of a cytokine signaling receptor construct is performed using LVV transfection.T Cell Differentiation

[0303] T cells may be differentiated in vitro from stem / progenitor cell populations. In one method, CD8+ T cells are differentiated from iPSC-derived CD34+ cells through controlled presentation of an immobilized Notch ligand, such as a Notch ligand immobilized on a microbead (Trotman-Grant et al., 2021, Carpenedo et al., 2024). In one method, a high level of Notch signaling is provided to induce CD8+ T cell differentiation (Carpenedo et al., 2024). In an alternate method, control of Notch signalling and CD3 activation is used to generate CD4+CD8+ double-positive cells and CD8+ T cells in a stepwise manner (Chandrasekaran et al., 2023, Martinez et al., 2023). Methods of generating CD34+ cells from iPSCs are known in the art, for example, differentiation with appropriate medium conditions (e.g., Trotman-Grant et al., 2021).T Cell Therapies

[0304] T cells have a broad range of therapeutic applications. T cells may be modified by, for example, conventional gene editing approaches such as nuclease editing or viral vector transduction, to express a chimeric antigen receptor (CAR), and / or an exogenous T Cell Receptor (TCR), to generate engineered T cell therapies (Weber et al., 2020). T cells derived from progenitor cells, including pluripotent stem cells, may be genetically engineered at the pluripotent, progenitor or derivative cell stage to comprise a nucleic acid encoding a CAR or TCR that may be expressed at the pluripotent cell, progenitor cell and / or derivative cell stage. Engineered T cell therapies have applicability in, for example, oncology and autoimmune disorders. In oncology, engineered T cell therapies have applicability in, for example, hematologic cancers, such as B cell lymphoma, B cell acute lymphoblastic leukemia and other B cellmalignancies, multiple myeloma and other hematologic cancers, as well as in solid tumours such as, for example, mesothelioma, adenocarcinomas, gliomas and sarcomas (Weber et al., 2020). In autoimmune disorders, engineered T cell therapies have applicability in, for example, Type I diabetes, rheumatoid arthritis, multiple sclerosis, lupus and other autoimmune disorders or conditions (Weber et al., 2020).

[0305] Engineered T cell therapies may target antigens known to be expressed on target cell types, including tumour cells or within tumour tissues. Chimeric antigen receptors (CARs) may be designed to target surface antigens or multivalent soluble antigens. The targeting extracellular domain of the CAR may be a single-chain variable fragment (scFv), single-domain antibodies (single variable domain on a heavy chain, VHH), nanoantibodies or other antigen-binding domain (Qu et al., 2022). CAR-T cell therapies may be directed towards multiple antigens using varying CAR designs or multiple CARs (Qu et al. 2022). Exemplary oncology antigens and corresponding cancer types for CAR-T cell therapies are listed in Table 1 below (Qu et al., 2022; Guha et al., 2022; Drougkas et al., 2023; Want et al., 2023).

[0306] TCR-T cell therapies target antigens expressed as peptide-human leukocyte antigen (HLA) complexes on the surface of a target cell. These targets may include tumour-associated antigens (TAAs) and tumour-specific antigens (TSAs) (Baulu et al., 2023). Exemplary oncology antigens and corresponding cancer types for TCR-T cell therapies are listed in Table 2 below (Baulu et al., 2023; Sun et al., 2021; Want et al., 2023).Cytokine Signalling Pathways

[0307] As known in the field, one of the largest groups of cytokines is those that signal through the Janus Kinase / signal transducer and activator of transcription (JAK / STAT) pathway (Morris et al., 2018). Signaling of these cytokines is induced by binding to a receptor, inducing activation of specific JAK(s) and STAT(s). IL2 family cytokines (IL2, IL4, IL7, IL9, IL15 and IL21) signal via receptors that contain the common gamma chain (gamma common, yc); IL4, IL7, IL9 and IL21 signal via heterodimeric receptors (yc and a receptor) whereas IL2 and IL15 require a third receptor subunit, IL2RP (Morris et al., 2018). Table 3 provides a summary of receptor components and downstream signalling for cytokines in this group.

[0308] Table 4 provides a summary of functions associated with these cytokines, including changes in apoptosis and proliferation of various primary T cell subsets.Engineered Cytokine Signaling Receptor Constructs

[0309] The present disclosure provides engineered constructs to induce STATS and STAT5 signaling in a cell, such as an iPSC, a lymphoid cell, a T cell or an iPSC-derived T cell. In an embodiment, provided is a STAT3 / STAT5 hybrid cytokine signaling receptor (Fig. 1A). This receptor includes an extracellular domain, such as a CD34 extracellular domain, expressed on the cell surface, a transmembrane domain and intracellular STAT3 and STAT5 signaling domains joined by a linker. In an embodiment, the transmembrane domain is modified to provide constitutive activation of the hybrid cytokine signaling receptor, such as, for example, a mutated IL7Ra transmembrane domain. In an embodiment, themutated IL7Ra transmembrane domain comprises a cysteine-proline-threonine (CPT) motif. In an embodiment, the intracellular STAT5 signaling domain is a portion of the IL7R intracellular domain, and the intracellular STAT3 signaling domain is a portion of the IL21R intracellular domain. In an embodiment, the transmembrane domain adjoins the STAT3 signaling domain, and the STAT5 domain is joined to the STAT3 signaling domain via a linker; alternatively, the transmembrane domain adjoins the STAT5 signaling domain, and the STAT3 domain is joined to the STAT5 signaling domain via a linker. When expressed in an engineered cell, the hybrid cytokine signaling receptor induces both STAT3 and STAT5 phosphorylation and downstream signaling (Fig. 1A).

[0310] In an alternate embodiment, provided is a combination of a STAT3 or STAT5 cytokine signaling receptor and a STAT5 or STAT3 cytokine (Fig. IB). For example, the combined engineering approach includes an engineered STAT5 cytokine receptor, including an extracellular domain, transmembrane domain, and intracellular STAT5 signaling domain, and a secreted STAT3 signaling cytokine such as, for example, IL21. The transmembrane domain of the engineered STAT5 cytokine signaling receptor may be modified to provide constitutive activation of the receptor, such as, for example, a mutated IL7Ra transmembrane domain. When expressed in an engineered cell, the STAT5 cytokine signaling receptor induces STAT5 phosphorylation and downstream signaling. The STAT3 signaling cytokine is expressed along with the STAT5 cytokine receptor as part of a bicistronic transgene. The STAT3 signaling cytokine contains the endogenous signal peptide at its N-terminus to direct its secretion from the cell. The STAT3 signaling cytokine activates an endogenous STAT3 signaling receptor of the cell to induce STAT3 phosphorylation and downstream signaling (Fig. IB). Alternatively, the combined engineering approach includes an engineered STAT3 cytokine receptor, including an extracellular domain, transmembrane domain, and intracellular STAT3 signaling domain, such as a portion of the IL21 receptor, and a secreted STAT5 signaling cytokine such as, for example, IL7. Both construct designs, as further described in Examples 2 and 3, therefore provide a combination of STAT3 and STAT5 signaling in the engineered cell. This combination of STAT3 and STAT5 signaling may improve cell proliferation, persistence and function (such as target cell clearance) of the engineered cell. Further, providing this cell-intrinsic cytokine support may avoid the need for toxic exogenous cytokine co-dosing, eliminate the manufacturing complexity of a CD4- and CD8-containing product and enable re-dosing of the cell product without patient lymphodepletion.

[0311] The present disclosure also provides regulation strategies to control the expression and activity of the STAT3 and STAT5 signaling construct in the cell. In an embodiment, the cytokine signaling receptor construct (e.g., a hybrid cytokine signaling receptor or a cytokine signaling receptor and acytokine) is integrated into a site in the genome of the cell associated with late-stage T cell lineage differentiation or T cell activation (Fig. 2A, top). The insertion of the cytokine signaling receptor construct may be monoallelic or biallelic. The hybrid cytokine signaling receptor, or the receptor and cytokine, are expressed under the control of the endogenous promoter at that site, in addition to the endogenous gene product (Fig. 2A, top). In an embodiment, the expression of the inserted hybrid cytokine signaling receptor, or the cytokine signaling receptor and cytokine, is controlled by an endogenous promoter associated with late-stage T lineage differentiation or T cell activation, such as, for example, a granzyme A (GZMA) promoter, a granzyme B (GZMB) promoter, a CD25 promoter, a GM- CSF promoter, a CD86 promoter or a CD70 promoter.

[0312] In an alternate embodiment, the cytokine signaling construct is inserted at an insertion site such as, for example, AAVS1, CIITA, or CD58, under the control of an exogenous inducible promoter inserted into the insertion site with the signaling construct (Fig. 2A, bottom). For example, the inducible promoter may comprise antigen-responsive elements that are activated by T cell antigen recognition. In an embodiment, the inducible promoter is an NFKB promoter, a synthetic antigen-responsive promoter ("ARP"), a GM-CSF promoter, an NFAT promoter, a GAS promoter, or a Nur77 promoter. The insertion of the cytokine signaling receptor construct may be monoallelic or biallelic.

[0313] Both strategies enable suppression of the expression and function of the cytokine signaling construct during T cell differentiation, and activation during T cell stimulation and antigen exposure (Fig. 2B).

[0314] The pharmaceutical composition provided herein may be administered to a subject in order to alleviate or ameliorate one or more symptoms of a disease; decrease the duration of time that one or more symptoms of a disease, are present in a subject; and increase the survival rate of a subject having a disease.

[0315] The pharmaceutical composition provided herein may be administered to a subject to treat cancer or autoimmune disorders in the subject.

[0316] The pharmaceutical composition provided herein may be administered to a subject in an effective amount or a therapeutically effective amount. A person of ordinary skill in the art would be able to determine such amounts based on such factors as the subject's size (e.g., weight), age and / or sex; the severity of the subject's symptoms; and the particular composition or route of administration selected. A person skilled the art would also know how to select the proper route of administration and to administer the compounds and compositions provided herein.

[0317] The dosage of the pharmaceutical composition of the disclosure varies depending on many factors, such as the pharmacodynamic properties of the composition, the mode of administration, the age, health and weight of the recipient, the nature and extent of the symptoms, the frequency of the treatment and the type of concurrent treatment, if any, and the clearance rate of the compound in the subject to be treated. One of skill in the art can determine the appropriate dosage based on the above factors. In some embodiments, the pharmaceutical composition is administered initially in a suitable dosage that is adjusted as required, depending on the clinical response.Kits

[0318] The invention also provides kits comprising the pharmaceutical composition described herein. Kits of the invention include one or more containers comprising the pharmaceutical composition described herein and instructions for use in accordance with any of the methods of the invention described herein. Generally, these instructions comprise a description of administration of the pharmaceutical composition for the above-described therapeutic treatments. In some embodiments, kits are provided for producing a single-dose administration unit.

[0319] The instructions relating to the use of the pharmaceutical composition generally include information as to dosage, dosing schedule, and route of administration for the intended treatment. The containers may be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses. Instructions supplied in the kits of the invention are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.

[0320] The disclosure is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the disclosure should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.EXAMPLE 1: Cytokine Support for iPSC-CD8+ Cells

[0321] The effect of supplementation with exogenous cytokines was investigated for iPSC-derived CD8+ TCR-T cells. Cells were generated by staged differentiation of iPSC-derived hematopoietic stem / progenitor cells, modified with an exogenous MAGEA4 TCR, to progenitor T cells and CD4+CD8+ cells (double-positive, DP) on an engineered Notch ligand support, followed by conversion to CD8+ T cells via CD3 activation (Chandrasekaran et al., 2023). Briefly, iPSC-derived CD34+ cells (Trotman-Grantet al., 2021) were seeded in well-plates at 5xl04cells / mL and differentiated to progenitor T cells for 10 days in progenitor expansion medium (serum-free expansion medium, SFEM II, and lymphoid progenitor expansion supplement, STEMCELL Technologies), with microbeads modified with DL4 and VCAM-l added at day 1, and half-media exchanges at days 4 and 7. Progenitor T cells were cultured with DL4 / VCAM microbeads for 18 days to allow for the emergence of mature late CD4+CD8+ DP cells (CD4+CD8A+CD8B+CD3+TCRaP+). Lastly, late DP cells were enriched for CD8 (CD8 Positive Selection kit, STEMCELL Technologies), and activated using anti-CD3 / CD28 microbeads for 7 days to allow for DP cell to CD8+ T cell conversion to occur. The iPSC-derived CD8+ TCR-T cells were responsive to IL2, IL15 and IL21 in the absence of antigen-presenting target cells (Fig. 3A-3D). The iPSC-derived CD8+ TCR-T cells were responsive to IL21 comparable to primary, donor-derived T cells (Fig. 3D). The iPSC-derived CD8+ TCR-T cells had a substantially lower pSTAT5 response to IL2, IL7 and IL15 in comparison to primary, donor-derived T cells (Fig. 3A-3C), and a reduced pSTAT3 response to IL21 in comparison to primary, donor-derived T cells (Fig. 3D).

[0322] A co-culture assay of iPSC-derived CD8+ TCR-T cells and antigen-presenting target cells with IL2, IL7 IL15 and IL21 over four rounds of antigen stimulation (i.e., serial antigen restimulation) resulted in strengthened response to IL2, IL7, and IL15 (Fig. 4A-4D, "post-stim 4" vs "baseline"). IL7 led to phosphorylation of STAT5 after four rounds of antigen stimulation of iPSC-derived TCR+ CD8+ cells, but not at baseline (Fig. 4C). Responses to IL2, IL15 and IL21 were increased after four rounds of antigen stimulation (Fig. 4A, 4B, 4D). IL21 leading to phosphorylation of STAT3 was critical for optimized proliferation of iPSC-derived CD8+ TCR-T cells (Fig. 5A, B). Serial antigen restimulation in the co-culture assay demonstrated a long-term benefit of IL7 signaling (Fig. 5B). IL21 mediated STAT3 activation complemented by a STAT5 signal from IL15, IL7, or IL2 was critical for the proliferation of iPSC-derived CD8+ TCR-T cells (Fig. 5B).

[0323] Next, a cytokine stimulation assay was performed to activate STAT3 and STAT5 in primary T cells (two independent donors #31, #33). Cells were cultured in cytokine-depleted media for four hours ("4 hrs starvation") and then transferred into media containing one or more of IL7, IL15 and IL21 and analyzed for STAT3 and STAT5 phosphorylation. Results are shown below in Table 5.

[0324] In an alternate differentiation protocol, CD8+ T cells were generated by staged differentiation of iPSC-derived hematopoietic stem / progenitor cells to progenitor T cells and CD8+ cells on an engineered Notch ligand support (Carpenedo et al., 2024). Briefly, iPSC-derived CD34+ cells (Trotman-Grant et al., 2021) were seeded in well-plates at 5xl04cells / mL and differentiated to progenitor T cells for 10 days in progenitor expansion medium (serum-free expansion medium, SFEM II, and lymphoid progenitor expansion supplement, STEMCELL Technologies), with 2.70 x 107beads / mL of microbeads modified with DL4 and VCAM-l added at day 1, and half-media exchanges at days 4 and 7. Cells were then re-seeded at 1.5xl06cells / mL and cultured with DL4 / VCAM microbeads at 1.62 x 108beads / mL for a further 11 days (for a total of 21 days from CD34+ cells); half-media exchanges were performed at days 12, 14, 16, and 19, and cells were re-seeded at 1.5x10scells / mL with DL4 / VCAM microbeads at 1.62 x 10sbeads / mL on day 17. At day 21 (from the CD34+ stage), cells were harvested and enriched for CD8 using the EasySep™ Human CD8 Positive Enrichment II kit (STEMCELL Technologies) (Carpenedo et al., 2024). These iPSC-derived CD8+ T cells were also expanded with single or combinations of cytokines in vitro. iPSC-derived CD8+ T cells were shown to be reliant on specific combinations of cytokines for proliferation in vitro. Cytokines were required for iPSC-CD8+ T cell survival (Fig. 6A). IL21 synergized with IL2 or IL15 (Fig. 6B). iPSC-derived CD8+ T cells did not proliferate in the absence of cytokine support (Fig. 6B); exogenous cytokines signaling through both STAT3 and STAT5 were required for proliferation.

[0325] Further, the effect of various cytokines on CD62L expression in primary and iPSC-derived CD8+ T cells was analyzed. IL21 strongly influenced CD62L expression on iPSC-derived CD8+ T cells differentiated via the two different protocols (Table 6). In the first protocol, control of Notch signalling and CD3 activation was used to generate CD4+CD8+ double-positive cells and CD8+ T cells in a stepwise manner (Chandrasekaran et al., 2023). In the second protocol, a high level of Notch signaling was provided to induce CD8+ T cell differentiation (Carpenedo et al., 2024).EXAMPLE 2: Design and Function of Cytokine Signaling Constructs

[0326] Cytokine signalling constructs were designed to augment signal-3 through STAT3 and / or STAT5 activation. Exemplary engineered cytokine signaling receptor constructs are shown in Figure 7 (STAT5 activating constructs: pNT466, pNT465, pNT468 (Fig. 7A); STAT3 activating constructs: pNT469 (Fig. 7 A); STAT5 and STAT3 activating constructs: pNT473, pNT547, pNT581, pNT548, pNT549, pNT729 (Fig. 7 A)). The IL7 receptor, activating predominantly STAT5, is proposed to allow for expansion of iPSC-derived CD8+ T cells with exogenous IL21 support. The IL7 design included naturally occurring IL7 receptor mutants previously shown to mediate constitutive IL7 signaling (indicated as IL7Ra*, Shum et al., 2017). A tyrosine-containing motif ("YLRQ") of the cytoplasmic domain of the IL21 receptor was engineered into several of the constructs for STAT3 activation (pNT473, pNT547, pNT548, pNT549, pNT581). The combined constructs require only one engineered solution and insertion site for both STAT5 and STAT3 activation (Fig. 7B).

[0327] The construct pNT465 (IL15-IL15Ra) was transduced into primary T cells using lentivirus (two independent donors #30, #31). Primary T cells were activated with Dynabeads™ prior to lentiviral transduction. The mean vector copy number was 0.01854 across two donor cell lines, significantly different from untransduced control. Increased expression of IL15Ra was detected by flow cytometry in transduced cells three days following transduction (data not shown). The construct pNT466 (IL7Ra) was also transduced into primary T cells. The mean vector copy number was 0.563536 for Donor 30 and 0.568294 for Donor 31 (0.565916 across cell lines). Construct expression (CD34 extracellular domain expression, as assessed by flow cytometry three days following transduction) was 71.7% for Donor 30 and 68.4% for Donor 31.

[0328] Twelve days following transduction, STAT5 and STAT3 phosphorylation were analyzed after four hours starvation of transduced primary T cells in media lacking exogenous cytokines. The constructs pNT465 (IL15-IL15Ra) and pNT466 (IL7Ra) induced STAT5 phosphorylation in primary T cells to a greater extent than the pNT473 construct (IL15-CD8 and I L21-B7-1), and the construct pNT469 (IL21-CD8) induced a low level of STAT3 phosphorylation (Table 7). The construct pNT468 was not analyzed in this assay.

[0329] Further analysis was performed following transduction of constructs in Dynabead™-activated primary T cells (two independent donors #31, #33). The vector copy number for transduction of pNT465, pNT466, pNT468, pNT469, pNT473 is shown in Table 8. Twelve days following transduction, STAT3 and STAT5 phosphorylation was analyzed for pNT465, pNT466, and pNT468 after four hours starvation in cytokine-free media. The constructs pNT466 and pNT468 induced constitutive STAT5 phosphorylation in primary T cells (Table 8). The constructs pNT469 and pNT473 were not analyzed in this assay.

[0330] The proliferation of transduced primary T cells was analyzed on day 7 following transduction in a cytokine withdrawal assay. Cells were cultured with no cytokines for the first 48 hours, followed by I L2- only supplementation for the following eight days. The construct pNT466 improved primary T cell persistence after cytokine withdrawal, with a lower change in cell number and increased viability compared to untransduced control (Fig. 8A-D).

[0331] The IL7Ra (pNT466) and IL7Ra / IL21R (pNT547, pNT548, pNT549) chimeric constructs showed surface CD34 expression in transduced primary T cells (two independent donors #33, #35) (Fig. 9A). The vector copy number (VCN, as determined by detection of the Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) sequence) was higher for Donor #35 for constructspNT466, NT548, and pNT549 (Fig. 9B). STAT5 and STATS phosphorylation was analyzed after four hours starvation in cytokine-free media. Adding IL21R motif to IL7Ra design resulted in combined STAT5 and STATS activation in primary T-cells (Fig. 9C). The YLRQ STATS signaling motif addition into the cytoplasmic domain of IL7Ra (pNT547, pNT548, pNT549) resulted in constitutive pSTATS and pSTAT5 induction in primary T cells. A shorter IL21R intracellular domain (pNT548, pNT549) resulted in better STATS induction (Fig. 9C). Further results are shown in Table 9 below.

[0332] Next, the function of the cytokine signaling receptor construct pNT466 was assessed in transduced iPSC-derived CD8+ CD19 CAR / MAGEA4 TCR T cells in a serial antigen restimulation assay. iPSC-derived CD8+ CAR-TCR T cells generated as described above from CAR- and TCR-modified iPSCs (Carpenedo et al., 2024) were co-cultured with target A549-CD19+ cells and replated with fresh target cells every 5 days for a total of three rounds of antigen stimulation, and assessed for cytokine signaling receptor expression, STAT5 phosphorylation and proliferation. The expression of the transduced constitutively active IL7 receptor variant (pNT466) increased from baseline following antigen stimulation (Fig. 10A), with a corresponding increase in pSTAT5 (Fig. 10B). The transduced cells also had increased STAT5 activation at baseline over untransduced sample (Fig. 10B). Transduced cells were selectively enriched over the course of three rounds of antigen stimulation, further increasing induction of STAT5 activation (Fig. 10B). The constitutively active IL7Ra design (pNT466) promoted proliferation of iPSC- derived CD8+ CAR-TCR T cells in the presence of IL21 alone (Fig. 10C).

[0333] In a subsequent serial antigen restimulation assay, additional IL21 exogenous support improved proliferation in iPSC-derived CD8+ CAR-TCR T cells transduced with pNT466 STAT5 construct (Fig. 11A), yet comparable cytotoxicity over one round of antigen stimulation was demonstrated in the absence of exogenous IL21 (Fig. 11B).

[0334] Next, the function of the pNT466 construct was compared to the pNT549 (SEQ ID NO: 1, SEQ ID NO:2) and pNT581 (SEQ ID NO:3, SEQ ID NO:4) constructs in transduced iPSC-derived CD8+ CAR-TCR T cells in the serial antigen restimulation assay. The dual STAT3 / STAT5 constructs (pNT549, pNT581) resulted in persistent proliferation and improved tumor control in comparison to the STAT5 only construct, pNT466 (Fig. 12).

[0335] The serial antigen restimulation assay was repeated for iPSC-derived CD8+ CAR-TCR T cells transduced with the pNT549 and pNT581 constructs in the presence or absence of exogenous cytokines. Cells transduced with the dual STAT3 / 5 constructs proliferated in the absence of exogenous cytokines, but proliferation was not as high as untransduced control with exogenous cytokine support (Fig. 13). Dual constructs allowed iPSC-derived CD8+ CAR-TCR T cells to proliferate nearly as well as exogenous STAT3 / STAT5 cytokine support (Fig. 13).

[0336] The function of the pNT549 and pNT581 cytokine signaling receptor constructs was also evaluated in iPSC-derived CD8+ TCR-T cells (engineered to express a MAGEA4 TCR). Transduced or untransduced cells were co-cultured with target A735 cells (MAGEA4+) for five days at several effector to target cell (E:T) ratios in the presence or absence of exogenous cytokines. Loss of tumor control was observed for iPSC-derived CD8+ TCR-T cells lacking cytokine signaling receptor construct in the absence of exogenous cytokines (Fig. 14B). Significant tumor growth inhibition was observed at a higher effector to target cell ratio (E:T) of 2:1 (Fig. 14B). An overall elevation in cytotoxicity was observed with the transduction of cytokine signaling receptor constructs (Fig. 14A, 14B).

[0337] Following the co-culture assay, granzyme A and perforin secretion were elevated in iPSC-derived CD8+ TCR-T cells transduced with the cytokine signaling receptor constructs (Fig. 15A, B). Further, the loss of granzyme B, GM-CSF, I FNy and TNFa secretion observed in untransduced cells was partially rescued by cytokine signaling receptor transduction (Fig. 15C-F).

[0338] Next, the proliferation of cytokine signaling receptor-transduced and untransduced iPSC-derived CD8+ TCR-T cells was assessed over the course of the co-culture assay as described above, in comparison to primary CD3+ T cells. Proliferation was improved by cytokine signaling receptor constructs in the absence of exogenous cytokine support but did not fully recapitulate exogenous cytokine support (Fig. 16A). Following the first round of stimulation, viability was improved with transduction of cytokine signaling receptor constructs in the absence of exogenous cytokine support (Fig. 16B). Untransduced iPSC-derived CD8+ TCR-T cells did not persist past 5 days without engineered or exogenous cytokine support (data not shown).

[0339] The cytotoxicity of transduced and untransduced iPSC-derived CD8+ CAR-T cells was also assessed in a serial restimulation assay with target A549 CD19+ cells. The transduction of cytokine signaling receptor constructs improved cytotoxicity in both the presence and absence of exogenous cytokines (Fig. 17A). Significant tumor growth inhibition was observed at a higher E:T ratio of 2:1. The cytokine signaling receptor construct co-transduced well with a CD19 CAR (highly expressed in CAR+ cells, Fig. 17C). Cytokine signaling receptor construct expression and gMFI declined over time in the assay (Fig. 17B, C).

[0340] In subsequent serial antigen stimulation assays, the transduction of cytokine signaling receptor constructs improved proliferation of iPSC-derived CD8+ TCR-T cells in the absence of exogenous cytokines (Fig. 18A-B, co-culture with MAGEA4+ A375 target cells), as well as the proliferation of iPSC- derived CD8+ CAR-T cells in the absence of exogenous cytokines (Fig. 18C, co-culture with A549-CD19+ target cells). Cells lacking cytokine signaling receptor constructs did not remain viable after the first round of antigen stimulation (five days) when in co-culture with target cells in the absence of cytokines (Fig. 18A-C, "Stim #2", "Stim #3"). Cytokine signaling receptor constructs maintained or showed improvement of proliferation throughout multiple rounds of stimulation in both TCR (Fig. 18A, B) and CAR (Fig. 18C) assays.

[0341] At the end of the serial antigen restimulation assay, the effect of cytokine signaling receptor construct transduction on target -activated secretion of granzyme A, granzyme B, perforin, GM-CSF, IFNy and TNFa was assessed for iPSC-derived CD8+ CAR-T cells. The results of the CAR-based assay (Fig. 19A- F) recapitulated those seen in TCR-based assay (Fig. 15A-F), with both the pNT549 and pNT581 cytokine signaling receptor constructs resulting in elevated secretion of granzyme A, granzyme B, perforin and IFNy in both the presence and absence of exogenous cytokines, with greater effects observed in the absence of exogenous cytokines.

[0342] Further, both a CD19 CAR and cytokine signaling receptor construct (pNT549 or pNT581) were co-transduced using lentiviral transduction into iPSC-derived CD8+ T cells. Increased proliferation in culture and comparable or slightly elevated viability was observed following transduction (Fig. 20A, B). Moderate transduction efficiency of the CAR construct was observed (Fig. 20C), and CAR-based enrichment also increased the percentage of cells positive for the cytokine signaling receptor constructs (Fig. 20D).

[0343] Next, these co-transduced cells were assessed in a serial restimulation assay with target cells in comparison to primary CAR-transduced CD3+ T cell controls. The cell phenotype at the start of the assay (Day 0) is shown in Table 10 below. In the absence of exogenous cytokines, iPSC-derived CD8+ CAR-Tcells did not proliferate beyond one round of stimulation (Fig. 21A, open squares). Both the pNT549 and pNT581 cytokine signaling receptor constructs improved proliferation of iPSC-derived CD8+ CAR-T cells in the absence of exogenous cytokines (Fig. 21A, B). As demonstrated in previous cell lines, secretion of I FNy and TNFa was increased in cells transduced with the pNT549 or pNT581 cytokine signaling receptor constructs (Fig. 21C, D).

[0344] The cells were further evaluated in an 18-day target cell co-culture assay to evaluate longer- term cell persistence (Fig. 22A, B). Cells were cultured with A549-CD19+ target cells at a 4:1 E:T ratio and re-exposed to fresh target cells on days 3, 5, 10, and 12. A similar trend across constructs was observed as in Fig. 21. The pNT549 construct outperformed pNT581 in the long-term assay in terms of both cell proliferation (Fig. 22A) and cytotoxicity (Fig. 22B).

[0345] Next, the cytotoxicity of cryopreserved (frozen) cells was compared to non-cryopreserved (fresh) cells. iPSC-derived CD8+ TCR-T cells transduced with the pNT581 construct were assessed in a coculture assay with MAGEA4+ A375 target cells at varying E:T ratios. Similar tumor growth inhibition (TGI) was observed for fresh or frozen cells in the presence or absence of cytokines, and in both conditions, transduction of the pNT581 construct improved cytotoxicity (Fig. 23A, B). Cells transduced with the cytokine signaling receptor construct had similar tumor growth inhibition in the presence and absence of exogenous cytokines (Fig. 23A, B). The cytokine signaling receptor construct also improved viability post-thaw (Fig. 23C). I FNy secretion was increased in cytokine signaling receptor construct-transduced cells in the absence of exogenous cytokines (Fig. 23D).

[0346] In a serial antigen restimulation assay, the dual STAT3 / STAT5 construct nominated for cell line engineering (pNT581) did not proliferate to the same degree as untransduced (UTD) iSPC-derived CD8+ CAR-T cells with exogenous cytokine support (Fig. 24). Exogenous cytokine did not substantially alter the proliferation of iPSC-derived CD8+ CAR-T cells transduced with the pNT581 construct. Transduction of the single STAT5 pNT466 construct resulted in lower proliferation in the absence of exogenous cytokines and the addition of exogenous IL21 increased proliferation comparable to untransduced, exogenous cytokine-supported cells.

[0347] Next, the expression and performance of the pNT729 cytokine signaling receptor construct (IL7Ra-T2A-IL21; SEQ ID NO:5, SEQ ID NO:6) was evaluated in comparison to the pNT466 and pNT581 cytokine signaling receptor constructs. This construct provides STAT5 signaling via the engineered STAT5 cytokine receptor (IL7Ra) and STATS signalling via a secreted STATS signaling cytokine ( I L21), more closely resembling the pNT466 and exogenous IL21 condition investigated above. Following transduction in primary T cells (two independent donors #1, #2), CD34 expression (extracellular domain of cytokine signaling receptor constructs) was comparable between pNT581 and pNT729 and highest in pNT466 (Fig. 25A). Construct designs and CD34 expression in primary T cells are shown in Fig. 6A and Table 13 below. The IL7R* notation indicates the inclusion of a naturally occurring mutation in the IL7Ra transmembrane domain to provide constitutive activation of the cytokine signalling receptor. Comparable VCNs of 0.5 - 0.8 were achieved across constructs (Fig. 25B). In primary T cells, the combination of a constitutive STAT5 signal (pNT466) and secretion of IL21 (pNT729) resulted in similar pSTAT3 and pSTAT5 MFIs compared to the benchmark, pNT466 and exogenous IL21 (Fig. 25C, D).

[0348] Further analysis of the effect of exogenous IL21 on iPSC-derived CD8+ cell proliferation and STAT3 / STAT5 phosphorylation was performed. In the context of exogenous IL2, an apparent lower bound for an observable effect of IL21 on proliferation and STAT3 / STAT5 phosphorylation was observed between 1 - lOng / mL (Fig. 26A-C). Primary T cells transduced with pNT729 (two independent donors #1, #2) secreted IL21 exceeding the quantity measured in supernatant from iPSC-derived CD8+ T cells cultured with 10 ng / mL exogenous IL21 (Fig. 26D).

[0349] iPSC-derived CD8+ CAR-T cells transduced with the cytokine signaling receptor constructs pNT466, pNT549 or pNT729 were evaluated in a serial antigen restimulation assay with tumor target cells (A549-CD19+ cells). Transduced cells or untransduced control cells were cultured with target cells at varying Erf ratios for 5 days, replaced with full media exchange onto fresh target cells for a further 5 days and again for a further 4 days. Untransduced control cells were supplemented with exogenous IL2,IL7 and I L21, and cells transduced with the pNT466 IL7Ra construct were supplemented with exogenous IL21. The pNT729-transduced cells (STAT5 / secreted IL21) showed proliferation and pSTAT3 and pSTAT5 signaling comparable to the pNT466 and IL21 condition following the first round of antigen stimulation (Fig. 27A-C). The pNT549-trasnduced cells had high levels of pSTAT3 in comparison to the pNT466 and IL21 condition (Fig. 27B). The pNT729-transduced cells had a proliferative advantage over the pNT549- transduced cells over the course of the assay at both 2:1 and 1:1 E:T, yet had reduced proliferation compared to the pNT466 and IL21 condition (Fig. 27D, E). The percentage of construct-transduced cells, as evaluated by CD34 extracellular domain expression, declined over the first two rounds of the assay and increased in the final round (Fig. 27F). For the pNT729-transduced cells, IL21 secretion substantially decreased in the second round of antigen stimulation, below the concentration of exogenous IL21 used in the pNT466 and IL21 condition (27.0 pg / mL for pNT729 vs. 130.1 pg / mL for pNT466 with exogenous IL21 supplementation).EXAMPLE 3: Design and Insertion of Cytokine Signaling Constructs in iPSC-CD8+ Cells

[0350] In one embodiment, the expression of the STAT3 / STAT5 signaling construct is controlled by insertion at a site in the genome of the cell associated with late-stage T cell lineage differentiation or T cell activation, capturing the endogenous promoter at that site and resulting in expression of both the cytokine signaling receptor and the target gene product (Fig. 2A, top). In an alternate embodiment, the expression of the STAT3 / STAT5 signaling construct is controlled by a synthetic, inducible promoter, such as an antigen-responsive promoter (Fig. 2A, bottom). Both strategies enable minimal expression of the cytokine signaling receptor during differentiation, followed by expression during T cell expansion and antigen exposure (Fig. 2B). An antigen-responsive inducible promoter may enable repeated increase in expression of the cytokine signaling receptor during repeated antigen exposure (Fig. 2B).

[0351] Several target insertion sites and promoters were selected as candidates to control expression of the cytokine signaling receptor constructs (Fig. 28). The cytokine signaling receptor construct control may be achieved by integration and expression under an endogenous promoter (Fig. 28A; sites: GZMA, GRZMB, MT2A, ITGB7, IL12RB2, IL21R, CD25, AGTRAP, CAPN2, PTPRJ, LINC00892, GM-CSF), or by expression under a synthetic inducible promoter (Fig. 28B, promoters: iSynPro, 6xNFAT, 5XNFKB, GM- CSF, GAS, Nur77).

[0352] The expression of the candidate insertion sites was analyzed in an iPSC-derived CD8+ TCR-T cell line. CD25 and IL21R were expressed in expanded cells, and minimally in unexpanded cells, asdetermined by flow cytometry (data not shown). IL12RP was also expressed in primary T cells on day 3 post-activation of I L12Rp. CD25 expression was further analyzed in iPSC-derived CD8+ CAR-TCR-T cells. CD25 expression was found to be highly upregulated upon expansion and CAR-based stimulation of CAR- TCR-T cells (data not shown).

[0353] The sustained presence of Granzyme A (Fig. 29A) and Granzyme B (Fig. 29B) through in vitro T cell differentiation (Carpenedo et al., 2024) was detected with a cytokine secretion assay (Meso Scale Discovery®, MSD®) in multiple iPSC-derived TCR-T cell lines. The GM-CSF site exhibited non-zero, yet minimal expression during differentiation (Fig. 29C).

[0354] The effect of constitutive expression of the STAT5 cytokine signaling receptor construct pNT466 on T cell lineage differentiation was investigated in a 21-day in vitro differentiation with bead-based Notch signalling ligand (Carpenedo et al., 2024). Briefly, iPSC-derived CD34+ cells (Trotman-Grant et al., 2021) were seeded in well-plates at 5xl04cells / mL and differentiated to progenitor T cells for 10 days in progenitor expansion medium (serum-free expansion medium, SFEM II, and lymphoid progenitor expansion supplement, STEMCELL Technologies), with 2.70 x 107beads / mL of microbeads modified with DL4 and VCAM-l added at day 1, and half-media exchanges at days 4 and 7. Cells were then re-seeded at 1.5xl06cells / mL and cultured with 3D ETN at 1.62 x 108beads / mL in for a further 11 days (for a total of 21 days from CD34+ cells) in lymphoid maturation medium (LMM, STEMCELL Technologies); halfmedia exchanges were performed at days 12, 14, 16 and 19, and cells were re-seeded at 1.5x10scells / mL with DL4 / VCAM microbeads at 1.62 x 10sbeads / mL on day 17 (Carpenedo et al., 2024).

[0355] Constitutively active STAT5 was shown to skew cells to a myeloid fate at early stages of differentiation. On Day 10, the iPSC-derived cells modified with the pNT466 construct displayed a large FSC / SSC-A profile in comparison to unedited WT iPSCs (29.55% FSC / SSC-Ahighvs. 5.07% FSC / SSC-Ahigh), were highly positive for CDllb (myeloid marker; 50.66% CDllb+ vs. 5.05% CDllb+) and had reduced CD7 levels (key lymphoid marker; 16.39% CD7hlghvs. 70.66% CD7hlgh). By Day 17, unedited iPSC-derived progenitor T (proT) cells or cells modified with an exogenous MAGEA4 TCR were fully lymphoid-lineage committed (no CDllb, >95% CD7+) whereas pNT466-modified cells retained mostly myeloid lineage markers (53.97% CDllb+, 13.95% CD7high).

[0356] The modulation of STAT5 / STAT3 signaling by exogenous I L15 or IL21 during in vitro differentiation also skewed cells to an NK-like phenotype. Transduction with pNT466 on day 14 or addition of exogenous IL15 (STAT5) at 25 ng / mL to the differentiation media reduced CD5+CD7+ coexpression (Fig. 30A) and increased CD16 and CD56 expression (Fig. 30B). Control cells were transduced with a MAGEA4 TCR. Similarly, modulation of STAT3 signaling by addition of exogenous IL21 skewedcells to a CD56hlgh, CD16+, NKp46+ NK-like phenotype (Fig. 30C, SOD). The timing of STAT signaling intervention correlated with the abundance of NK-like cells, as day 10 IL15-supplemented cells had in the highest number of NK-like cells, followed by day 14 transduced cells, then day 17 IL15-supplemented cells. The day 0 IL21-supplementated cells were equivalent to day 10 IL21-supplemented cells and had a higher number of NK-like cells than day 17 IL21-supplemented cells or the no IL21 control. Unregulated expression of a STAT3 / STAT5 cytokine signaling receptor may therefore be undesirable for generation of iPSC-derived lymphoid cells, such as CD8+ T cells.

[0357] Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITEseq) data (mRNA and surface protein expression at single cell resolution, Stoeckius et al., 2017) was analyzed to generate data-driven identification of genes transcribed at late stages of T cell differentiation (Fig. 31). The gene signature enrichment was calculated with single-sample gene set enrichment analysis (ssGSEA) at a single-cell level using Gene Set Variation Analysis (GSVA 1.40.1, (Hanzelmann et al., 2013). Overall enrichment patterns were summarized as a bubble plot, with the area of each circle representing the percentage of cells expressing the signatures and the shading representing the average expression level (Fig. 31). CD25 (IL2RA) and CD86 were nominated based on high protein expression observed or predicted in end-stage CD8 SP cells (Fig. 31). The known function of genes analyzed in Fig. 31 is shown in Table 11 below.

[0358] Next, homology-directed repair (HDR) plasmid templates were designed for promoter capture constructs (Fig. 32).

[0359] Flow cytometry analysis of iPSC-derived CD8+ T cells further deprioritized PTPRJ, ITGB7, CAPN2, and IL12RB2 as candidate insertion sites. PTPRJ, ITGB7, CAPN2 and IL12RB2 sites were deprioritized based on protein expression data in iPSC-derived CD8+ T cells and / or during differentiation (Table 12). CD25 and CD86 were prioritized based on protein expression data in iPSC-derived CD8+ cells and / or during differentiation (Table 12). Due to a promising expression profile, IL21R was also moved forward.

[0360] Next, the expression of candidate insertion sites in iPSC-derived cells was examined. CD25, CD70, and CD86 exhibited desired expression profiles through differentiation at a well-plate scale (Carpenedo et al., 2024), as determined by flow cytometry (Fig. 33A, n=9 CD19 CAR-EBV TCR lines) and RT-PCR of gene expression (Fig. 33B, NTX4H27 CD19 CAR-EBV TCR cell line). CD25, CD70 and CD86 expression were suppressed during differentiation. Low CD25 and CD86 expression was corroborated by RT-PCR. ITGB7 was used as a positive control for expression during differentiation. The low expression of CD25 and CD86 was also observed in an alternate stirred-tank reactor differentiation format (Carpenedo et al., 2024), as determined by flow cytometry (Fig. 33C, NTX4H20 CAR-TCR line) and RT-PCR of gene expression (Fig. 33D, NTX4H20 CAR-TCR line).

[0361] Phenotyping of iPSC-derived CD8+ CAR-TCR-T cells further informed selection of endogenous and inducible promoter strategies (Fig. 34A, B). Cells were analyzed pre- and post-expansion ("Unexp.","Exp.");as well as post-antigen stimulation with target cells ("Post-Stiml"). The expression of CD25, CD70, and CD86 was promising post-expansion and post-antigen stimulation (Fig. 34A). IL21R exhibited poor expression. MHC class II transactivator, CIITA, as analyzed by MHC Class II expression (HLA-DR, HLA-DP, HLA-DQ) and CD58 expression were promising post-expansion; however, MHC class II expression was lower post-antigen stimulation (Fig. 34A). CD25, CD70, and CD86 exhibited desired protein flow cytometry profiles post-activation (Fig. 34B).

[0362] Further, MSD data were collected to determine cytokine signaling receptor regulation by endogenous and inducible GM-CSF promoters. The cell line information is shown in Table 14 below. Secreted GM-CSF concentrations remained low through D10-D21, particularly for the CAR-TCR cell line NTX4H27 (Fig. 35A). Granzyme B and GM-CSF were secreted at high levels in unexpanded iPSC-derived CD8+ T cells (Fig. 35B). GM-CSF secretion post antigen-stimulation was specific and high. Granzyme B and GM-CSF secretion decreased significantly in expanded cells (Fig. 35B).

[0363] The choice of insertion site provides an additional mechanism to regulate transgene expression through differentiation. Site-specific insertion (SSI) into a region of open chromatin may provide increased expression of an inserted cytokine signaling receptor in comparison with insertion at a region of closed chromatin. Transgene expression from the CIITA locus exhibited the desired trend for dampening basal cytokine signaling receptor expression through differentiation (Fig. 36A). Shown is representative transgene insertion of an EBV TCR at the indicated loci (Fig. 36A). CIITA mRNA expression was suppressed through differentiation (Fig. 36B) and MHC Class II expression (HLA-DR, HLA-DP, HLA- DQ) was induced upon activation (Fig. 36C). CIITA and MHC Class II expression were promising through differentiation and post-activation / expansion and antigen stimulation. Insertion of a cytokine signaling construct with an inducible promoter at the CIITA locus may therefore further dampen basal activity of the cytokine signaling receptor (or receptor and secreted cytokine) through differentiation and further shield the inducible promoter from transcription factors that would otherwise induce expression of the construct.

[0364] Regulated cytokine signaling constructs were designed to include a candidate inducible promoter and the STAT3 / STAT5 cytokine signaling receptor construct pNT581. The inducible promoters evaluated were: an NFKB promoter containing five NFKB transcriptional response elements (TREs) and the IL2 minimal promoter ("NFKB", SEQ ID NO: 7), an engineered GM-CSF promoter containing a GM-CSF enhancer and GM-CSF promoter ("GM-CSFp", SEQ ID NO:8) and a synthetic antigen-responsive promoter containing eight antigen-responsive TREs and the IL2 minimal promoter ("ARP", Wei and Jensen, 2023, SEQ ID NO:9), as well as the control EFla constitutive promoter (Fig. 37).

[0365] Next, promoter activity was evaluated in LVV-transduced primary T cells. The stringency of expression was assessed on the basis of percent CD34 expression (Fig. 38A), and strength of expression was assessed on the basis of CD34 MFI (Fig. 38B). The background expression observed in the "Unstim" condition may be lower in an SSI context because LVV preferentially integrates into "open" loci. In addition, this experimental process required CD3 / CD28 stimulation for transduction, which may have contributed to background promoter activity.

[0366] The synthetic antigen-responsive promoter "ARP" and engineered GM-CSF promoter exhibited stringent inducibility of expression in iPSC-derived CD8+ T cells, as shown in Table 15. iPSC-derived CD8+ T cells were stimulated with Dynabeads™ (1:1 cell to bead ratio) in the presence of I L7 / 15 / 21 and I L18 and anti-41BB for 15 hours.

[0367] Following 36 hours of antigen stimulation under varying stimulation conditions (effector cells only, A549-CD19+ target cells, CD19- WT cells, or Dynabeads™), the dynamic range of expression of the NFKB promoter, the synthetic antigen-responsive promoter "ARP" and the engineered GM-CSF promoter (GM-CSFp) under antigen-dependent and cytokine-dependent simulation was evaluated (Fig. 39). The inducible promoters all showed a degree of antigen-dependent activation with reduced expression in effector cell-only culture or culture with CD19- A549 WT cells. Expression was highest in the NFKB promoter line in all conditions (Fig. 39).

[0368] Next, transduced cells were treated with A549-CD19+ target cells ("36h") and then removed from targets ("48h") (Fig. 40A-D). Separately, cells were treated with Dynabeads™ and anti-41BB("36h") and then continued in culture with Dynabeads™ and anti-41BB ("84h") (Fig. 40E-H). The expression of promoters was measured by CD34+ expression by flow cytometry. The EFla and NFKB promoters exhibited continued expression after target removal (Fig. 40A, B) and persistent expression (Fig. 40E, F). The synthetic antigen-responsive promoter and engineered GM-CSF promoter exhibited promising "off" dynamics (Fig. 40C, D) and persistence of expression (Fig. 40G, H).

[0369] The function of the inducible promoters was evaluated in an in vitro persistence assay in the presence of exogenous cytokines. Transduced iPSC-derived CD8+ T cells were cultured with A549-CD19+ target cells at a 4:1 E:T ratio with exogenous cytokines, and re-exposed to fresh target cells at a 1:2 split on days 3, 5, 10, and 12. The assay was ended on day 18. Tumor growth inhibition was comparable among the inducible promoters at the early stages of the assay, with a modest benefit of the NFKB promoter or synthetic antigen-responsive promoter ("ARP") observed during the last round of stimulation (Fig. 41A). The inducible promoter-driven engineered cytokine receptors augmented iPSC- derived CD8+ T cell proliferation under repeated antigen exposure in comparison to the constitutive promoter (Fig. 41B). The assay analysis was complicated by lower transduction of the inducible promoter constructs, resulting in a lower percentage of transduced cells following expansion in comparison to the constitutive promoter, yet antigen-dependent expansion was still observed (Fig. 41C). At the end of the persistence assay, the antigen-responsive promoter exhibited desirable antigendependent cytokine signaling receptor expression (Fig. 41D). Antigen-dependent expression was also observed for the NFKB and engineered GM-CSF promoters, with higher expression in the absence of antigen in comparison to the synthetic antigen-responsive promoter ("ARP").

[0370] In a repeat persistence assay conducted in the absence of exogenous cytokines, cytotoxicity profiles were similar between experimental conditions (Fig. 42A). The inducible promoters outperformed EFla in proliferation, with a decline in live cells observed for all conditions over the course of the assay (Fig. 42B). A higher percentage of CD56+CD8a- cells was observed at the end of the two-week persistence assay for the EFla promoter-transduced cells in comparison to the inducible promoter groups (Table 16).

[0371] The inducible promoters were also evaluated in a serial restimulation assay. Fold expansion over the course of the assay was comparable between the constitutive promoter and inducible promoter- transduced cells (Fig. 43A). The percentage of cells expressing the cytokine signaling receptor was initially low for cells transduced with the inducible promoter constructs and increased over the course of the assay (Fig. 43B). The inclusion of an inducible promoter provided improved target specificity, with reduced antigen-negative target cell killing observed for the three inducible promoter constructs (Fig. 43C). The constitutive promoter construct, EFla, also provided benefit in comparison to untransduced cells supplemented with exogenous cytokines (Fig. 43C).

[0372] Next, the bulk integration efficiencies of cytokine signaling receptor constructs into iPSCs were analyzed for several late stage-specific sites (GZMA, GZMB 3' and 5', GM-CSF, CD70 5' and 3', CD865' and 3', CD25 3' and 5', IL21R 3' and 5', IL12RB2 3' and 5', IGTB7, AGTRAP). For each site at least two guides were screened for SSI VCN in iPSCs after editing with either MAD7 or Cas9 nuclease as indicated; only the data point of the selected guide is shown (Fig. 44A). For insertion of the pNT581 construct, eleven out of sixteen sites met or surpassed a bulk integration threshold of 0.2 VCN (Fig. 44A).

[0373] For insertion of the cytokine signaling receptor construct (pNT581) and an inducible promoter, bulk integration efficiency was assessed following insertion into CIITA and AAVS1 via MAD7 or Cas9 nuclease as indicated (Fig. 44B). Among nine inducible promoter constructs and site combinations, five surpassed a bulk integration threshold of 0.2 VCN. Further, at the CIITA locus, five out of six constructs (pNT581 and indicated promoters) passed the bulk integration threshold of 0.2 VCN (Fig. 44C).

[0374] Lines with insertion and construct combinations above the VCN threshold were advanced into clone production. In total, eleven out of twelve pNT581 constructs resulted in successful clone production (Fig. 45). Clone production was assessed by determining the percentage of clones without additional copies (Fig. 45A, C) and the number of bi-al lei ic clones that passed all screening assays (Fig. 45B, D; criteria: VCN SSI approximately at or above 2, internal VCN approximately 2, 1 band on agarose gel at expected size). Seven out of eight endogenous promoters and four out of four inducible line productions were successfully completed with at least one clone without additional copy number. As expected, none of the designs showed any expression of cytokine receptors (CD34 extracellular domain expression) in iPSC (data not shown).

[0375] Next, combinations of the IL7R-2A-I L21 (pNT729) construct and endogenous or inducible promoters were also generated. For expression under an endogenous promoter, four out of six constructs generated with Cas9 nuclease passed the bulk integration threshold of 0.2 VCN (Fig. 45E). For expression under an inducible promoter at the CIITA locus, four out of five constructs generated with MAD7 nuclease surpassed the bulk integration threshold of 0.2 VCN (Fig. 45F).

[0376] Assessment of the endogenous or exogenous promoter, insertion site and cytokine signaling receptor combinations were based on five criteria. First, the cytokine signaling receptor expression should be minimal during in vitro differentiation, such as CD8+ T cell differentiation. Second, this minimal expression should be coupled with minimal effector molecule secretion during differentiation (such as, for example, granzyme, perforin, interferon secretion). Third, the cytokine receptor expression should be induced upon the appropriate stimulation, such as TCR-based stimulation or antigen-based stimulation. Fourth, the cytokine receptor expression and activation upon the appropriate stimulation should induce cell proliferation in the absence of exogenous cytokine support. Finally, the cytokine signaling receptor activity should also provide a functional benefit for the cells, such as, for example, improved and sustained tumor control in an in vitro assay in the absence of exogenous cytokine support.

[0377] An iPSC line with the pNT581 (IL7Ra-IL21R) cytokine signaling receptor construct ("CyR") inserted at the GZMA locus was assessed for CD8+ T cell differentiation capacity on immobilized Notch ligand as described above (Carpenedo et al., 2024). During iPSC to CD34 differentiation, lower yields and unusual CD34 hematopoietic progenitor cell phenotypic profiles were observed in three out of four GZMA-CyR SSI clones (Table 17). PCE0377 was the only clone that differentiated successfully without major complications. GZMA expression for this clone was 0.17% and 3.24xl05cells / well were harvested at day 9.

[0378] The GZMA CyR SSI line, indicated as "GZMA CyR", showed normal phenotypic progression during differentiation: greater than 95% CD5+CD7+, approximately 50% CD8a+CD8P+, a 2:1 ratio of CD8aP+ to CD8aa+, greater than 65% CD8a+, a similar CD4+ profile as parental control line (i.e. 50:50 split between CD4+CD8+ DP cells and CD4+ or CD8+ SP cells), and decreasing CD56+CD7+ alternative lineagepopulations over time (Fig. 46A-D). No myeloid commitment was observed at the Day 10 ProT stage (88.1% CD7hi and 4.38% CDllb+ for "GZMA CyR" line vs. 88.99% CD7hi and 2.93% CDllb+ for parental control "No CyR" line) and NK lineage markers remained low over time (Fig. 46E). Switching the cytokine signaling receptor insertion site to late-stage genes, such as granzyme A (GZMA) enabled lymphoid commitment and T-lineage specification to occur. The granzyme A cytokine signaling receptor line also displayed normal growth kinetics, comparable to parental control line (Fig. 46F).

[0379] The granzyme A cytokine signaling receptor line showed similar surface expression patterns for CD3, EB TCR and CD19 CAR as the parental control line (Fig. 47 A, B, "GZMA CyR" vs. "No CyR"). Low intracellular granzyme A and surface cytokine signaling receptor (CD34 extracellular domain) background expression was detected throughout the course of HBD differentiation (Fig. 47C, D). This suggests that low levels of cytokine signaling receptor expression / signaling are tolerable during in vitro differentiation without altering key lineage commitment steps.

[0380] Next, the in vitro function of the granzyme A cytokine signaling receptor construct line was assessed. The construct (GZMA SSI pNT581, indicated as "GZMA CyR") was expressed following coculture with antigen-positive target cells (Fig. 48A) or I L7 / I L15 / I L21 expansion protocol (Fig. 48B). Cells were either unexpanded and directly activated with A549 CD19+ target cells or activated by Dynabead™-based expansion in I L7 / I L15 / IL21 or IL7 alone. Following activation with target cells, construct expression ("GZMA CyR") correlated with granzyme A-positive cells (Fig. 48A). Following activation by Dynabead™-based expansion, construct expression ("GZMA CyR", as determined by CD34 extracellular domain expression by flow cytometry) was induced to a lesser extent with IL7 expansion as compared to the previous protocol (Fig. 48B). Further, pSTAT3 / pSTAT5 (after four hour cytokine washout) was elevated in construct positive cells (Fig. 48C, D).

[0381] Comparable tumor growth inhibition was observed with and without the cytokine signaling receptor construct at 1:2 and 2:1 E:T ratios in the presence and absence of exogenous cytokines (Fig. 49A). The initial proliferation was similar between cell lines in the presence of cytokines, possibly due to an initial lack of expression of the construct (Fig. 49B). After the construct was expressed by day 5, proliferation was lower in construct positive cells in the presence of cytokines as compared to the parental control line (Fig. 49C). Tumor growth inhibition was assessed during the initial 5 days of stimulation (unexpanded cells; Fig. 49D) and in a subsequent persistence assay (unexpanded cells; Fig. 49E). Cells were re-seeded at 4:1 E:T and split at 1:4 with fresh target cells for each following round. The parental control unexpanded line ("No CyR") failed to control tumor in the absence of cytokines (Fig.49E). The GZMA SSI unexpanded line ("GZMA CyR") initially continued to control tumor during the persistence assay without exogenous cytokines (Fig. 49E).

[0382] The in vitro function of expanded cells with and without cytokine signaling receptor at the GZMA locus was also assessed in the presence (Fig. 50A, B) or absence (Fig. 50C, D) of exogenous cytokines. The IL7 condition for expansion generated iPSC-derived CD8+ T cells with short-term persistence in absence of cytokines (Fig. 50D). The parental control line ("No CyR") was more potent than the cytokine signaling receptor ("GZMA CyR") line out of expansion (Fig. 50B, D).

[0383] iPSC lines containing the IL7RQ-IL21R hybrid cytokine signaling receptor (pNT581 construct) inserted at the granzyme B, CD25 or GM-CSF loci, under the control of the endogenous granzyme B, CD25, or GM-CSF promoters, were also evaluated. During the 21-day in vitro T cell differentiation as described above, granzyme B and the cytokine signaling receptor at the GZMB locus were expressed at day 17 and day 21, whereas CD25, GM-CSF, and their respective cytokine signaling receptors were not expressed (data not shown). The cytokine signaling receptor at the GM-CSF locus was expressed in approximately 6-9% of cells following CD8+ T cell activation but was no longer expressed during expansion in the absence of antigen stimulation (data not shown). At day 21 of differentiation, comparable CD5 and CD7 expression profiles, CD4 and CD8a expression profiles and CD56 expression to the parental control line was observed in all endogenous promoter lines; elevated NKp44 expression was observed in the granzyme B endogenous promoter line and GM-CSF endogenous promoter line in comparison to the parental control line (data not shown). Functional assessment by in vitro serial antigen stimulation did not demonstrate a proliferative or cytotoxic benefit of the cytokine signaling receptor under the control of the granzyme B, CD25 or GM-CSF endogenous promoter in the generated iPSC-derived CD8+ T cell lines (data not shown).

[0384] Next, the in vitro CD8 T cell differentiation, activation and expansion of iPSCs modified with exogenous inducible promoters were evaluated. iPSCs were modified with the IL7RQ-IL21R hybrid cytokine signaling receptor (pNT581 construct) inserted at the CIITA locus under the control of the exogenous NFKB inducible promoter (PCE434, PCE436), engineered GM-CSF inducible promoter (PCE438, "GM-CSFp") or inducible synthetic antigen-responsive promoter (PCE481, "ARP") as shown in Fig. 37. Expansion and viability in the NFKB inducible promoter lines were similar to parental control line (Fig. 51). Higher cumulative expansion and viability was observed for the engineered GM-CSF inducible promoter line ("GM-CSFp") in comparison to the parental control line; lower cumulative expansion was observed for the antigen-responsive promoter line ("ARP") in comparison to parental control line (Fig. 51).

[0385] Normal phenotypic attributes were observed in all inducible promoter lines on day 21 of differentiation, including CD5 and CD7 expression profiles (Fig. 52A), CD56 expression (Fig. 52B), CD4 and CD8a expression profiles (Fig. 52C) and CD8a and CD8P expression profiles (Fig. 52D). At day 21, NK lineage marker expression was also similar in all inducible promoter lines to the parental control line (Fig. 53).

[0386] Following differentiation, iPSC-derived CD8+ T cells were activated with CD3 / CD28 Dynabeads™ and exogenous cytokines for three days and expanded with IL7 for a further four days. NFKB inducible promoter lines had improved growth kinetics during activation and expansion (Fig. 54A). The engineered GM-CSF inducible promoter line ("GM-CSFp") and antigen-responsive promoter line ("ARP") had similar growth kinetics to the parental control line (Fig. 54A). All inducible promoter lines, irrespective of growth kinetics, followed expected activation marker (CD25) kinetics during activation and expansion (Fig. 54B). A higher CD25 MFI was observed in the NFKB inducible promoter lines during activation (days 0-3) and expansion (days 3-7) stages as compared to other inducible promoter lines tested (Fig. 54B). The NFKB inducible promoter lines displayed progressive incremental expression of the cytokine signaling receptor during activation and expansion (as assessed by CD34 extracellular domain expression, Fig. 55A), whereas the engineered GM-CSF inducible promoter line ("GM-CSFp") and antigen-responsive promoter line ("ARP") did not display robust cytokine signaling receptor expression (Fig. 55A, B).

[0387] The in vitro function of the inducible promoter iPSC-derived CD8+ T cell lines was evaluated in a persistence assay with CD19 target cells as described above. Proliferation after one round of antigen stimulation was improved in all inducible promoter lines in comparison to the parental control line in the absence of exogenous cytokines, with higher proliferation observed for the NFKB inducible promoter lines (Fig. 56A). Parental control line cells did not persist after three rounds of antigen stimulation in the absence of exogenous cytokines (Fig. 56B). In contrast, proliferation was observed for all inducible promoter lines at seven days, following the third round of antigen stimulation, with higher proliferation observed for the NFKB inducible promoter lines (Fig. 56B). The specificity of tumor growth inhibition was evaluated by comparing antigen-positive and antigen-negative target cell killing. In the absence of exogenous cytokines, on-target tumor growth inhibition (TGI) was reduced in the parental control line at lower effector to target cell ratios (E:T) (Fig. 57A). Specificity was better maintained for inducible promoter cell lines, comparable to parental control line supplemented with exogenous cytokines (Fig. 57A). Tumor growth inhibition was maintained over multiple rounds of antigen stimulation in the NFKBinducible promoter lines, comparable to the parental control line supplemented with exogenous cytokines (Fig. 57B).EXAMPLE 4: In vivo Function of Cytokine Signaling Constructs in iPSC-CD8+ Cells

[0388] The function of the cytokine signaling constructs were evaluated in an in vivo A549-CD19+ disseminated NSG mouse model. The study design is outlined in Table 18. iPSC-derived CD8+ CAR-TCR T cells (CD19 CAR and EBV TCR) were generated and transduced with the pNT549 cytokine signaling receptor construct ( I L7 Ra-I L21R) under the EFla constitutive promoter via lentiviral vector. Cells with the cytokine signaling receptor were compared to untransduced iPSC-derived CD8+ CD19 CAR-TCR T cells, as well as primary CD4+ and CD8+ (pan-T cell) CD19 CAR-T control cells. The disseminated A549 model was established by intravenous (i.v.) injection of tumor cells, with a further rechallenge of tumor cells injected at day 14. The cell characteristics prior to in vivo transplantation are shown in Table 19.

[0389] Tumor growth inhibition was assessed in vivo via bioluminescence imaging for an initial 14-day period, and then for a further 14 days upon rechallenge of tumor cells. Comparable tumor growth inhibition was observed in all groups receiving CAR-T cells at 14 days, with slower control of tumor growth observed for untransduced iPSC-derived CD8+ T cells in comparison to cells transduced with the pNT549 cytokine signaling receptor (Fig. 58). Transduction of the pNT549 cytokine signaling receptor induced significantly greater tumor growth inhibition upon rechallenge in comparison to untransducediPSC-derived CD8+ T cells (Fig. 58). No signs of acute toxicity were observed in any groups following cell infusion (data not shown).

[0390] Transduction of the cytokine signaling receptor resulted in increased detection of infused cells in the lungs at end-of-life, as assessed by human CD45 (hCD45) expression (Fig. 59A). Comparable CAR and CD3 expression in detected cells was observed for iPSC-derived CD8+ T cells transduced with the cytokine signaling receptor and primary CD4 / CD8 T cells (Fig. 59B, C). The high co-expression of CD3 and cytokine signaling receptor (as assessed by CD34 extracellular domain expression) in cytokine signaling receptor-transduced cells in the lung tissue indicates preferential persistence of cells expressing the cytokine signaling receptor, providing an advantage over untransduced cells (Fig. 60A). Detected iPSC- derived CD8+ T cells transduced with the cytokine signaling receptor were primarily CD8a+CD8P-, in contrast to primary CD4 / CD8 CAR-T cells that were primarily CD8a-CD4+ (Fig. 60B, C). Detected CAR+ iPSC-derived CD8+ T cells also had greater CD56 expression in comparison to detected primary CD4 / CD8 CAR-T cells (data not shown); these CD56+ cells were highly NKp44-negative.

[0391] The biodistribution of infused cells was also assessed at end-of-life in the lung, liver, spleen and bone marrow by digital droplet PCR (ddPCR). Without transduction of the cytokine receptor, no detection of infused iPSC-derived CD8+ CAR+ T cells was observed in these tissues (Fig. 61A, B). Transduction of the cytokine signaling receptor resulted in persistence of infused iPSC-derived CD8+ CAR+ T cells in all tissues (Fig. 61A), with comparable relative concentration in the bone marrow to primary CD4 / CD8 CAR-T cells (Fig. 61B, normalized to lung tissue).EXAMPLE 5: In vitro and In vivo Function of Cytokine Signaling Constructs in iPSC-CD8+ Cells

[0392] The in vitro performance of iPSC-derived CD8+ CAR-T cells, generated as described above in Example 1, with exogenous STAT3 and / or STAT5 cytokine support was investigated. STAT3 cytokine support was provided by IL21, and STAT5 cytokine support was provided by IL2 and IL7. iPSC-derived CD8+ CAR-T cells required STAT3 and STAT5 cytokine support for long-term persistence and cytotoxicity (Fig. 62A, 62B). In the presence of STAT5 and STAT3 cytokine support, iPSC-derived CD8+ CAR-T cells controlled A549 CD19+ tumor target cells over a duration of two weeks, while in the absence of cytokines, cells were unable to completely clear tumors (Fig. 62B). In the absence of cytokine support, iPSC-derived CD8+ CAR-T cells failed to proliferate over a duration of two weeks, while in the presence of cytokines that drive STAT5 and STAT3 signaling the cells demonstrated robust proliferation (Fig. 62C).

[0393] As described above in Example 2, a STAT5 engineered cytokine signaling receptor construct (pNT466) and a STAT3 / STAT5 engineered cytokine signaling receptor construct (pNT581) wereengineered into primary T cells by L\Z\Z transduction. Primary T cells transduced with CytR L\Z\Z induced pSTAT3 and pSTAT5 (Fig. 63A, 63B). Next, as described above, the engineered cytokine signaling receptor constructs were transduced into iPSC-derived CD8+ T cells. iPSC-derived CD8+ T cells alone or transduced with a STAT5-only CytR construct (pNT466) were unable to sustain tumor control in a serial rechallenge assay with A549 CD19+ target cells, whereas transduction with the dual STAT3 / STAT5 CytR construct (pNT581) enabled tumor control over repeated challenges (Fig. 63B). Similarly, only transduction with the dual STAT3 / 5 CytR construct (pNT581) enabled iPSC-derived CD8+ T cell proliferation in the absence of exogenous cytokines (Fig. 63C). iPSC-derived CD8+ T cells alone or those transduced with a STAT5-only CytR construct (pNT466) failed to proliferate over the course of the serial rechallenge assay (Fig. 63C).

[0394] As described above in Example 3, regulating engineered cytokine receptor through differentiation was shown to be critical for T-lineage commitment. Stringent regulated expression of the engineered cytokine receptor was found to be critical for preserving the phenotype of cells through differentiation. Ideally, engineered cytokine receptor expression would be repressed through differentiation and upregulated upon CD8SP activation (Fig. 64A, line "3"). Regulation of cytokine receptor expression was achieved through the combination of promoter choice and site of gene integration. The engineered cytokine receptor gene cassette was integrated into the site of a gene with the desired expression profile with a synthetic, inducible promoter or coupled to the endogenous promoter. Premature cytokine signaling receptor expression during differentiation resulted in suboptimal T-lineage commitment and emergence of off-target populations (Fig. 64A, line "1", insertion of the mutated IL7Ra cysteine-proline-threonine, CPT, motif (Shum et al., 2017) into the endogenous transmembrane domain of the IL7R gene or line "2", insertion of the pNT581 IL7Ra-IL21R hybrid cytokine signaling receptor at the GZMB locus under the control of the endogenous GZMB promoter). Cytokine signaling receptor expression at the CD34 HPC stage led to aberrant myeloid (CDllb) / lymphoid (CD7) lineage fate decisions, compromising subsequent T-cell progenitor specification and commitment stages of differentiation (Fig. 64A, line "1", Fig. 64B, 64C). Upregulation of CytR at intermediate developmental stages restored proper lymphoid commitment during T cell differentiation (Fig. 64A, line "2", Fig. 64D). However, expression of alternative innate lymphoid lineage markers (i.e. CD56, NKp44, CD16, etc.) was observed and could derail the course of downstream a T cell lineage commitment and maturation (Fig. 64E). Temporal regulation of CytR expression during later stages of T cell development (Fig. 64A, line "3", insertion of the pNT581 IL7Ra-IL21R hybrid cytokine signaling receptor at the CIITAlocus under the control of the exogenous NFKB promoter) was an essential step for the establishment of proper cell-fate decisions and generation of CD8aP T cells in vitro (Fig. 64F, 64G).

[0395] iPSC-derived CD34+ HPCs modified with a cytokine signaling receptor (as in Example 3, pNT581 IL7Ra-IL21R hybrid cytokine signaling receptor, inserted at the CIITA locus under the control of the exogenous NFKB promoter) were differentiated to CD5+CD7+ lymphoid-progenitor cells and matured into CD8aP+ T cells using a bead-based differentiation system as described above in Example 1 (Carpenedo et al., 2024). Assessment of key T-lineage markers (CD7, CD5, CD56, CD8a, CD8P) and CAR expression was performed throughout T cell differentiation using flow cytometry (Fig. 65A-65D, n=2 biological replicates). Representative flow cytometry plots of iPSC-derived T cells are shown in Figure 65E. Stage-specific induction of engineered cytokine receptor expression was demonstrated during T cell expansion (Fig. 65F). Engineered CytR surface expression was routinely monitored across multiple timepoints during T cell differentiation ("Early", "Mid", "Late") and expansion ("Post-Activation", "Exp. (Mid)" and "Exp. (End)") using flow cytometry (Fig. 65F, n=2 biological replicates).

[0396] Next, iPSC-derived CD8+ CAR-T cells harboring the engineered cytokine receptor demonstrated sustained effector function, long-term persistence, and antigen-dependent expression in vitro. In a single stimulation assay iPSC-derived CD8+ CAR-T cells with an engineered cytokine receptor (as in Example 3, pNT581 IL7Ra-IL21R hybrid cytokine signaling receptor, inserted at the CIITA locus under the control of the exogenous NFKB promoter) exhibited comparable tumor growth inhibition to cells that receive optimal exogenous cytokine support against A549-CD19 and Raji target cells across multiple effector to target ratios (E:T, Fig. 66A, 66B). Tumor growth inhibition (TGI) after a 5-day coculture at multiple E:Ts was determined by lncucyte®-based monitoring of target-cell fluorescence. Optimal cytokine secretion from iPSC-derived CD8+ CAR-T cells was also comparable between cells receiving exogenous cytokine support and cells modified with an engineered cytokine receptor measured at 48 hours after CD19 antigen stimulation, as measured by a Meso Scale Discovery (MSD®, CellCarta) assay (Fig. 66C). Engineered cytokine receptor constructs activated STAT5 or both STAT3 and STAT5 similar to exogenous cytokine support as measured by intracellular cytometry (Fig. 66D).

[0397] Cells were cultured in a serial tumor challenge assay with A549-CD19 target cells, as described in Example 2, or in a serial tumor challenge assay with CD19+ Raji target cells. In the Raji target cell assay, cells were cultured at an initial 4:1 E:T ratio, then split at a 1:2 ratio at 3, 5, 7, 10, and 12 days and replated onto fresh Raji target cells. In both serial tumor challenge assays, iPSC-derived CD8+ CAR-T cells engineered with the cytokine receptor exhibited comparable tumor cell control relative to cells with the optimal exogenous cytokine cocktail and primary blood-derived CD3 CAR-T cells (Fig. 67A, 67B). Incontrast, iPSC-derived CD8+ CAR-T cells lacking cytokine support were unable to control target cells over the duration of the assay (Fig. 67A, 67B). iPSC-derived CD8+ CAR-T cells with an engineered cytokine receptor also demonstrated sustained proliferation over the duration of the serial challenge assays, like that of cells supported with exogenous cytokines or primary CD3 T cells (Fig. 67C, 67D). iPSC-derived CD8+ CAR-T cells exhibited antigen-dependent cytokine receptor expression (Fig. 67E). Following initial A549 CD19+ target cell challenge, cytokine receptor expression was upregulated. Following target cell clearance, cytokine receptor expression returned to near baseline levels. Cytokine receptor expression remained low until T cells were rechallenged with target cells. Upon co-culture with A549 CD19- target cells, cytokine receptor expression was not upregulated beyond baseline levels (Fig. 67E). Consistent with antigen-dependent cytokine receptor expression, iPSC-derived CD8+ CAR-T cells did not demonstrate cytolytic activity when co-cultured with A549 CD19- target cells and cytotoxicity was specific to A549 CD19+ target cells in culture (Fig. 67F).

[0398] iPSC-derived CD8+ CAR-T cells harboring the engineered cytokine receptor induced complete tumor clearance comparable to primary CD3 CAR-T cells in vivo. A549 CD19+ cells co-expressing luciferase were implanted into 6-7-week-old NOD.Cg-PrkdcSCIDH2rgtmlWjl / SzJ (NSG) mice through lateral tail vein injection, three days before the first CAR T injections. A single dose of 3xl06iPSC-derived CD8+ CAR-T cells with a regulated cytokine receptor (as described in Example 3, pNT581 IL7Ra-IL21R hybrid cytokine signaling receptor, inserted at the CIIT / \ locus under the control of the exogenous NFKB promoter) were administered intravenously. A single dose of 3xl06primary CD3+ CAR-Ts was used as a positive control. A single maximum dose of lxlO7of iPSC-derived CD8+ CAR-T without cytokine support was administered as a control. On Day 14, all treatment groups were rechallenged with additional A549 CD19+ cells injected through the lateral tail vein. Tumor burden was monitored bi-weekly by bioluminescence imaging using MS® Lumina III through intraperitoneal injection of D-luciferin substrate in sterile phosphate-buffered saline. Quantification of tumor burden as region of interest (ROI) flux values are shown in Fig. 68A, with each dot representing mean of the treatment cohorts and error bars representing standard error of the mean (SEM). Percent change in body weight was monitored throughout the study for assessment of acute toxicity (Fig. 68B).

[0399] As demonstrated in this Example, iPSC-derived CD8+ CAR-T cells required both STAT5 and STAT3 for long-term cytotoxicity and proliferation. It was demonstrated that an engineered cytokine receptor can provide cell-intrinsic support, eliminating the need for toxic and costly exogenous cytokines and opening an opportunity to eliminate lymphodepleting chemotherapy. Leveraging genomic sites that are regulated through T cell differentiation combined with context-specific regulatory sequences to regulatecytokine receptor expression was critical to ensure appropriate T-lineage commitment. Engineered iPSC- derived CD8+ T cells demonstrated tumor growth inhibition and persistence in the absence of exogenous cytokines with efficacy comparable to primary CD3+ T cells. These findings support the development of scalable, potent, uniform, and persistent allogeneic CAR-T cell therapies for the treatment of oncology and autoimmune patients.

[0400] Although the disclosure has been described with reference to certain specific embodiments, various modifications thereof will be apparent to those skilled in the art. Any examples provided herein are included solely for the purpose of illustrating the disclosure and are not intended to limit the disclosure in any way. Any drawings provided herein are solely for the purpose of illustrating various aspects of the disclosure and are not intended to be drawn to scale or to limit the disclosure in any way. The scope of the claims appended hereto should not be limited by the preferred embodiments set forth in the above description, but should be given the broadest interpretation consistent with the present specification as a whole. The disclosures of all art recited herein are incorporated herein by reference in their entirety.DOCUMENTS CITED1. Baulu, E., et al. TCR-engineered T cell therapy in solid tumors: State of the art and perspectives. Sci. Adv. 9, eadf3700 (2023).2. 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US11,851,649 (2023).SEQ ID NO: 1 pNT549 (DNA)ATGACTATCCTTGGCACCACTTTTGGGATGGTGTTCTCACTGCTGCAAGTGGTTAGCGGCGAGAGCCTCGACAATA ACGGCACAGCTACCCCCGAACTGCCTACACAAGGCACGTTCAGTAACGTGAGCACCAACGTGAGCTACCAAGAGA CTACCACCCCTAGCACACTGGGCTCTACAAGCTTGCACCCAGTGAGCCAGCACGGCAACGAGGCCACCACCAATAT CACCGAGACAACCGTGAAGTTCACTAGCACCAGCGTGATCACCAGCGTGTACGGCAACACCAATTCTAGCGTGCAGTCCCAAACCTCCGTGATCTCCACCGTGTTCACCACACCCGCCAACGTGTCTACACCCGAAACCACACTGAAGCCTA GCCTTTCACCCGGCAACGTGAGCGATCTCAGCACAACCTCTACCAGCTTGGCCACTTCTCCAACCAAGCCCTACACC AGCTCTAGCCCTATCCTCAGCGATATCAAGGCCGAGATCAAGTGCAGCGGAATCCGGGAAGTGAAGCTGACCCAA GGGATTTGCCTCGAGCAAAACAAGACCAGCAGTTGCGCCGAGTTCAAGAAGGATAGAGGCGAGGGCCTTGCTAGAGTGCTGTGTGGCGAGGAACAAGCCGACGCTGACGCTGGAGCCCAAGTGTGTTCACTGCTGCTGGCACAGTCCG AAGTTAGACCCCAGTGCTTGCTGTTGGTCCTCGCAAACCGGACCGAAATCAGCAGCAAGCTGCAGCTGATGAAGA AGCACCAGTCCGATCTTAAGAAACTGGGAATCCTCGACTTCACCGAGCAAGACGTGGCAAGCCATCAGTCCTACA GCCAAAAGACACTGCTCACTTGCCCCACAATCTCTATCTTGAGCTTCTTTAGCGTGGCCCTTCTCGTGATCCTTGCTTGCGTGCTGTGGAAGAAGCGGATCAAGCCAATCGTGTGGCCCTCACTGCCCGATCACAAGAAGACCCTTGAGCACT TGTGCAAGAAGCCCCGCAAGAACCTCAACGTGTCCTTCAACCCCGAGTCCTTCCTCGACTGCCAAATCCATAGAGT GGACGATATCCAAGCCCGGGACGAAGTGGAAGGCTTCCTCCAAGATACTTTCCCACAGCAGCTGGAGGAATCCGA GAAGCAAAGGCTGGGCGGAGACGTGCAGTCTCCAAATTGCCCTTCCGAGGACGTGGTGATTACTCCCGAGAGCTTTGGAAGGGACTCTTCACTGACTTGCCTTGCCGGAAACGTGAGCGCTTGTGACGCCCCTATACTGAGCTCCTCTAGG TCACTGGACTGTAGGGAAAGCGGCAAGAACGGACCTCACGTGTACCAAGACCTCTTGCTGAGCCTTGGAACCACC AACTCTACACTGCCTCCACCCTTCAGCCTCCAAAGTGGGATCCTCACACTGAATCCAGTGGCCCAAGGCCAGCCTAT CTTGACTAGCCTCGGCAGCAATCAAGAGGAGGCCTACGTGACAATGAGCAGCTTCTACCAAAACCAGGGAGGCGGTGGGAGCGGAGGCGGTGGGAGCGGTGGACTTCCTTGGGGCGGAAGATCTCCAGGCGGAGTGTCTGAGTCTGA AGCCGGTTCTCCACTGGCCGGCCTGGACATGGATACCTTCGATTCTGGCTTCGTGGGCAGCGACTGTAGCAGCCCT GTGGAATGCGACTTCACAAGCCCTGGCGACGAGGGCCCACCTAGAAGCTATCTGAGACAGTGGGTCGTGATCCCT CCACCTCTGTCTAGTCCTGGACCTCAGGCTTCTTAGSEQ ID NO: 2 pNT549 (Protein)MTILGTTFGMVFSLLQVVSGESLDNNGTATPELPTQGTFSNVSTNVSYQETTTPSTLGSTSLHPVSQHGNEATTNITETT VKFTSTSVITSVYGNTNSSVQSQTSVISTVFTTPANVSTPETTLKPSLSPGNVSDLSTTSTSLATSPTKPYTSSSPILSDIKAEI KCSGIREVKLTQGICLEQNKTSSCAEFKKDRGEGLARVLCGEEQADADAGAQVCSLLLAQSEVRPQCLLLVLANRTEISSK LQLMKKHQSDLKKLGILDFTEQDVASHQSYSQKTLLTCPTISILSFFSVALLVILACVLWKKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDWITPESFGRDSSL TCLAGNVSACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAY VTMSSFYQNQGGGGSGGGGSGGLPWGGRSPGGVSESEAGSPLAGLDMDTFDSGFVGSDCSSPVECDFTSPGDEGPP RSYLRQWWIPPPLSSPGPQASSEQ ID NO: 3 pNT581 (DNA)ATGACTATCCTTGGCACCACTTTTGGGATGGTGTTCTCACTGCTGCAAGTGGTTAGCGGCGAGAGCCTCGACAATAACGGCACAGCTACCCCCGAACTGCCTACACAAGGCACGTTCAGTAACGTGAGCACCAACGTGAGCTACCAAGAGACTACCACCCCTAGCACACTGGGCTCTACAAGCTTGCACCCAGTGAGCCAGCACGGCAACGAGGCCACCACCAATATCACCGAGACAACCGTGAAGTTCACTAGCACCAGCGTGATCACCAGCGTGTACGGCAACACCAATTCTAGCGTGCAGTCCCAAACCTCCGTGATCTCCACCGTGTTCACCACACCCGCCAACGTGTCTACACCCGAAACCACACTGAAGCCTAGCCTTTCACCCGGCAACGTGAGCGATCTCAGCACAACCTCTACCAGCTTGGCCACTTCTCCAACCAAGCCCTACACCAGCTCTAGCCCTATCCTCAGCGATATCAAGGCCGAGATCAAGTGCAGCGGAATCCGGGAAGTGAAGCTGACCCAAGGGATTTGCCTCGAGCAAAACAAGACCAGCAGTTGCGCCGAGTTCAAGAAGGATAGAGGCGAGGGCCTTGCTAGAGTGCTGTGTGGCGAGGAACAAGCCGACGCTGACGCTGGAGCCCAAGTGTGTTCACTGCTGCTGGCACAGTCCGAAGTTAGACCCCAGTGCTTGCTGTTGGTCCTCGCAAACCGGACCGAAATCAGCAGCAAGCTGCAGCTGATGAAGAAGCACCAGTCCGATCTTAAGAAACTGGGAATCCTCGACTTCACCGAGCAAGACGTGGCAAGCCATCAGTCCTACAGCCAAAAGACACTGCTCACTTGCCCCACAATCTCTATCTTGAGCTTCTTTAGCGTGGCCCTTCTCGTGATCCTTGCTTGCGTGCTGTGGAAGAAGCGGATCAAGCCAATCGTGTGGCCCTCACTGCCCGATCACAAGAAGACCCTTGAGCACTTGTGCAAGAAGCCCCGCAAGAACCTCAACGTGTCCTTCAACCCCGAGTCCTTCCTCGACTGCCAAATCCATAGAGTGGACGATATCCAAGCCCGGGACGAAGTGGAAGGCTTCCTCCAAGATACTTTCCCACAGCAGCTGGAGGAATCCGAGAAGCAAAGGCTGGGCGGAGACGTGCAGTCTCCAAATTGCCCTTCCGAGGACGTGGTGATTACTCCCGAGAGCTTTGGAAGGGACTCTTCACTGACTTGCCTTGCCGGAAACGTGAGCGCTTGTGACGCCCCTATACTGAGCTCCTCTAGGTCACTGGACTGTAGGGAAAGCGGCAAGAACGGACCTCACGTGTACCAAGACCTCTTGCTGAGCCTTGGAACCACCAACTCTACACTGCCTCCACCCTTCAGCCTCCAAAGTGGGATCCTCACACTGAATCCAGTGGCCCAAGGCCAGCCTATCTTGACTAGCCTCGGCAGCAATCAAGAGGAGGCCTACGTGACAATGAGCAGCTTCTACCAAAACCAGGGAGGCGGTGGGAGCGGAGGCGGTGGGAGCAGCCCTGTGGAATGCGACTTCACAAGCCCTGGCGACGAGGGCCCACCTAGAAGCTATCTGAGACAGTGGGTCGTGATCCCTCCACCTCTGTCTAGTCCTGGACCTCAGGCTTCTTAGSEQ ID NO: 4 pNT581 (Protein)MTILGTTFGMVFSLLQVVSGESLDNNGTATPELPTQGTFSNVSTNVSYQETTTPSTLGSTSLHPVSQHGNEATTNITETTVKFTSTSVITSVYGNTNSSVQSQTSVISTVFTTPANVSTPETTLKPSLSPGNVSDLSTTSTSLATSPTKPYTSSSPILSDIKAEIKCSGIREVKLTQGICLEQNKTSSCAEFKKDRGEGLARVLCGEEQADADAGAQVCSLLLAQSEVRPQCLLLVLANRTEISSKLQLMKKHQSDLKKLGILDFTEQDVASHQSYSQKTLLTCPTISILSFFSVALLVILACVLWKKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDVVITPESFGRDSSL TCLAGNVSACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAY VTMSSFYQNQGGGGSGGGGSSPVECDFTSPGDEGPPRSYLRQWVVIPPPLSSPGPQASSEQ ID NO: 5 pNT729 (DNA)ATGACTATCCTTGGCACCACTTTTGGGATGGTGTTCTCACTGCTGCAAGTGGTTAGCGGCGAGAGCCTCGACAATAACGGCACAGCTACCCCCGAACTGCCTACACAAGGCACGTTCAGTAACGTGAGCACCAACGTGAGCTACCAAGAGACTACCACCCCTAGCACACTGGGCTCTACAAGCTTGCACCCAGTGAGCCAGCACGGCAACGAGGCCACCACCAATATCACCGAGACAACCGTGAAGTTCACTAGCACCAGCGTGATCACCAGCGTGTACGGCAACACCAATTCTAGCGTGCAGTCCCAAACCTCCGTGATCTCCACCGTGTTCACCACACCCGCCAACGTGTCTACACCCGAAACCACACTGAAGCCTAGCCTTTCACCCGGCAACGTGAGCGATCTCAGCACAACCTCTACCAGCTTGGCCACTTCTCCAACCAAGCCCTACACCAGCTCTAGCCCTATCCTCAGCGATATCAAGGCCGAGATCAAGTGCAGCGGAATCCGGGAAGTGAAGCTGACCCAAGGGATTTGCCTCGAGCAAAACAAGACCAGCAGTTGCGCCGAGTTCAAGAAGGATAGAGGCGAGGGCCTTGCTAGAGTGCTGTGTGGCGAGGAACAAGCCGACGCTGACGCTGGAGCCCAAGTGTGTTCACTGCTGCTGGCACAGTCCGAAGTTAGACCCCAGTGCTTGCTGTTGGTCCTCGCAAACCGGACCGAAATCAGCAGCAAGCTGCAGCTGATGAAGAAGCACCAGTCCGATCTTAAGAAACTGGGAATCCTCGACTTCACCGAGCAAGACGTGGCAAGCCATCAGTCCTACAGCCAAAAGACACTGCTCACTTGCCCCACAATCTCTATCTTGAGCTTCTTTAGCGTGGCCCTTCTCGTGATCCTTGCTTGCGTGCTGTGGAAGAAGCGGATCAAGCCAATCGTGTGGCCCTCACTGCCCGATCACAAGAAGACCCTTGAGCACTTGTGCAAGAAGCCCCGCAAGAACCTCAACGTGTCCTTCAACCCCGAGTCCTTCCTCGACTGCCAAATCCATAGAGTGGACGATATCCAAGCCCGGGACGAAGTGGAAGGCTTCCTCCAAGATACTTTCCCACAGCAGCTGGAGGAATCCGAGAAGCAAAGGCTGGGCGGAGACGTGCAGTCTCCAAATTGCCCTTCCGAGGACGTGGTGATTACTCCCGAGAGCTTTGGAAGGGACTCTTCACTGACTTGCCTTGCCGGAAACGTGAGCGCTTGTGACGCCCCTATACTGAGCTCCTCTAGGTCACTGGACTGTAGGGAAAGCGGCAAGAACGGACCTCACGTGTACCAAGACCTCTTGCTGAGCCTTGGAACCACCAACTCTACACTGCCTCCACCCTTCAGCCTCCAAAGTGGGATCCTCACACTGAATCCAGTGGCCCAAGGCCAGCCTATCTTGACTAGCCTCGGCAGCAATCAAGAGGAGGCCTACGTGACAATGAGCAGCTTCTACCAAAACCAGAGGGCTAAGAGGGGAAGTGGAGAAGGACGCGGATCACTGCTGACTTGTGGCGACGTGGAGGAAAACCCCGGCCCAATGAGGAGCAGCCCCGGCAATATGGAACGGATCGTGATTTGCTTGATGGTGATCTTCCTCGGGACACTGGTGCACAAATCTAGCTCCCAAGGACAAGACCGGCATATGATTAGAATGAGACAGCTGATCGATATTGTGGACCAGCTCAAGAACTACGTGAACGACCTCGTGCCCGAATTCTTGCCAGCTCCCGAAGATGTGGAGACAAACTGCGAGTGGAGCGCCTTTAGCTGCTTCCAAAAGGCCCAGCTGAAGAGCGCCAATACCGGGAACAACGAGAGGATTATTAACGTGTCAATTAAGAAGCTGAAGCGGAAACCCCCAAGCACAAACGCTGGCCGGAGGCAAAAGCATAGGCTGACTTGTCCCAGCTGTGATAGCT ACGAGAAGAAGCCTCCTAAGGAGTTCCTCGAGAGGTTTAAGAGCCTACTGCAAAAGATGATCCACCAGCATCTTA GCTCTAGAACCCACGGCAGCGAGGACAGCTAGSEQ ID NO: 6 pNT729 (Protein)MTILGTTFGMVFSLLQVVSGESLDNNGTATPELPTQGTFSNVSTNVSYQETTTPSTLGSTSLHPVSQHGNEATTNITETTVKFTSTSVITSVYGNTNSSVQSQTSVISTVFTTPANVSTPETTLKPSLSPGNVSDLSTTSTSLATSPTKPYTSSSPILSDIKAEIKCSGIREVKLTQGICLEQNKTSSCAEFKKDRGEGLARVLCGEEQADADAGAQVCSLLLAQSEVRPQCLLLVLANRTEISSKLQLMKKHQSDLKKLGILDFTEQDVASHQSYSQKTLLTCPTISILSFFSVALLVILACVLWKKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSSFYQNQRAKRGSGEGRGSLLTCGDVEENPGPMRSSPGNMERMCLMVIFLGTLVHKSSSQGQDRHMIRMRQL IDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTC PSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSSEQ ID NO: 75XN FKB (DNA)GGGGACTTTCCACTGGGGACTTTCCACTGGGGACTTTCCACTGGGGACTTTCCACTGGGGACTTTCCACTTCTAGA CATTTTGACACCCCCATAATATTTTTCCAGAATTAACAGTATAAATTGCATCTCTTGTTCAAGAGTTCCCTATCACTCT CTTTAATCACTACTCACAGTAACCTCAACTCCTGCSEQ ID NO: 8GM-CSFp (DNA)TTGCTGAGAGTGGCTGCAGTCTCGCTGCTGGATGTGCACATGGTGGTCATTCCCTCTGCTCACAGGGGCAGGGGTCCCCCCTTACTGGACTGAGGTTGCCCCCTGCTCCAGGTCCTGGGTGGGAGCCCATGTGAACTGTCAGTGGGGCAGGTCTGTGAGAGCTCCCCTCACACTCAAGTCTCTCACAGTGGCCAGAGAAGAGGAAGGCTGGAGTCAGAATGAGG CACCAGGGCGGGCATAGCCTGCCCAAAGGCCCCTGGGATTACAGGCAGGATGGGGAGCCCTATCTAAGTGTCTCC CACGCCCCACCCCAGCCATTCCAGGCCAGGAAGTCCAAACTGTGCCCCTCAGAGGGAGGGGGCAGCCTCAGGCCC ATTCAGACTGCCCAGGGAGGGCTGGAGAGCCCTCAGGAAGGCGAGTGGGTGGGCTGTCGGTTCTTGGAAAGGTT CATTAATGAAAACCCCCAAGCCTGACCACCTAGGGAAAAGGCTCACCGTTCCCATGTGTGGCTGATAAGGGCCAG GAGATTCCACAGTTCAGGTAGTTCCCCCGCCTCCCTGGCATTTTGTGGTCACCATTAATCATTTCCTCTGTGTATTTA AGAGCTCTTTTGCCAGTGAGCCCAGTACACAGAGAGAAAGGCTAAAGTTCTSEQ ID NO: 9 ARP (DNA)TCGAATGAGTCACATCGATCTCCGCCCCCTCTTCGAGGGGGCGGGGTCGAGGAGGAAAAACTCGAATGAGTCACA TCGACCCTTTGATCTTCGAGGGGACTTTCCGGGGTGGAGCAAGCGTGACAAGTCCACGTATGACCCGACCGACGA TATCGAAGCCTACGCGCTGAACGCCAGCCCCGATCGACCCCGCCCCCTCGATTTCCAAGAAATCGAATGACATCAT CTTTCGAATGACATCATCTTTCGAGGGGACTTTCCTCGAACTTCCTTCGAGGGGACTTTCCTCGAGGGGACTTTCCT CGAGGAGGAAAAACTCGAGTAGAGTCTAGACATTTTGACACCCCCATAATATTTTTCCAGAATTAACAGTATAAAT TGCATCTCTTGTTCAAGAGTTCCCTATCACTCTCTTTAATCACTACTCACAGTAACCTCAACTCCTGC

Claims

CLAIMSWe claim:

1. An engineered lymphoid cell comprising a STAT3 and STAT5 hybrid cytokine signaling receptor; wherein the hybrid cytokine signaling receptor is expressed at the cell surface and induces both STAT3 and STAT5 signaling in the engineered cell; and wherein the cell, in the absence of the hybrid cytokine signaling receptor, has attenuated STAT5 signaling in comparison to a primary CD8+ T cell.

2. The engineered lymphoid cell of claim 1, wherein the cell is a CD8+ T cell.

3. The engineered lymphoid cell of claim 1 or claim 2, wherein the hybrid cytokine signaling receptor is constitutively active.

4. The engineered lymphoid cell of any one of claims 1 to 3, wherein the hybrid cytokine signaling receptor is under the control of a promoter associated with T cell activation or late-stage T cell differentiation.

5. The engineered lymphoid cell of claim 4, wherein the promoter is an endogenous promoter.

6. The engineered lymphoid cell of claim 4 or claim 5, wherein the promoter is a granzyme A promoter, a granzyme B promoter, a granulocyte-macrophage colony-stimulating factor (GM-CSF) promoter, a CD25 promoter, a CD86 promoter or a CD70 promoter.

7. The engineered lymphoid cell of any one of claims 1 to 3, wherein the hybrid cytokine signaling receptor is under the control of an inducible promoter.

8. The engineered lymphoid cell of claim 7, wherein the inducible promoter comprises an antigen- responsive element activated by T cell antigen recognition.

9. The engineered lymphoid cell of claim 7, wherein the promoter is a NFKB promoter, a NFAT promoter or a granulocyte-macrophage colony-stimulating factor (GM-CSF) promoter.

10. The engineered lymphoid cell of any one of claims 1 to 9, wherein the STAT3 and STAT5 hybrid cytokine signaling receptor comprises an extracellular domain; a transmembrane domain coupled to the extracellular domain; a STAT5 signaling domain coupled to the transmembrane domain; and a STAT3 signaling domain attached to the STAT5 signaling domain via a linker.

11. The engineered lymphoid cell of claim 10, wherein the STAT5 signaling domain comprises an IL7 receptor alpha (IL7Ra) signaling domain.

12. The engineered lymphoid cell of claim 10 or claim 11, wherein the STAT3 signaling domain comprises an IL21 receptor signaling domain.

13. The engineered lymphoid cell of claim 12, wherein the IL21 receptor signaling domain comprises a tyrosine-containing motif.

14. The engineered lymphoid cell of any one of claims 10 to 13, wherein the transmembrane domain comprises an IL7 receptor alpha (IL7Ra) transmembrane domain.

15. The engineered lymphoid cell of claim 14, wherein the IL7 receptor alpha (IL7Ra) transmembrane domain is constitutively active.

16. The engineered lymphoid cell of claim 1, wherein the hybrid signaling receptor comprises a polypeptide encoded by SEQ ID NO: 2 or SEQ ID NO: 4.

17. The engineered lymphoid cell of any one of claims 1 to 9, wherein the STAT3 and STAT5 hybrid cytokine signaling receptor comprises an extracellular domain; a transmembrane domain coupled to the extracellular domain; a STAT3 signaling domain coupled to the transmembrane domain; and a STAT5 signaling domain attached to the STAT3 signaling domain via a linker.

18. The engineered lymphoid cell of any one of claims 1 to 17, wherein the engineered cell is derived from a pluripotent stem cell.

19. The engineered lymphoid cell of any one of claims 1 to 18, further comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR) and / or a nucleic acid sequence encoding an exogenous T cell receptor (TCR).

20. An engineered lymphoid cell comprising: an exogenous STAT5 signaling receptor; and an exogenous STAT3 signaling cytokine, wherein the STAT5 signaling receptor is expressed on the cell surface and the STAT3 signaling cytokine is secreted from the cell, and wherein the engineered cell, in the absence of the signaling receptor and the cytokine, has attenuated STAT5 signaling in comparison to a primary CD8+ T cell.

21. The engineered lymphoid cell of claim 20, wherein the STAT5 signaling receptor and the STAT3 signaling cytokine are inserted into the same insertion site of the engineered cell.

22. The engineered lymphoid cell of claim 20 or claim 21, wherein the STAT5 signaling receptor is constitutively active.

23. The engineered lymphoid cell of any one of claims 20 to 22, wherein the STAT5 signaling receptor and the STAT3 signaling cytokine are under the control of an endogenous or exogenous promoter associated with T cell activation or late-stage T cell differentiation.

24. The engineered lymphoid cell of any one of claims 20 to 22, wherein the STAT5 signaling receptor and the STAT3 signaling cytokine are under the control of an inducible promoter.

25. The engineered lymphoid cell of claim 20, wherein the STAT5 signaling receptor and the STAT3 signaling cytokine are expressed from a single construct, and wherein the construct comprises the nucleic acid sequence of SEQ ID NO: 5.

26. The engineered lymphoid cell of any one of claims 20 to 25, further comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR) and / or a nucleic acid sequence encoding an exogenous T cell receptor (TCR).

27. A STATS and STAT5 hybrid cytokine signaling receptor construct, comprising: a promoter; an extracellular domain; a transmembrane domain; a STATS signaling domain; and a STAT5 signaling domain; wherein the promoter is a synthetic promoter associated with T cell activation or late-stage T cell differentiation.

28. The construct of claim 27, wherein the promoter is a NFKB promoter or a granulocyte-macrophage colony-stimulating factor (GM-CSF) promoter.

29. An engineered CD8+ T cell population comprising a STATS and STAT5 hybrid cytokine signaling receptor, wherein the hybrid cytokine signaling receptor is expressed at the cell surface and induces both STATS and STAT5 signaling in the engineered cell, and wherein the engineered CD8+ T cell population is derived in vitro from a pluripotent stem cell.

30. The engineered CD8+ T cell population of claim 29, wherein the STATS and STAT5 cytokine signaling receptor comprises: an extracellular domain; a transmembrane domain coupled to the extracellular domain; a STAT5 signaling domain coupled to the transmembrane domain; and a STATS signaling domain attached to the STAT5 signaling domain via a linker.

31. The engineered CD8+ T cell population of claim 29 or claim 30 wherein the STATS and STAT5 hybrid cytokine signaling receptor is constitutively active.

32. The engineered CD8+ T cell population of any one of claims 29 to 31, wherein the STATS and STAT5 hybrid cytokine signaling receptor is under the control of a promoter associated with T cell activation, or late-stage T cell differentiation, or under the control of an inducible promoter.

33. The engineered CD8+ T cell population of claim 29, wherein the STATS and STAT5 hybrid cytokine signaling receptor is a polypeptide encoded by SEQ ID NO: 2 or SEQ ID NO: 4.

34. The engineered CD8+ T cell population of any one of claims 29 to 33, further comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR) and / or a nucleic acid encoding an exogenous T cell receptor (TCR).

35. A use of the engineered CD8+ T cell population of any one of claims 29 to 34 in the treatment of a disease or a condition in a subject.

36. An engineered CD8+ T cell population comprising an exogenous STAT5 signaling receptor; and an exogenous STATS signaling cytokine,wherein the STAT5 signaling receptor is expressed on the cell surface and the STAT3 signaling cytokine is secreted from the cell, and wherein the engineered CD8+ T cell population is derived in vitro from a pluripotent stem cell.

37. The engineered CD8+ T cell population of claim 36, wherein the STAT5 signaling receptor and the STAT3 signaling cytokine are inserted into the same insertion site of the engineered cell population.

38. The engineered CD8+ T cell population of claim 36 or claim 37, wherein the STAT5 signaling receptor is constitutively active.

39. The engineered lymphoid cell of any one of claims 36 to 38, wherein the STAT5 signaling receptor and the STAT3 signaling cytokine are under the control of a promoter associated with T cell activation, or late-stage T cell differentiation, or under the control of an inducible promoter.

40. The engineered CD8+ T cell population of any one of claims 36 to 39, wherein the STAT5 signaling receptor comprises an extracellular domain, a transmembrane domain and a STAT5 signaling domain.

41. The engineered CD8+ T cell population of claim 40, wherein the STAT5 signaling domain comprises an IL7 receptor alpha (IL7Ra) signaling domain.

42. The engineered CD8+ T cell population of any one of claims 36 to 41, wherein the STAT3 signaling cytokine is IL21.

43. The engineered CD8+ T cell population of claim 36, wherein the STAT5 signaling receptor and the STAT3 signaling cytokine are expressed from a single construct, and wherein the construct comprises the nucleic acid sequence of SEQ ID NO: 5.

44. The engineered CD8+ T cell population of any one of claims 36 to 43, further comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR) and / or a nucleic acid encoding an exogenous T cell receptor (TCR).

45. A use of the engineered CD8+ T cell population of any one of claims 36 to 44 in the treatment of a disease or a condition in a subject.

46. An induced pluripotent stem cell (iPSC) comprising a STAT3 and STAT5 hybrid cytokine signaling receptor construct, the construct comprising: a promoter; an extracellular domain; a transmembrane domain; a STAT5 signaling domain; and a STAT3 signaling domain coupled to the STAT5 signaling domain via a linker.

47. The induced pluripotent stem cell of claim 46, wherein the promoter is associated with T cell activation, or late-stage T cell differentiation, or is an inducible promoter.

48. The induced pluripotent stem cell of claim 46 or claim 47, wherein the STAT5 signaling domain is an IL7 receptor alpha (IL7Ra) signaling domain and the STAT3 signaling domain is an IL21 receptor signaling domain.

49. An induced pluripotent stem cell (iPSC) comprising a STAT3 and STAT5 hybrid cytokine signaling receptor construct, the construct comprising:a promoter; - a STAT5 signaling receptor; and a STATS signaling cytokine.

50. The induced pluripotent stem cell of claim 49, wherein the promoter is associated with T cell activation, or late-stage T cell differentiation, or is an inducible promoter.

51. The induced pluripotent stem cell of claim 49 or claim 50, wherein the STAT5 signaling receptor comprises an IL7 receptor alpha (IL7Ra) signaling domain, and the STATS signaling cytokine is IL21.

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