Engineered allogeneic cells and uses thereof

Abstract

The present disclosure provides engineered allogeneic cells, such as iPSC-derived CD8+ T cells, that include a combination of knock-in of exogenous factors and knockout or knockdown of endogenous genes to confer allogeneic protection. The engineered allogeneic cells exhibit increased viability in the presence of natural killer cells in comparison to non-engineered cells. The present disclosure also proves methods of use of the engineered allogeneic cells.

Description

Engineered Allogeneic Cells and Uses ThereofFIELD

[0001] The present invention relates generally to methods of engineering cells and uses thereof.BACKGROUND OF THE DISCLOSURE

[0002] Rejection by host T cells and natural killer (NK) cells is a critical consideration for allogeneic cell therapies, including adult donor-derived cell therapies and iPSC-derived cell therapies incorporating human leukocyte antigen (HLA) class I disruption. Previous approaches to mitigate patient T-cell mediated rejection include knockout (KO) of HLA class I and class II (e.g., Riolobos et al., 2013, Kagoya et al., 2020). However, a lack of consensus remains around the optimal design for avoiding missing-self NK responses resulting from HLA class I knockout. A common approach for NK cell evasion includes knock-in (KI) of HLA-E, a non-classical class I molecule with limited polymorphism that inhibits NK cells via binding to an inhibitory receptor NKG2A (Gornalusse et al., 2017). HLA-E can also stimulate NK cell subsets expressing the activating receptor NKG2C, leading to accelerated allogeneic clearance of HLA-E engineered cells.

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

[0004] In a first aspect of the disclosure, provided is an engineered cell comprising an exogenous HLA single-chain trimer construct (HLA-SCT), a knockout of the endogenous gene encoding CD58, a knockout of the endogenous gene encoding ICAM3 and a knockout of at least one gene encoding a protein or regulator of HLA class I or HLA class II complex.

[0005] In an embodiment of the first aspect of the disclosure, the knockout of the at least one gene encoding a protein or regulator of the HLA class I complex is a knockout of the gene encoding P2M.

[0006] In an embodiment of the first aspect of the disclosure, the knockout of the at least one gene encoding a protein or regulator of the HLA class II complex is a knockout of the gene encoding CIITA.

[0007] In an embodiment of the first aspect of the disclosure, the knockout of at least one gene encoding a protein or regulator of the HLA class I or HLA class II complex comprises a knockout of a gene encoding P2M and a knockout of a gene encoding CIITA.

[0008] In an embodiment of the first aspect of the disclosure, the HLA-SCT encodes a non-classical HLA molecule.

[0009] In an embodiment of the first aspect of the disclosure, the HLA-SCT encodes a molecule comprising HLA-E.

[0010] In an embodiment of the first aspect of the disclosure, the HLA-SCT nucleic acid sequence is SEQ ID NO:1 or SEQ ID NO:3.

[0011] In an embodiment of the first aspect of the disclosure, the HLA-SCT encodes a protein with the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4.

[0012] In an embodiment of the first aspect of the disclosure, the HLA-SCT is expressed at the GAPDH locus under the control of the endogenous GAPDH promoter.

[0013] In an embodiment of the first aspect of the disclosure, the HLA-SCT is expressed at the CIITA locus under the control of a CAG promoter.

[0014] In an embodiment of the first aspect of the disclosure, the insertion of the HLA-SCT into the cell is monoallelic or biallelic.

[0015] In an embodiment of the first aspect of the disclosure, the cell is an induced pluripotent stem cell (iPSC).

[0016] In an embodiment of the first aspect of the disclosure, the cell is derived from an iPSC.

[0017] In an embodiment of the first aspect of the disclosure, the cell derived from the iPSC is a T cell.

[0018] In an embodiment of the first aspect of the disclosure, the 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).

[0019] In an embodiment of the first aspect of the disclosure, the cell exhibits increased viability in the presence of natural killer (NK) cells compared to a non-engineered cell.

[0020] In an embodiment of the first aspect of the disclosure, the cell exhibits increased viability in the presence of natural killer (NK) cells compared to a cell lacking one or more of the exogenous HLA-SCT, the knockout of the endogenous gene encoding CD58, and the knockout of the endogenous gene encoding ICAM3.

[0021] In a second aspect of the disclosure, provided is a population of cells comprising the engineered cells of the first aspect of the disclosure. The engineered cells comprise an exogenous HLA single-chain trimer construct (HLA-SCT), a knockout of the endogenous gene encoding CD58, a knockout of the endogenous gene encoding ICAM3 and a knockout of at least one gene encoding a protein or regulator of HLA class I or HLA class II complex.

[0022] In a third aspect of the disclosure, provided is a use of the engineered cell of the first aspect of the disclosure in the treatment of a disease or a condition in a subject. The engineered cell comprises anexogenous HLA single-chain trimer construct (HLA-SCT), a knockout of the endogenous gene encoding CD58, a knockout of the endogenous gene encoding ICAM3 and a knockout of at least one gene encoding a protein or regulator of HLA class I or HLA class II complex.

[0023] In a fourth aspect of the disclosure, provided is a pharmaceutical composition comprising the engineered cell of the first aspect of the disclosure and a pharmaceutically acceptable carrier. The engineered cell comprises an exogenous HLA single-chain trimer construct (HLA-SCT), a knockout of the endogenous gene encoding CD58, a knockout of the endogenous gene encoding ICAM3 and a knockout of at least one gene encoding a protein or regulator of HLA class I or HLA class II complex.

[0024] In a fifth aspect of the disclosure, provided is an engineered cell comprising an exogenous HLA single-chain trimer construct (HLA-SCT), a knockout or knockdown of the endogenous gene encoding CD58, a knockout or knockdown of the endogenous gene encoding ICAM3 and a knockout or knockdown of at least one gene encoding a protein or regulator of HLA class I or HLA class II complex.

[0025] In an embodiment of the fifth aspect of the disclosure, the knockout or knockdown of the at least one gene encoding a protein or regulator of the HLA class I complex is a knockout of the gene encoding P2M.

[0026] In an embodiment of the fifth aspect of the disclosure, the knockout or knockdown of the at least one gene encoding a protein or regulator of the HLA class II complex is a knockout of the gene encoding CIITA.

[0027] In an embodiment of the fifth aspect of the disclosure, the knockout or knockdown of at least one gene encoding a protein or regulator of HLA class I or HLA class II complex comprises a knockout or knockdown of a gene encoding P2M and a knockout or knockdown of a gene encoding CIITA.

[0028] In an embodiment of the fifth aspect of the disclosure, the HLA-SCT encodes a non-classical HLA molecule.

[0029] In an embodiment of the fifth aspect of the disclosure the HLA-SCT encodes a molecule comprising HLA-E.

[0030] In an embodiment of the fifth aspect of the disclosure, the HLA-SCT nucleic acid sequence is SEQ ID NO:1 or SEQ ID NO:3.

[0031] In an embodiment of the fifth aspect of the disclosure, the HLA-SCT encodes a protein with the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4.

[0032] In an embodiment of the fifth aspect of the disclosure, the HLA-SCT is expressed at the GAPDH locus under the control of the endogenous GAPDH promoter.

[0033] In an embodiment of the fifth aspect of the disclosure, the HLA-SCT is expressed at the CIITA locus under the control of a CAG promoter.

[0034] In an embodiment of the fifth aspect of the disclosure, the insertion of the HLA-SCT into the cell is monoallelic or biallelic.

[0035] In an embodiment of the fifth aspect of the disclosure, the cell is an induced pluripotent stem cell (iPSC).

[0036] In an embodiment of the fifth aspect of the disclosure, the cell is derived from an iPSC.

[0037] In an embodiment of the fifth aspect of the disclosure, the cell derived from the iPSC is a T cell.

[0038] In an embodiment of the fifth aspect of the disclosure, the 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).

[0039] In an embodiment of the fifth aspect of the disclosure, the cell exhibits increased viability in the presence of natural killer (NK) cells compared to a non-engineered cell.

[0040] In an embodiment of the fifth aspect of the disclosure, the cell exhibits increased viability in the presence of natural killer (NK) cells compared to a cell lacking one or more of the exogenous HLA-SCT, the knockout or knockdown of the endogenous gene encoding CD58, the knockout or knockdown of the endogenous gene encoding ICAM3.

[0041] In a sixth aspect of the disclosure, provided is a population of cells comprising the engineered cells of the fifth aspect of the disclosure. The engineered cells comprise an exogenous HLA single-chain trimer construct (HLA-SCT), a knockout or knockdown of the endogenous gene encoding CD58, a knockout or knockdown of the endogenous gene encoding ICAM3 and a knockout or knockdown of at least one gene encoding a protein or regulator of HLA class I or HLA class II complex.

[0042] In a seventh aspect of the disclosure, provided is a use of the engineered cell of the fifth aspect of the disclosure in the treatment of a disease or a condition in a subject. The engineered cell comprises an exogenous HLA single-chain trimer construct (HLA-SCT), a knockout or knockdown of the endogenous gene encoding CD58, a knockout or knockdown of the endogenous gene encoding ICAM3 and a knockout or knockdown of at least one gene encoding a protein or regulator of HLA class I or HLA class II complex.

[0043] In an eighth aspect of the disclosure, provided is a pharmaceutical composition comprising the engineered cell of the fifth aspect of the disclosure and a pharmaceutically acceptable carrier. The engineered cell comprises an exogenous HLA single-chain trimer construct (HLA-SCT), a knockout or knockdown of the endogenous gene encoding CD58, a knockout or knockdown of the endogenous geneencoding ICAM3 and a knockout or knockdown of at least one gene encoding a protein or regulator of HLA class I or HLA class II complex.BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order that the subject matter may be readily understood, embodiments are illustrated by way of non-limiting examples in the accompanying drawings.

[0045] FIG. 1A is a heatmap of the number of HLA-E molecules per cell for unmodified primary CD8+ T cells (wild-type, WT), primary CD8+ T cells including a P2M knockout (P2M KO), or primary CD8+ T cells including a P2M knockout and a low-, mid-, or high-level expression of an HLA-E single-chain trimer (SCT) NT568 or NT363 (Table 3).

[0046] FIG. IB is a heatmap of the ratio of NKG2A to NKG2C expression on donor NK cells during coculture with primary CD8+ T cells. Unmodified (WT) primary CD8+ T cells, P2M knockout (P2M KO) primary CD8+ T cells, or P2M knockout primary CD8+ T cells with low-, mid- or high-level expression of NT568 or NT363 HLA-E SCT were cultured with NK cells from independent donors as indicated on the y- axis.

[0047] FIG. 1C is a heatmap of NK cell clearance, measured as reduction in the area under the curve (AUC) for cell viability of primary CD8+ T cells in NK cell co-culture. Unmodified (WT) primary CD8+ T cells, P2M knockout (P2M KO) primary CD8+ T cells, or P2M knockout primary CD8+ T cells with low-, mid- or high-level expression of NT568 or NT363 HLA-E SCT were cultured with NK cells from independent donors as indicated on the y-axis.

[0048] FIG. 2A is a graph of cell phenotype during CD8+ T cell differentiation for iPSC-derived TCR+ cells with and without CIITA knockout and modified to express an HLA-E SCT with lentivirus. Cell lines included: unmodified (PCE209 CIITA WT), CIITA knockout, NT363 HLA-E-transduced (PCE275 CIITA KO NT363) and two CIITA KO, NT568 HLA-E-transduced lines (PCE283 CIITA KO NT568, PCE285 CIITA KO NT568. Indicated timepoints represent days since initiation of differentiation from CD34+ cells.

[0049] FIG. 2B is a graph of cumulative fold expansion during CD8+ T cell differentiation for iPSC- derived TCR+ cells with and without CIITA knockout and modified to express an HLA-E SCT with lentivirus. Cell lines included: unmodified (PCE209 CIITA WT), CIITA knockout, NT363 HLA-E-transduced (PCE275 CIITA KO NT363 and two CIITA KO and NT568 HLA-E-transduced lines (PCE283 CIITA KO NT568, PCE285 CIITA KO NT568). Indicated timepoints represent days since initiation of differentiation from CD34+ cells.

[0050] FIG. 3A is a graph of HLA-E expression during CD8+ T cell differentiation for iPSC-derived TCR+ cells with and without CIITA knockout and modified to express an HLA-E SCT with lentivirus. Cell lines included: unmodified (PCE209 CIITA WT), CIITA knockout, NT363 HLA-E-transduced (PCE275 CIITA KO NT363) and two CIITA KO and NT568 HLA-E-transduced lines (PCE283 CIITA KO NT568, PCE285 CIITA KO NT568). Indicated timepoints represent days since initiation of differentiation from CD34+ cells.

[0051] FIG. 3B is a graph of the binding ratio of recombinant NKG2A-CD94 heterodimer and NKG2C- CD94 heterodimer to the HLA-E SCT constructs NT363 and NT568 on iPSC-derived CD8+ T cells (NTX6B10).

[0052] FIG. 4A is a graph of the HLA-E molecules per cell for expanded iPSC-derived CD8+ T cells, either unmodified (WT), P2M knockout (P2M KO), P2M knockout and knock-in of HLA-E SCT NT363 at the GAPDH locus (GAPDH HLA-E), P2M knockout and knock-in of HLA-E SCT NT363 at the 62M locus (P2M HLA-E), or P2M knockout and knock-in of HLA-E SCT NT363 at the CIITA locus (CIITA HLA-E SCT).

[0053] FIG. 4B is a graph of HLA-E molecules per cell pre- and post -antigen stimulation following expansion. Shown are iPSC-derived CD8+ T cells modified with P2M knockout and knock-in of HLA-E SCT NT363 at the GAPDH locus (GAPDH HLA-E), P2M knockout and knock-in of HLA-E SCT NT363 at the 62M locus (P2M HLA-E), or P2M knockout and knock-in of HLA-E SCT NT363 at the CIITA locus (CIITA HLA-E).

[0054] FIG. 4C is a graph of HLA-E molecules per cell pre- and post-antigen stimulation following expansion. Shown are iPSC-derived CD8+ T cells modified with P2M knockout and knock-in of HLA-E SCT NT363 at the GAPDH locus (GAPDH HLA-E) or P2M knockout and knock-in of HLA-E SCT NT363 at the CIITA locus (CIITA HLA-E).

[0055] FIG. 4D is a graph of the HLA-E molecules per cell for expanded iPSC-derived CD8+ T cells, either with P2M knockout and knock-in of HLA-E SCT NT363 at the GAPDH locus (GAPDH HLA-E), P2M knockout and knock-in of HLA-E SCT NT363 at the 62M locus (P2M HLA-E), or P2M knockout and knock- in of HLA-E SCT NT363 at the CIITA locus (CIITA HLA-E SCT). Lines and error bars represent mean ± standard deviation (SD) for n = 3 independent experiments; **P < 0.01, **P < 0.001 (ordinary one-way ANOVA with Tukey correction).

[0056] FIG. 5A is a graph of NK cell clearance of expanded iPSC-derived CD8+ T cells, either unmodified (NTX4B24 (WT)), P2M knockout (NTX4H27 (P2M KO)), P2M knockout and knock-in of HLA-E SCT NT363 at the GAPDH locus (NTX4I36 (GAPDH HLA-E NT363)), P2M knockout and knock-in of HLA-E SCT NT363 at the 62M locus (NTX4I37 (P2M HLA-E NT363)), or P2M knockout and knock-in of HLA-E SCT NT363 at the CIITA locus (NTX4I38 (CIITA HLA-E SCT NT363)). Clearance by three NK cell donors (individualsymbols) was evaluated as the percent reduction in the area under the curve (AUC) of cell viability in the NK cell co-culture (1:1 target cell to NK cell ratio) after 72 h.

[0057] FIG. 5B is a graph of NK cell clearance of expanded iPSC-derived CD8+ T cells, either unmodified (NTX4B24 (WT)), P2M knockout (NTX4H27 (P2M KO)), P2M knockout and knock-in of HLA-E SCT NT363 at the GAPDH locus (NTX4I36 (GAPDH HLA-E NT363)), P2M knockout and knock-in of HLA-E SCT NT363 at the 62M locus (NTX4I37 (P2M HLA-E NT363)), or P2M knockout and knock-in of HLA-E SCT NT363 at the CIITA locus (NTX4I38 (CIITA HLA-E SCT NT363)). Clearance by one NK cell donor with lower NKG2C expression (a relative NKG2A to NKG2C expression ratio of greater than 10, black circles) and three NK cell donors with higher NKG2C expression (a relative NKG2A to NKG2C expression ratio of less than 3, gray symbols) was evaluated as the percent reduction in the area under the curve (AUC) of cell viability in the NK cell co-culture (1:1 target cell to NK cell ratio) after 72 h.

[0058] FIG. 6A is a graph of cumulative fold proliferation during an in vitro persistence assay with A549 CD19+ target cells. Cell lines tested were cells modified with P2M knockout (NTX4H27 (P2M TCR)), P2M knockout and knock-in of HLA-E SCT NT568 at the CIITA locus (NTX4I32 (P2M TCR | CIITA HLA-E NT568)), P2M knockout and knock-in of HLA-E SCT NT363 at the 62M locus (NTX4I33 (CIITA TCR | P2M HLA-E NT568)), P2M knockout and knock-in of HLA-E SCT NT363 at the GAPDH locus (NTX4I36 (P2M TCR | GAPDH HLA-E NT363)), P2M knockout and knock-in of HLA-E SCT NT363 at the 62M locus (NTX4I37 (CIITA TCR | P2M HLA-E NT363)) or P2M knockout and knock-in of HLA-E SCT NT363 at the CIITA locus (NTX4I38 (P2M TCR | CIITA HLA-E NT363)).

[0059] FIG. 6B is a graph of fold proliferation between day 0 and day 14 of the persistence assay of Fig. 6A. Three technical replicates for each cell line are shown.

[0060] FIG. 6C is a graph of cytotoxicity during the in vitro persistence assay of Fig. 6A.

[0061] FIG. 7A is a graph of GAPDH enzymatic activity on day 13 of the persistence assay of Fig. 6 for iPSC-derived CD8+ T cells modified with P2M knockout (NTX4H27 P2M TCR), P2M knockout and knock- in of HLA-E SCT NT363 at the GAPDH locus (NTX4I36 P2M TCR | GAPDH HLA-E NT363) or 62M knockout and knock-in of HLA-E SCT NT363 at the CIITA locus (NTX4I38 P2M TCR | CIITA HLA-E NT363).

[0062] FIG. 7B is a graph of GAPDH mRNA expression level, as assessed by reverse transcriptase digital droplet polymerase chain reaction (RT-ddPCR) using a probe targeting Exons 2-3 of GAPDH. GAPDH mRNA expression was assessed on day 13 of the persistence assay of Fig. 6 for iPSC-derived CD8+ T cells modified with P2M knockout (NTX4H27 P2M TCR), P2M knockout and knock-in of HLA-E SCT NT363 at the GAPDH locus (NTX4I36 P2M TCR | GAPDH HLA-E NT363) or P2M knockout and knock-in of HLA-E SCT NT363 at the CIITA locus (NTX4I38 P2M TCR | CIITA HLA-E NT363).

[0063] FIG. 8A is a graph of expansion following activation of iPSC-derived CD8+ CAR-TCR T cells. Cell lines shown are iPSC-derived CD8+ CD19 CAR+ and EBV TCR+ T cells modified with P2M knockout (NTX4H27 (P2M TCR)), P2M knockout and biallelic knock-in of HLA-E SCT NT363 at the GAPDH locus (NTX4I36 (GAPDH 363)) or P2M knockout and biallelic knock-in of HLA-E SCT NT363 at the CIITA locus (NTX4I38 (CIITA 363)).

[0064] FIG. 8B is a graph of viability following activation of iPSC-derived CD8+ CAR-TCR T cells. Cell lines shown are iPSC-derived CD8+ CD19 CAR+ and EB TCR+ T cells modified with P2M knockout (NTX4H27 (P2M TCR)), P2M knockout and biallelic knock-in of HLA-E SCT NT363 at the GAPDH locus (NTX4I36 (GAPDH NT363)) or P2M knockout and biallelic knock-in of HLA-E SCT NT363 at the CIITA locus (NTX4I38 (CIITA NT363)).

[0065] FIG. 8C is a graph of HLA-E molecules per cell post-activation and expansion for iPSC-derived CD8+ CD19 CAR+ and EB TCR+ T cells modified with P2M knockout (NTX4H27 (P2M TCR)), P2M knockout and biallelic knock-in of HLA-E SCT NT363 at the GAPDH locus (NTX4I36 (GAPDH NT363)) or P2M knockout and biallelic knock-in of HLA-E SCT NT363 at the CIITA locus (NTX4I38 (CIITA NT363)).

[0066] FIG. 9A is a graph of tumor growth inhibition (TGI) in an A549 CD19+ target cell co-culture assay with varying effector to target cell (E:T) ratios as indicated. Cell lines shown are iPSC-derived CD8+ CD19 CAR+ and EBV TCR+ T cells modified with P2M knockout (NTX4H27 (P2M TCR)), P2M knockout and biallelic knock-in of HLA-E SCT NT363 at the GAPDH locus (NTX4I36 (GAPDH NT363)) or P2M knockout and biallelic knock-in of HLA-E SCT NT363 at the CIITA locus (NTX4I38 (CIITA NT363)).

[0067] FIG. 9B is a graph of proliferation over the co-culture assay of Fig. 9A at varying E:T as indicated. Cell lines shown are iPSC-derived CD8+ CD19 CAR+ and EBV TCR+ T cells modified with P2M knockout (NTX4H27 (P2M TCR)), P2M knockout and biallelic knock-in of HLA-E SCT NT363 at the GAPDH locus (NTX4I36 (GAPDH NT363)) or P2M knockout and biallelic knock-in of HLA-E SCT NT363 at the CIITA locus (NTX4I38 (CIITA NT363)).

[0068] FIG. 9C is a graph of GAPDH enzymatic activity in iPSC-derived CD8+ CD19 CAR+ and EBV TCR+ T cells modified with P2M knockout (NTX4H27 (P2M TCR)), P2M knockout and biallelic knock-in of HLA-E SCT NT363 at the GAPDH locus (NTX4I36 (GAPDH NT363)) or P2M knockout and biallelic knock-in of HLA-E SCT NT363 at the CIITA locus (NTX4I38 (CIITA NT363)).

[0069] FIG. 9D is a graph of GAPDH mRNA expression in iPSC-derived CD8+ CD19 CAR+ and EBV TCR+ T cells modified with P2M knockout (NTX4H27 (P2M TCR)), P2M knockout and biallelic knock-in of HLA-E SCT NT363 at the GAPDH locus (NTX4I36 (GAPDH NT363)) or P2M knockout and biallelic knock-in of HLA-E SCT NT363 at the CIITA locus (NTX4I38 (CIITA NT363)).

[0070] FIG. 10A is a graph of NK cell clearance of iPSC-derived CD8+ TCR-T cells, unmodified (WT) or modified with P2M knockout (P2M KO). Clearance by three NK cell donors (individual symbols) was evaluated as the percent reduction in the area under the curve (AUC) of cell viability in the NK cell coculture.

[0071] FIG. 10B is a graph of NK cell clearance of iPSC-derived CD8+ TCR-T cells, untransduced (WT) or transduced with HLA-SCT constructs NT363 or NT568. Clearance by three NK cell donors(individual symbols) was evaluated as the percent reduction in the area under the curve (AUC) of cell viability in the NK cell co-culture.

[0072] FIG. 10C is a graph of the expression of the HLA-SCT NT363 construct in LVV-transduced iPSC- derived CD8+ TCR-T cells during the NK cell co-culture assay of Fig. 9B ("+NK"), or in culture without NK cells ("-NK").

[0073] FIG. 10D is a graph of the expression of the HLA-SCT NT568 construct in LVV-transduced iPSC- derived CD8+ TCR-T cells during the NK cell co-culture assay of Fig. 9B ("+NK"), or in culture without NK cells ("-NK").

[0074] FIG. 11 is a graph of NK cell clearance of stimulated iPSC-derived CD8+ TCR-T cells without P2M knockout, from two independent donor background cell lines (NTX6B10 (WT)and NTX4B3 (WT)) and stimulated iPSC-derived CD8+ CAR-T cells with P2M knockout (NTX4H20 (P2M KO)). Clearance by three NK cell donors(individual symbols) was evaluated as the percent reduction in the area under the curve (AUC) of cell viability in the NK cell co-culture (1:1 target cell to NK cell ratio) after 96h.

[0075] FIG. 12 is a plot of expression of various cell markers, as indicated, on iPSC-derived CD8+ T cells and primary CD8+ T cells as detected by flow cytometry. The percent expression on a population basis is represented by the size of the circle, while the expression intensity is represented by the shading.

[0076] FIG. 13A is a graph of NK cell clearance of iPSC-derived CD8+ TCR-T cells, untransduced (UTD), transduced with the HLA-SCT construct NT363 (NT363 LVV), untransduced with PVR knockout by CRISPR-Cas9 (UTD PVR KO), or transduced with the HLA-SCT construct NT363 and with PVR knockout (NT363 LVV PVR KO). Clearance by three NK cell donors (individual symbols) was evaluated as the percent reduction in the area under the curve (AUC) of cell viability in the NK cell co-culture.

[0077] FIG. 13B is a graph of NK cell clearance of iPSC-derived CD8+ TCR-T cells alone or with the addition of a CD2 blocking antibody (+ CD2 NK block), a CDlla / CD18 blocking antibody (+CDlla / CD18 NK Block), or both CD2 and CDlla / CD18 blocking antibodies (+CD2 / CDlla / CD18 NK Block). Clearance by two NK cell donors in two independent experiments (individual symbols) was evaluated as the percent reduction in the area under the curve (AUC) of cell viability in the NK cell co-culture.

[0078] FIG. 14A is a representative flow cytometry plot of CD2 expression on CD3-CD56+ NK cells by cytomegalovirus (CMV) serostatus of peripheral blood mononuclear cell (PBMC) donors. FMO, fluorescence minus one gating control.

[0079] FIG. 14B is a graph of the percentage of CD2hi CD3-CD56+ NK cells, as assessed by flow cytometry, for cells sourced from CMV-positive (CMV+) or CMV-negative (CMV-) donors (n = 5-6 donors per serostatus). Lines represent mean ± SD.

[0080] FIG. 14C is a graph of CDlla Mean Fluorescence Intensity (MFI) and the percentage of NKG2A- NKG2C+ NK cells, as assessed by flow cytometry, in 11 NK cell donors (black dots represent independent donors).

[0081] FIG. 14D is a graph of CD18 MFI and the percentage of NKG2A-NKG2C+ NK cells, as assessed by flow cytometry, in 11 NK cell donors (black dots represent independent donors).

[0082] FIG. 15A is a graph of HLA-E, P2M and CD58 expression on iPSC-derived CD8+ T cells after editing. Cells were unmodified or modified to express the HLA-E SCT construct NT363 or NT568 (HLA-E NT363 KI, HLA-E NT568 KI), and / or to knockout (KO) one or both of P2M and CD58 as indicated ("+" = including modification, = lacking modification).

[0083] FIG. 15B is a flow cytometry plot of residual CD58 expression 7 days after editing.

[0084] FIG. 15C is a graph of ICE analysis of InDei species for the CD58 sgRNA.

[0085] FIG. 16 is a graph of NK cell clearance of iPSC-derived CD8+ T cells, unmodified or modified to express the HLA-E SCT construct NT363 or NT568 (NT363 KI, NT568 KI), and / or to knockout (KO) one or both of P2M and CD58 as indicated ("+" = including modification,= lacking modification). Clearance by NK cells from independent donors (individual symbols) was evaluated as the percent reduction in the area under the curve (AUC) of cell viability in the NK cell co-culture after 72 h. The lower solid line represents the clearance of P2M-intact cells (first column), and the upper dashed line represents the clearance of P2M-deficient cells (second column).

[0086] FIG. 17A is a graph of CD54 and CD58 expression on iPSC-derived CD8+ T cells after editing. Cells were modified to knockout (KO) one or both of CD54 and CD58 as indicated ("+" = including modification, = lacking modification).

[0087] FIG. 17B is a graph of CD54, ICAM2 and ICAM3 expression on iPSC-derived CD8+ T cells after editing. Cells were modified to knockout (KO) one or more of CD54, ICAM2 and ICAM3 as indicated ("+" = including modification, = lacking modification).

[0088] FIG. 18A is a graph of NK cell clearance of iPSC-derived CD8+ T cells, unmodified or modified to knockout (KO) one or both of CD54 and CD58 as indicated ("+" = including modification, = lackingmodification) and with the addition of a CD58 blocking antibody (aCD58 Block). Clearance by three NK cell donors (individual symbols) was evaluated as the percent reduction in the area under the curve (AUC) of cell viability in the NK cell co-culture after 48 h.

[0089] FIG. 18B is a graph of NK cell clearance of iPSC-derived CD8+ T cells, unmodified or modified to knockout (KO) one or more of CD54, ICAM2 and ICAM3 as indicated ("+" = including modification,= lacking modification). Clearance by three NK cell donors (individual symbols) was evaluated as the percent reduction in the area under the curve (AUC) of cell viability in the NK cell co-culture after 48 h.

[0090] FIG. 19A is a flow cytometry plot of HLA-E expression for untransduced iPSC-derived CD8+ T cells or cells transduced with HLA-E SCT NT363 L\Z\Z, with knockout (KO) of one or more of P2M, CD58 and ICAM3 by CRISPR-Cas9 editing as indicated ("+" = including modification, = lacking modification).

[0091] FIG. 19B is a graph of editing efficiency of iPSC-derived CD8+ T cells, as assessed by expression of P2M, CD58 and ICAM3. Cells were untransduced or transduced with HLA-E SCT NT363, with knockout (KO) of one or more of P2M, CD58 and ICAM3 by CRISPR-Cas9 editing as indicated ("+" = including modification, = lacking modification).

[0092] FIG. 20A is a graph of NK cell clearance of iPSC-derived CD8+ T cells, untransduced or transduced with HLA-E SCT NT363 (HLA-E KI), with knockout (KO) of one or more of P2M, CD58 and ICAM3 by CRISPR-Cas9 editing, as indicated ("+" = including modification,= lacking modification). Clearance by three NK cell donors (individual symbols) was evaluated as the percent reduction in the area under the curve (AUC) of cell viability in the NK cell co-culture after 72 h. The upper solid line represents the level of clearance of P2M-deficient cells (second column), while the lower dashed line represents the level of clearance of P2M-intact cells (first column).

[0093] FIG. 20B is a graph of NK cell clearance of iPSC-derived CD8+ T cells transduced with HLA-E SCT NT363 (P2M KO + HLA-E KI), with knockout (KO) of one or more of P2M, CD58 and ICAM3 as indicated, relative to the clearance of P2M KO-only cells. The difference between the added effects of separate CD58 or ICAM3 KO (Sum of Parts) and the clearance observed in the combined HLA-E KI, P2M KO, CD58 KO and ICAM3 KO group (Whole) was also reported.

[0094] FIG. 21A is a graph of iPSC-derived CD8+ TCR-T cell proliferation in a three-way co-culture assay with A375 target cells at a 2:1 iPSC-CD8+ to target cell ratio and NK cells at a range of NK to iPSC-CD8+ cell ratios. Proliferation was assessed on day 5 of co-culture for unmodified iPSC-derived CD8+ TCR-T cells and cells modified with P2M knockout or P2M and CD58 knockout and transduction with an HLA-E SCT (iPSC TCR-T P2M / CD58KO HLA-E KI).

[0095] FIG. 21B is a graph of iPSC-derived CD8+ TCR-T cell proliferation in a three-way co-culture assay with A375 target cells at a 1:1 iPSC-CD8+ to target cell ratio and NK cells at a range of NK to iPSC-CD8+ cell ratios. Proliferation was assessed on day 5 of co-culture for unmodified iPSC-derived CD8+ TCR-T cells and cells modified with P2M knockout or P2M and CD58 knockout and transduction with an HLA-E SCT (iPSC TCR-T P2M / CD58KO HLA-E KI).

[0096] FIG. 22A is a graph of iPSC-derived CD8+ CAR-T cell cytotoxicity in a three-way co-culture assay with NK cells and Raji target cells at a 4:4:1 NK cell to iPSC CAR-T cell to Raji cell ratio. iPSC-derived CD8+ CAR-T cells were unmodified or modified with P2M knockout (KO) or P2M and CD58 KO and transduction with an HLA-E SCT (iPSC CAR-T P2M / CD58KO + HLA-E KI).

[0097] FIG. 22B is a graph of proliferation of iPSC-derived CD8+ CAR-T cells in a three-way co-culture assay with NK cells and Raji target cells at day 7, at varying cell ratios as indicated. iPSC-derived CD8+ CAR-T cells were unmodified or modified with P2M knockout (KO) or P2M and CD58 KO and transduction with an HLA-E SCT (iPSC CAR-T P2M / CD58KO + HLA-E KI).

[0098] FIG. 23A is a graph of HLA-E molecules per cell for iPSC-derived CD8+ CAR-T cells, unmodified or modified with transduction of an HLA-E SCT (HLA-E KI), P2M knockout (KO), CD58 KO and / or ICAM3 KO as indicated ("+" = including modification,= lacking modification).

[0099] FIG. 23B is a graph of editing efficiency, as measured by CD58 and ICAM3 expression by flow cytometry for iPSC-derived CD8+ CAR-T cells modified with transduction of an HLA-E SCT (HLA-E KI), P2M knockout (KO), CD58 KO and / or ICAM3 KO as indicated ("+" = including modification, = lacking modification).

[0100] FIG. 24 is a graph of NK cell clearance of iPSC-derived CD8+ CAR-T cells, unmodified or modified with transduction of an HLA-E SCT (KHLA-E KI), P2M knockout (KO), CD58 KO and / or ICAM3 KO as indicated ("+" = including modification, = lacking modification). Clearance by three NK cell donors (individual symbols) was evaluated as the percent reduction in the area under the curve (AUC) of cell viability in the NK cell co-culture after 72 h.

[0101] FIG. 25A is a graph of tumor growth inhibition (TGI) of unmodified, CD58 KO, or CD58 and ICAM3 KO iPSC-derived CD8+ CAR-T cells. Specificity of TGI was assessed against CD19+ Raji cells and CD19 KO Raji cells at varying effector to target (E:T) cell ratios as indicated.

[0102] FIG. 25B is a graph of proliferation of unmodified, CD58 KO, or CD58 and ICAM3 KO iPSC-derived CD8+ CAR-T cells during the co-culture assay of Fig. 23A at varying E:T ratios as indicated.

[0103] FIG. 25C is a graph of cell phenotype pre- and post-assay for unmodified, CD58 KO, or CD58 and ICAM3 KO iPSC-derived CD8+ CAR-T cells.

[0104] FIG. 26A is a graph of CD58 expression intensity (MFI) on iPSC-derived CD8+ CAR-TCR T cells modified as indicated. Cells modified with CD58 KO (+CD58 KO) or CD58 and ICAM3 KO (+CD58 / ICAM3 KO) also included insertion of a TCR at the 62M locus (P2M TCR) and insertion of an HLA-E SCT at the GAPDH locus (P2M TCR GAPDH HLA-E).

[0105] FIG. 26B is a graph of CD58 and ICAM3 expression on iPSC-derived CD8+ CAR-TCR T cells modified as indicated. Cells modified with CD58 KO (+CD58 KO) or CD58 and ICAM3 KO (+CD58 / ICAM3 KO) also included insertion of a TCR at the 62M locus (P2M TCR) and insertion of an HLA-E SCT at the GAPDH locus (P2M TCR GAPDH HLA-E).

[0106] FIG. 26C is a graph of HLA class II expression on iPSC-derived CD8+ CAR-TCR T cells modified as indicated. Cells modified with CD58 KO (+CD58 KO) or CD58 and ICAM3 KO (+CD58 / ICAM3 KO) also included insertion of a TCR at the 62M locus (P2M TCR) and insertion of an HLA-E SCT at the GAPDH locus (P2M TCR GAPDH HLA-E).

[0107] FIG. 26D is a graph of HLA-E molecules per cell for iPSC-derived CD8+ CAR-TCR T cells modified as indicated. Cells modified with CD58 KO (+CD58 KO) or CD58 and ICAM3 KO (+CD58 / ICAM3 KO) also included insertion of a TCR at the 62M locus (P2M TCR) and insertion of an HLA-E SCT at the GAPDH locus (P2M TCR GAPDH HLA-E).

[0108] FIG. 27 is a graph of NK cell clearance of iPSC-derived CD8+ CAR-TCR T cells modified as indicated. Cells modified with CD58 KO (NTX4K36, (+CD58 KO)) or CD58 and ICAM3 KO (NTX4M36 (+CD58 / ICAM3 KO)) also included insertion of a TCR at the 62M locus (NTX4H27 (P2M TCR)) and insertion of an HLA-E SCT at the GAPDH locus (NTX4I36 (P2M TCR GAPDH HLA-E)). Clearance by one NKG2A-prominent donor (black symbol, "NKG2A / NKG2C >10 donor") and five NKG2C-prominent donors (gray symbols, "NKG2A / NKG2C <3 donors") was evaluated as the percent reduction in the area under the curve (AUC) of cell viability in the 1:1 NK to iPSC-derived cell ratio co-culture after 72 h.

[0109] FIG. 28A is a graph of CD58 expression in differentiated iPSC lines modified with insertion of an HLA-E SCT at the CIITA or GAPDH locus and knockout of CD58 or CD58 and ICAM3, as indicated. All cell lines included a CD19 CAR and an EBV TCR.

[0110] FIG. 28B is a graph of ICAM3 expression in differentiated iPSC lines modified with insertion of an HLA-E SCT at the CIITA or GAPDH locus and knockout of CD58 or CD58 and ICAM3, as indicated. All cell lines included a CD19 CAR and an EBV TCR.

[0111] FIG. 28C is a graph of intensity of HLA-E expression (MFI) on differentiated iPSC lines modified with insertion of an HLA-E SCT at the CIITA or GAPDH locus and knockout of CD58 or CD58 and ICAM3, as indicated. All cell lines included a CD19 CAR and an EBV TCR.

[0112] FIG. 29A is a graph of tumor growth inhibition (TGI) for iPSC-derived CD8+ CAR-T cells cocultured with Raji CD19- cells ("KO") or Raji CD19+ tumor cells ("WT"). iPSC-derived cells were modified with: insertion of a TCR at the 62M locus (PCE309 (P2M TCR)); insertion of a TCR at the 62M locus and insertion of an HLA-E SCT at the GAPDH locus (PCE402 (+GAPDH HLA-E)); insertion of a TCR at the 62M locus and insertion of an HLA-E SCT at the CIITA locus (PCE425, (+CIITA HLA-E)), insertion of a TCR at the 62M locus, insertion of an HLA-E SCT at the GAPDH locus and CD58 KO or CD58 and ICAM3 KO (PCE496 (+GAPDH HLA-E | CD58 KO)), (PCE488 (+GAPDH HLA-E | CD58 / ICAM3 KO)); or insertion of a TCR at the 62M locus, insertion of an HLA-E SCT at the CIITA locus and CD58 KO or CD58 and ICAM3 KO (PCE539 (+CIITA HLA-E | CD58 KO)), (PCE545 (+CIITA HLA-E | CD58 / ICAM3 KO)).

[0113] FIG. 29B is a graph of Raji cell proliferation during co-culture with iPSC-derived CD8+ CAR-T cells, for either Raji CD19- cells ("KO") or Raji CD19+ tumor cells ("WT"). iPSC-derived cells were modified with: insertion of a TCR at the 62M locus (PCE309 (P2M TCR)); insertion of a TCR at the 62M locus and insertion of an HLA-E SCT at the GAPDH locus (PCE402 (+GAPDH HLA-E)); insertion of a TCR at the 62M locus and insertion of an HLA-E SCT at the CIITA locus (PCE425, (+CIITA HLA-E)); insertion of a TCR at the 62M locus, insertion of an HLA-E SCT at the GAPDH locus and CD58 KO or CD58 and ICAM3 KO (PCE496 (+GAPDH HLA-E | CD58 KO)), (PCE488 (+GAPDH HLA-E | CD58 / ICAM3 KO)); or insertion of a TCR at the 62M locus, insertion of an HLA-E SCT at the CIITA locus and CD58 KO or CD58 and ICAM3 KO (PCE539 (+CIITA HLA-E | CD58 KO)), PCE545 (+CIITA HLA-E | CD58 / ICAM3 KO)).

[0114] FIG. 29C is a graph of iPSC-derived CD8+ CAR-T cell proliferation, modified as indicated, during co-culture with Raji CD19- cells ("KO") or Raji CD19+ tumor cells ("WT"). iPSC-derived cells were modified with: insertion of a TCR at the 62M locus (PCE309 (P2M TCR)); insertion of a TCR at the 62M locus and insertion of an HLA-E SCT at the GAPDH locus (PCE402 (+GAPDH HLA-E)); insertion of a TCR at the 62M locus and insertion of an HLA-E SCT at the CIITA locus (PCE425, (+CIITA HLA-E)); insertion of a TCR at the 62M locus, insertion of an HLA-E SCT at the GAPDH locus and CD58 KO or CD58 and ICAM3 KO (PCE496 (+GAPDH HLA-E | CD58 KO)), PCE488 (+GAPDH HLA-E | CD58 / ICAM3 KO)); or insertion of a TCR at the 62M locus, insertion of an HLA-E SCT at the CIITA locus and CD58 KO or CD58 and ICAM3 KO (PCE539 (+CIITA HLA-E | CD58 KO)), PCE545 (+CIITA HLA-E | CD58 / ICAM3 KO)).

[0115] FIG. 30A is a graph of NK cell clearance of expanded clonal iPSC-derived CD8+ CAR-TCR T cells. NTX4J26 and NTX4H27 cells represent P2M-intact (CIITA TCR) and P2M-deficient (P2M TCR) controls, respectively, whereas NTX4I36, NTX4K36, and NTX4M36 lines are P2M-deficient experimental groups that have insertion of an HLA-E SCT at the GAPDH locus without (P2M TCR | GAPDH HLA-E) and with additional layering of a CD58 KO alone (P2M TCR | GAPDH HLA-E | CD58KO) or CD58 and ICAM3 KO(P2M TCR | GAPDH HLA-E | CD58 / ICAM3 KO) as indicated. Clearance by 10 NK donors (denoted by different symbols) at a 1:1 NK to iPSC-derived cell ratio was evaluated as the percent reduction in the area under the curve (AUC) of cell viability after 96 h. Lines represent the mean; n = 10 unique NK donors; ns > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (RM one-way ANOVA with Holm- Sidak correction). Unique NK donors colored according to their CMV serostatus (gray, CMV+; black, CMV-).

[0116] FIG. 30B is a graph of the ratio of % NKG2A+NKG2C- expression over % NKG2A-NKG2C+ expression on CD3-CD56+ NK cells from 10 donors used in the NK clearance assay of Fig. 30A, as measured by flow cytometry. Line represents the mean; n = 10 unique NK donors denoted by different symbols and colored according to their CMV serostatus as in Fig. 30C (gray, CMV+; black, CMV-).

[0117] FIG. 31 is a schematic of mechanisms of allogeneic cell clearance by patient NK cells, CD8+ T cells, CD4+ T cells, and B cells, and cell engineering strategies to address allogeneic cell clearance. Specific engineering approaches to mediate NK cell clearance of 2M KO iPSC-derived T cells are shown in the right-hand insert.

[0118] FIG. 32A is a flow cytometry plot of HLA-ABC (Class I) expression on expanded iPSC-derived T cells with clonal P2M KO, CIITA KO, or P2M KO and CIITA double KO.

[0119] FIG. 32B is a flow cytometry plot of HLA-DR / DP / DQ (Class II) expression on expanded iPSC- derived T cells with clonal P2M KO, CIITA KO, or P2M KO and CIITA double KO.

[0120] FIG. 32C is a graph of proliferation of primary donor CD4+ T cells following co-culture with P2M or CIITA single KO or P2M and CIITA double KO iPSC-derived CD8+ T cells as indicated, in comparison with primary CD4+ T cells alone ("T cells Only"). Proliferation of CD4+ T cells was tracked by % CellTrace™ Violet (CTV) dilution.

[0121] FIG. 32D is a graph of proliferation of primary donor CD8+ T cells following co-culture with P2M or CIITA single KO or P2M and CIITA double KO iPSC-derived CD8+ T cells as indicated, in comparison with primary CD8+ T cells alone ("T cells Only"). Proliferation of CD8+ T cells was tracked by % CTV dilution.

[0122] FIG. 32E is a graph of the lysis of P2M or CIITA single KO or P2M and CIITA double KO iPSC- derived CD8+ T cells, as indicated, after co-culture with allogeneic CD4+ and CD8+ T cells. Data points and error bars represent the mean ± standard deviation (SD); n = 5 T cell donors. ***p < 0.001 and ****P < 0.0001 (repeated measures (RM) one-way ANOVA with Dunnett's test).

[0123] FIG. 33A is a graph of HLA-E expression of iPSC-derived cell lines through differentiation and CD8+ T cell expansion, for iPSC-derived cells with HLA-E insertion at the GAPDH locus, the / 32M locus or the CIITA locus. "Post-Exp.", following CD8+ T cell expansion.

[0124] FIG. 33B is a graph of the correlation of NKG2A- NKG2C+ phenotype on CD3-CD56+ NK cells, from n = 7 donors, with NK lysis of expanded P2M KO iPSC-derived T cells containing targeted integration of HLA-E at the GAPDH locus.

[0125] FIG. 34A is a graph of expression of HLA-E, CD58 and ICAM3 on iPSCs, CD34+ hematopoietic progenitor cells (HPCs), progenitor ? cells (ProTs), unexpanded (CD8 T) and expanded (Post-Exp.T) T cells derived from iPSCs modified with HLA-E KI at the GAPDH locus and CD58 and ICAM3 double KO, as measured by flow cytometry (n = 2 iPSC clones)

[0126] FIG. 34B is representative flow cytometry plots of key phenotypic attributes of unexpanded iPSC-derived T cells modified with HLA-E KI at the GAPDH locus and CD58 and ICAM3 double KO.

[0127] FIG. 35A is a graph of the percentage of Raji cells after a 7-day persistence assay of Raji CD19+ target cells and iPSC-derived CD8+ T cells, modified with HLA-E KI at the GAPDH locus and CD58 and ICAM3 double KO. At the end of each stimulation, as indicated by the vertical dashed lines, the percentage of Raji cells was determined by manual counting and flow cytometry.

[0128] FIG. 35B is a graph of iPSC-derived T cell proliferation in a co-culture after a 7-day persistence assay of Raji CD19+ target cells and iPSC-derived CD8+ T cells, modified with HLA-E KI at the GAPDH locus and CD58 and ICAM3 double KO. At the end of each stimulation, as indicated by the vertical dashed lines, iPSC-derived CD8+ T cell proliferation was determined by manual counting and flow cytometry.

[0129] FIG. 36A is a graph of Raji TGI after a 5-day co-culture of iPSC-derived CD8+ T cells, modified with HLA-E KI at the GAPDH locus and CD58 and ICAM3 double KO, with allogeneic NK and CD8+ T cells at a 1:1:0.5:1 NK:CD8T:iPSC:Raji ratio. TGI was determined by manual counting and flow cytometry to distinguish the different cell types. Lines represent the mean; n = 4 unique NK donors, ns > 0.05, *P < 0.05 (RM one-way ANOVA with Dunnett correction).

[0130] FIG. 36B is a graph of iPSC-derived CD8+ T cell proliferation after a 5-day co-culture of iPSC- derived CD8+ T cells, modified with HLA-E KI at the GAPDH locus and CD58 and ICAM3 double KO, with allogeneic NK and CD8+ T cells at 1:1:0.5:1 NK:CD8T:iPSC:Raji ratio. iPSC-derived CD8+ T cell proliferation was determined by manual counting and flow cytometry to distinguish the different cell types. Lines represent the mean; n = 4 unique NK donors, ns > 0.05, *P < 0.05 (RM one-way ANOVA with Dunnett correction).DETAILED DESCRIPTION OF THE DISCLOSUREDefinitions

[0131] As used herein, the term "HLA single-chain trimer", or "HLA-SCT" refers to a polypeptide comprised of a P2M molecule covalently linked to a binding peptide and an HLA class I heavy chain, such as, for example, HLA-E.

[0132] As used herein, the term "knockout" or "KO" refers to the inactivation of a gene, or disruption of expression of the gene, through perturbation of the genomic DNA, such as, for example, removal of a portion of the DNA sequence of the gene, mutation of the DNA sequence of the gene, introduction of an exogenous DNA fragment.

[0133] As used herein, the term "knockdown" or "KD" refers to the partial disruption, reduction of expression or inhibition of a gene, for example, through perturbation of the genomic DNA, such as removal of a portion of the DNA sequence of the gene, mutation of the DNA sequence of the gene, or introduction of an exogenous DNA fragment, or through suppression of the activity of a gene through antibody or small molecule inhibition, expression or delivery of short interfering RNA (siRNA) or short hairpin RNA (shRNA), or inactivation or disruption of a regulator of a gene.

[0134] As used herein, the term "knock-in" or "KI" refers to the insertion of an exogenous DNA sequence into a cell genome.

[0135] As used herein, the term "NK cell clearance" refers to the killing of allogeneic cells by NK cells, such as, for example, in an in vitro co-culture assay or following in vivo transplantation of the allogeneic cells.

[0136] 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.

[0137] 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+).

[0138] 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.

[0139] 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) or CD4+CD8- (CD4 single-positive, CD4SP) stages. CD8 may 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).

[0140] 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.

[0141] 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 (toprevent 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.

[0142] 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.

[0143] The term "effective amount" or "therapeutically effective amount", for example an effective amount or therapeutically effective amount of a T cell lineage population as used herein is an amount sufficient to bring about any one or more beneficial or desired 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.

[0144] 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.

[0145] 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, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, PA, 1990; and Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing, 2000).

[0146] 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.

[0147] 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.

[0148] 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") canrefer, 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.

[0149] 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%.

[0150] 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.

[0151] 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.

[0152] 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

[0153] 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.

[0154] 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, 3rd. ed., 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 Editing

[0155] 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). The SSI may be biallelic or monoallelic.

[0156] In an embodiment, SSI of an HLA-SCT 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.

[0157] Partial or complete knockout (KO) or knockdown of a gene may be achieved with nuclease editing systems such as MAD7 or CRISPR-Cas (including Cas9 nuclease) (Ellis et al., 2021). In an embodiment, KO of CD58 and / or ICAM3 is carried out using a MAD7 nuclease and gRNA targeting a region of the CD58 and / or ICAM3 genes. In an embodiment, the KO is performed in an iPSC.

[0158] Alternatively, suppression of the activity of an endogenous factor, such as CD58 or ICAM3, may be achieved with antibody or small molecule inhibition (Rolle et al., 2016), short interfering RNA (siRNA), short hairpin RNA (shRNA), or other conventional methods.T Cell Differentiation

[0159] 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

[0160] 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 cell, progenitor cell, or T cell stage to comprise a nucleic acid encoding a CAR or TCR that may be expressed at the pluripotent cell, progenitor cell, or T 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 cell malignancies, 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, and other autoimmune disorders or conditions (Weber et al., 2020).

[0161] 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 ectodomain of the CAR may be a single-chain variable fragment (scFv), single-domain antibodiy (single variable domain on a heavy chain,VHH), nanoantibody, 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).

[0162] B-cell targeting CAR-T cell therapies, such as CD19 CAR-T cells, or CAR-T cells targeting other B cell antigens, have demonstrated clinical efficacy in the treatment of systemic lupus erythematosus and have applicability to the treatment of other autoimmune conditions and disorders such as antisynthetase syndrome, pemphigus vulgaris, scleroderma, myasthenia gravis, rheumatoid arthritis, Type 1 diabetes, multiple sclerosis, and organ rejection (Arnold, 2024).

[0163] 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).Allogeneic Engineered Cells

[0164] Provided herein is an engineered cell modified with knockout or knockdown of HLA class I, HLA class II, CD58 and ICAM3, and knock-in of an HLA-SCT. In an embodiment, the HLA class I and HLA class II knockout is achieved via knockout of P2M and CIITA. P2M, or P2 microglobulin, is a protein that is encoded by the P2M gene and forms heterodimers with HLA class I proteins in the HLA class I complex. CIITA, or class II major histocompatibility complex transactivator, is a regulator of class II major histocompatibility complex gene transcription and is essential for all HLA class II expression. In an embodiment, the HLA-SCT is an HLA-E SCT, an HLA-F SCT, or an HLA-G SCT.

[0165] In an embodiment, the engineered cell is an iPSC or an iPSC-derived cell, such as, for example, an iPSC-derived T cell, an iPSC-derived CD34+ hematopoietic stem / progenitor cell, an iPSC-derived progenitor T cell, an iPSC-derived NK cell, an iPSC-derived neural cell, an iPSC-derived islet cell or islet cell progenitor, an iPSC-derived cardiac cell or cardiac progenitor, an iPSC-derived B cell, or an iPSC- derived macrophage.

[0166] In an embodiment, the engineered cell is a donor-derived cell such as, for example, a primary donor T cell or a primary donor NK cell.

[0167] It is contemplated that engineered cells and the cell populations provided herein may be contained in pharmaceutical compositions.

[0168] 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.

[0169] The pharmaceutical composition provided herein may be administered to a subject to treat cancer or autoimmune disorders in the subject.

[0170] 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.

[0171] 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

[0172] 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.

[0173] 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. Thecontainers 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.

[0174] 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: HLA-SCT Presentation

[0175] Two HLA-E single-chain trimer constructs (SCTs) were designed to protect P2M knockout cells from T cell clearance in the context of allotransplantation. The construct designs and expression percentage and intensity (geometric mean fluorescence intensity, gMFI) in P2M knockout primary CD8+ T cells are shown in Table 3 below. The constructs differed in their binding peptide sequence and HLA-E allele (SEQ ID NO: 1-4).

[0176] Cell lines were generated with high-, mid-, and low-level expression of the NT363 and NT568 HLA-SCT constructs. Cells with a wide range of number of HLA molecules per cell were therefore generated (Fig. 1A). Next, the effect of the expression level of the HLA-E SCTs on NK cell clearance was investigated. Engineered cells were labelled with carboxyfluorescein succinimidyl ester (CFSE) and cocultured with donor NK cells for 96 hours. Viability was assessed daily over the course of the assay and the change in NK cell clearance between groups was determined as the percent reduction in the area under the curve (AUC), for the difference between the background viability of the engineered cells (i.e., cultured alone, without NK cells) and the viability of the engineered cells in NK cell co-culture.

[0177] NK donor cells were analyzed for relative expression of NKG2A and NKG2C. Each donor had a different NKG2A / NKG2C ratio, with consistent trends across co-culture with the different engineered cell groups (Fig. IB). Analysis of NK cell clearance by donor revealed that HLA-E SCT expression level is an important parameter that affected NK clearance, especially in the context of NKG2A / NKG2C expressionratio (Fig. 1C). For NK donors with a high NKG2A / NKG2C ratio (e.g. donors A, B), low levels of HLA-E SCT on P2M KO cells were insufficient for complete protection (Fig. 1C). For NK donors with a low NKG2A / NKG2C ratio (e.g. donor D), high levels of HLA-E SCT on P2M KO cells resulted in loss of protection. Medium levels of HLA-E expression driven by NT363 were shown to provide an optimal balance for protection. The level of NK cell clearance by NK cells expressing higher levels of NKG2C (lower NKG2A / NKG2C ratio), even of cells engineered with NT363, indicated that further engineering of the cells, as described in Example 2, in addition to knock-in of an HLA-E SCT construct may be necessary for more robust protection.

[0178] iPSC-derived cell lines were generated comprising the NT363 or NT568 constructs, and cell phenotype, viability, and fold expansion during in vitro CD8+ T cell differentiation was evaluated. Briefly, iPSC-derived P2M knockout (P2M - / -) TCR+ CD34+ cells, with and without knockout of CIITA , were transduced with the NT363 or NT568 HLA-SCT constructs. Cells were then differentiated to CD8+ T cells using staged presentation of immobilized Notch ligand and CD3 activation (Iriguchi et al., 2021; Chandrasekaran et al., 2023). Lack of P2M and CIITA did not impair differentiation kinetics or progression to a CD8aP+ phenotype (Fig. 2A). Viability was similar between cell lines (data not shown). Similar growth trajectories, with differences in the magnitude of growth, were observed across cell lines (Fig. 2B).

[0179] The expression of HLA-E in the iPSC-derived cell lines over time was also analyzed. The clone transduced with the HLA-E NT363 construct (PCE275) had more stable HLA-E surface expression across multiple stages of differentiation as compared to clones that were transduced with the HLA-E NT568 construct (PCE283 and PCE285) (Fig. 3A).

[0180] The generated cell lines were expanded in vitro following CD8+ T cell differentiation (Iriguchi et al., 2021). Post-expansion viability was 90% or higher, and similar across cell lines (data not shown).

[0181] The binding ratio of recombinant NKG2A-CD94 and NKG2C-CD94 heterodimeric proteins to the NT363 and NT568 HLA-E SCT constructs was also evaluated in iPSC-derived CD8+ T cells. Cells expressing NT363 and NT568 similarly bound NKG2A over NKG2C at a single concentration tested (0.3pM) (Fig. 3B).

[0182] The effect of the insertion site selection for the HLA-E SCT on HLA-E SCT expression level was evaluated in iPSC-derived CD8+ CAR-T cells. Cells were modified with a CD19 CAR and P2M knockout (P2M KO), P2M knockout and knock-in of HLA-E SCT NT363 at the GAPDH locus (GAPDH HLA-E), P2M knockout and knock-in of HLA-E SCT NT363 at the 62M locus (P2M HLA-E), or P2M knockout and knock- in of HLA-E SCT NT363 at the CIITA locus (CIITA HLA-E). Following cell expansion, the expression level ofHLA-E was highest in the P2M HLA-E line, with medium and low levels of expression in the CIITA HLA-E and GAPDH HLA-E lines, respectively (Fig. 4A).

[0183] After cell expansion, cells were co-cultured with CD19+ A549 target cells in a persistence assay. Briefly, the expanded iPSC-derived CD8+ CAR-T cells were co-cultured with Nuclight™ green-labeled A549 CD19+ target cells at a 4:1 E:T ratio and re-seeded at a 1:4 dilution on fresh target cells every 2-3 days for up to 2 weeks (6 stimulations total). An Incucyte® culture system (Sartorius) was used to fluorescently monitor target cytotoxicity. One group of cells was cultured without target cells for up to 9 days following one round of antigen stimulation with target cells (Fig. 4B, 4C, post-stimulation round one (PSI) day 5, PSI day 7, PSI day 9). A second group of cells was serially restimulated with target cells six times (Fig. 4B, 4C, post-stimulation round six (PS6)). Following expansion and antigen stimulation, HLA-E expression levels in the SSI-modified iPSC-derived cells peaked on 5-7 days post-stimulation (PSI Day 5, PSI Day 7 vs. PS6, Fig. 4B). The GAPDH HLA-E SSI line maintained the lowest HLA-E expression for all timepoints sampled following the first stimulation, close to the "ideal range" identified from primary T cell studies above (Fig. 4B, 4C). The number of HLA-E molecules per cell driven by GAPDH, CIITA or 62M targeted integration on expanded iPSC-derived CD8+ CAR-T cells was determined by BD Quantibrite™ phycoerythrin (PE) analysis. Across three independent experiments with expanded iPSC-derived CD8+ CAR-T cells, insertion of the HLA-E SCT at GAPDH locus resulted in significantly lower HLA-E expression than insertion at CIITA and 62M loci, which may be advantageous for mitigating NKG2C-driven NK clearance (Fig. 4D; **P < 0.01, ***p < 0.001 (ordinary one-way ANOVA with Tukey correction)).Accordingly, targeted integration of HLA-E at GAPDH locus was selected for further testing.

[0184] NK cell clearance of the HLA-E SSI lines was evaluated in an in vitro co-culture assay with donor NK cells. All expanded HLA-E SSI lines mitigated NK clearance to a similar extent (Fig. 5A). The NK clearance cell assay was repeated with NKG2C-prominent NK donor cells (having a relative NKG2A to NKG2C expression ratio of less than 3). Assessing protection from NKG2C-prominent NK cells is particularly important in the context of cancer treatment, especially for leukemia and lymphoma patients. Adaptive CD56dim NKG2C+ NK cells (Moretta, 2010) are enriched after cytomegalovirus (CMV) infection, and cancer patients, such as leukemia and lymphoma patients, commonly experience CMV reactivation after receiving HSC transplantation or lymphodepletion. NKG2C-prominent NK donors (having a relative NKG2A to NKG2C expression ratio of less than 3) displayed more clearance against the iPSC-derived HLA-E SSI lines (Fig. 5B, "NKG2A / NKG2C <3 donors", light symbols) in comparison to NKG2A-prominent NK donors (having a relative NKG2A to NKG2C expression ratio of greater than 10,Fig. 5B, "NKG2A / NKG2C >10", black circles). The GAPDH HLA-E NT363 line (NTX4I36) showed the most protection against these NK donors (Fig. 5B), and the level of NK clearance of HLA-E SSI clonal lines correlated with the level of HLA-E expression of each line.

[0185] The impact of insertion site selection on in vitro iPSC-derived CD8+ CAR-T cell function was also examined in the above lines as well as two lines generated with P2M knockout and knock-in of HLA-E SCT NT568 at the 62M locus (NTX4I32) or P2M knockout and knock-in of HLA-E SCT NT568 at the CIITA locus (NTX4I33). The expanded iPSC-derived CD8+ T cells were initially seeded on A549 CD19+ target cells at a 1:4 effector (CD8+ T cell) to target cell ratio (E:T) and split 1:4 every 2-3 days on fresh target cells for 6 stimulations total over 14 days. All HLA-E SSI clonal lines, including the biallelic GAPDH HLA-E line (NTX4I36), demonstrated relatively comparable proliferation to the P2M knockout control line (NTX4H27) during the co-culture assay (Fig. 6A, 6B). Similar cytotoxicity against A549 CD19+ target cells over 6 rounds of antigen stimulation was also observed across all lines (Fig. 6C).

[0186] Next, the impact of insertion of the HLA-E SCT NT363 on the expression and activity of GAPDH was assessed. On day 13 of the persistence assay, GAPDH enzymatic activity was measured using a GAPDH activity assay kit (abeam). The activity was normalized by total protein extracted from each lysate as quantified by BCA assay (Thermo Fisher Scientific). Insertion of the HLA-E SCT NT363 into the GAPDH site resulted in reduced GAPDH activity (Fig. 7A). Intracellular expression of GAPDH was also quantified by flow cytometry. The magnitude of GAPDH expression was also measured by RT-ddPCR using a probe targeting Exons 2-3 of GAPDH. The mRNA quantity was normalized by the total RNA mass per RT-ddPCR reaction, quantified by a TapeStation HS-RNA kit (Agilent). The RNA Integrity Number (RIN) scores were all above 9. A reduced GAPDH mRNA level was observed in the GAPDH HLA-E SCT line (NTX4I36) (Fig. 7B).

[0187] Biallelic insertion of the HLA-SCT 363 at the GAPDH or CIITA loci was evaluated in comparison to control cells without HLA-SCT insertion. Biallelic SSI of HLA-E at the GAPDH locus did not impact the expansion of iPSC-derived CD8+ CAR-TCR cells in comparison to control cells without HLA-SCT insertion (Fig. 8A), with similar viability during expansion, suggesting that insertion of the HLA-SCT was well- tolerated (Fig. 8B). Expression of HLA-E from the biallelic GAPDH insertion yielded approximately 19,000 HLA-E molecules per cell post-expansion, which was within the desired range for expression as determined above (Fig. 8C).

[0188] The in vitro function of cells modified with insertion of the HLA-SCT constructs was evaluated in a co-culture assay with A549 CD19+ target cells. Across a range of E:T ratios, SSI of the HLA-E SCT NT363at the GAPDH or CIITA loci did not impact the function of iPSC-derived CD8+ CAR-TCR cells in comparison to the control line (Fig. 9A). Similar proliferation was observed between the NT363 lines and the control line at all E:T ratios (Fig. 9B).

[0189] GAPDH enzymatic activity and mRNA level was assessed in these cell lines as described above. The GAPDH HLA-E NT363 line (NTX4I36) showed similar levels of GAPDH activity at higher E:T ratios, and activity was maintained across all E:T ratios (Fig. 9C). A similar trend, with more subtle differences between lines, was observed at the mRNA level (Fig. 9D).

[0190] In summary, to ensure effective inhibition of NK cells by engagement of NKG2A while minimizing activating NK cells via NKG2C, HLA-E expression levels were optimized by regulating expression of HLA-E by site-specific integration under the control of endogenous and exogenous promoters. It was shown that surface expression level of HLA-E was critical to balance engagement of NKG2A while minimizing activation of NK cells via NKG2C.EXAMPLE 2: NK Cell Clearance of Engineered Allogeneic Cells

[0191] The NK cell clearance of unmodified iPSC-derived CD8+ TCR-T cells (expressing a MAGEA4 TCR) was compared to clearance of P2M- / - iPSC-derived CD8+ TCR-T cells. NK cell clearance was reported as the percent reduction in the area under the curve (AUC) for cell viability, with and without NK cell coculture; a greater percent reduction in AUC indicating higher NK cell killing. iPSC-derived CD8SPs lacked immune tolerance to allogeneic NK cells independent of HLA class I status, with a higher level of clearance of P2M- / - cells (Fig. 10A). In P2M+ / + iPSC-derived CD8+ TCR-T cells, lentiviral transduction of the HLA-SCT constructs NT363 or NT568 did not prevent NK cell clearance (Fig. 10B). Further, the expression of the NT363 and NT568 HLA-E SCTs were not enriched on HLA class I intact iPSC-derived CD8+ TCR-T cells after co-culture with NK cells, suggesting that the HLA-E SCTs did not provide selective protection (Fig. 10C, 10D). These data indicate that an additional HLA-independent mechanism, beyond NK cell activation by lack of class I presentation, was responsible for clearance of the iPSC-derived cells.

[0192] NK cell clearance of multiple iPSC-derived CD8+ T cell lines was investigated. iPSC-derived CD8+ MAGE-A4 TCR-T cells (NTX6B10), or iPSC-derived CD8+ CD19 CAR-T cells generated from independent donor cell material, both with and without P2M knockout (NTX4H20 and NTX4B3, respectively), were stimulated for 5 days with A549 CD19+ target cells (NTX4H20, NTX4B3) or A375 MAGE-A4+ target cells (NTX6B10) before co-culture with NK cells (at 1:1 NK to iPSC-derived cell ratio). Both TCR and CAR lines had similar levels of HLA-independent NK cell clearance, lower than the P2M knockout line control (NTX4H20 P2M KO) (Fig. 11).

[0193] Flow cytometry phenotyping of iPSC-derived CD8+ T cells was performed to identify potential NK cell activating and inhibitory ligands. HLA expression on unmodified iPSC-derived cells was high and comparable to primary T cells (Fig. 12), further explaining why insertion of HLA-E SCTs did not confer additional protection. CD155.PVR and CD58 were identified as highly expressed on iPSC-derived CD8+ T cells in comparison to primary cells (Fig. 12). CD47 was not differentially expressed on iPSC-derived CD8+ T cells (data not shown).

[0194] The effect of PVR knockout on NK cell clearance, with and without insertion of an HLA-E SCT, was investigated in iPSC-derived CD8+ TCR-T cells. Cells were activated with CDS stimulation and transduced with the HLA-E SCT NT363 construct by LVV. Transduced or untransduced cells were then expanded and cells were electroporated to introduce a Cas9 ribonucleic protein (RNP) complex targeting PVR. Cells were then evaluated in a NK cell co-culture assay with three independent NK cell donors. The transduction efficiency of the HLA-E SCT NT363 was above 30%, and the efficiency of PVR knockout was above 80% (Table 4). Knockout of PVR, with or without HLA-E insertion, did not mitigate HLA- independent clearance of iPSC-derived CD8+ T cells (Fig. 13A).

[0195] Additional screening was performed on NK cell activating ligands. Antibody-based blocking of CD2, CDlla / CD18, NKG2D, TRAIL and FasL on NK cells during clearance assays with iPSC-derived CD8+ TCR-T cells was performed. Only CDlla / CD18 blocking substantially inhibited NK clearance, although the addition of CD2 blocking boosted protection (Fig. 13B).

[0196] The expression of adhesion ligands on iPSC-derived CD8+ T cells was assessed in comparison to primary CD8+ T cells. CD58 and CD54 (ICAM1) were upregulated on iPSC-derived CD8+ T cells (5-fold, and 3-fold, respectively) in comparison to primary T cells. ICAM2, ICAM3, and FUR were not upregulated, as determined by flow cytometry (data not shown).

[0197] CD2, CDlla and CD18 expression on both CMV+ and CMV- NK donor cells (CD3-CD56+) was investigated. NK cells from CMV+ donors expressed higher levels of CD2 in comparison to cells from CMV- donors (Fig. 14A, 14B, n = 5-6 donors per serostatus, P = 0.0009, two-tailed t-test), demonstrating the promise of a CD58 KO for added protection against NK clearance in CMV seropositive (CMV+) patients. CMV seropositivity is associated with an increase in the percentage of NKG2C+ NK cell (Moretta et al., 2010). Across 11 donor NK cell lines, the magnitude of CDlla and CD18 expression correlated positively with NKG2C expression on NK cells (Fig. 14C, 14D), highlighting the potential of ICAM disruption for mitigating NKG2C-driven NK clearance of HLA-E KI iPSC-derived CD8+ T cells.

[0198] Activated iPSC-derived CD8+ T cells were transduced with an HLA-E SCT (NT363 or NT568) LVV construct and subsequently modified to knockout one or both of P2M and CD58. Cells were activated with CD3 / CD28 Dynabeads™ with cytokine supplementation and transduced 2 hours later with the LVV constructs. Cells were then expanded and electroporated with Cas9 RNP targeting P2M and / or CD58 on day 5 post-transduction. An HLA-E enrichment was performed, and cells were re-activated with CD3 / CD28 Dynabeads™ for 3 days and expanded for a further 2 days before analysis and initiation of an NK cell clearance assay. Efficient editing and HLA-E enrichment yielded highly pure populations, with approximately 95% HLA-E+ / P2M- cells for the P2M single KO group and 80% HLA-E+ / P2M- / CD5810cells for the P2M and CD58 double KO group (Fig. 15A). Despite extended culture time following editing, incomplete loss of CD58 protein from the cell surface was observed (Fig. 15B). Inference of CRISPR Edits (ICE) analysis of insertion-deletion (InDei) species suggested this was not due to incomplete editing (Fig. 15C) but may have been due to a slow turnover rate of CD58 protein in the cell.

[0199] The modified iPSC-derived CD8+ T cells were then evaluated in an NK cell clearance assay. The combination of HLA-E SCT expression and CD58 KO provided significantly improved protection from HLA-dependent NK clearance compared to P2M knockout alone (Fig. 16).

[0200] Next, the individual and combinatorial effects of CD58, CD54, ICAM2 and ICAM3 knockout were investigated in P2M-intact cells. iPSC-derived CD8+ T cells were activated with CD3 / CD28 Dynabeads™ with cytokine supplementation. Cells were then expanded and electroporated with Cas9 RNP targeting CD58, CD54, ICAM2 and / or ICAM3 on day 5. Cells were re-activated with CD3 / CD28 Dynabeads™ on day 7 post-electroporation for 3 days and expanded for a further 2 days before analysis and initiation of an NK cell clearance assay on day 12. The multiplexed Cas9 editing was highly efficient, generating cells with two or three adhesion molecule knockouts (Fig. 17A, 17B). The efficiencies of single knockout, double knockout, and triple knockout were 87-94%, 71-85%, and 64%, respectively (Fig. 17A, 17B).Additional anti-CD58 antibody blocking was performed during NK clearance assays to artificially reduce residual CD58 expression after knockout to near-control levels (Fluorescence-Minus-One (FMO) gating control, Table 5).

[0201] CD58 knockout (with additional antibody blockade, as described above) provided a small but significant reduction in HLA-independent NK clearance at 48 h (Fig. 18A). CD54 knockout did not exhibit any protection alone or in combination with CD58 (Fig. 18A). Of the samples with multiple ICAM knockouts, only those that had an ICAM3 knockout displayed mitigation of HLA-independent clearance at 48 h (Fig. 18B). The combination of CD54 and ICAM2 knockout did not show protective effects across all 3 donors, suggesting that ICAM3 knockout was the main driver of the protection observed in the CD54 / ICAM2 / ICAM3 triple knockout cells (Fig. 18B).

[0202] Next, iPSC-derived CD8+ T cells were modified with LVV transduction and CRISPR-Cas9 editing as described above to express the NT363 HLA-E SCT construct and knockout P2M, CD58 and / or ICAM3. In this editing process, cells were cultured for an additional 2 days before initiation of the NK clearance assay (14 days in total). As described above, iPSC-derived CD8+ T cells were co-cultured with primary NK cells from n = 3 donors at a 1:1 ratio for 72 h. Viability of iPSC-derived CD8+ T cells in the presence and absence of NK cells was monitored daily by flow cytometry, analyzed as reduction in the area under the curve, and normalized to a P2M KO only control to measure % reduction in NK lysis. Due to slow turnover in CD58, a CD58 blocking antibody was included in CD58 knockout NK cell co-cultures to reduce uncleared CD58. Cells with combinations of P2M knockout, HLA-E knock-in, CD58 knockout and / or ICAM3 knockout had high editing efficiency (Fig. 19A, 19B), with greater than 96% HLA-E+ cells following enrichment (Fig. 19A) and approximately 75% triple knockout efficiency for P2M, CD58 and ICAM3 (Fig. 19B). The knockout of either CD58 or ICAM3 alone in a P2M-deficient background was not effective in mitigating NK clearance (Fig. 20A). The knock-in of an HLA-E SCT (HLA-E KI) was effective at mitigating HLA-dependent NK clearance (Fig. 20A, difference between second and sixth group). The combination of CD58 and ICAM3 knockouts with knock-in of an HLA-E SCT provided the most effective mitigation of HLA-independent clearance in comparison to separate knockouts of CD58 or ICAM3 (Fig. 20A). This combinatorial effect of HLA-E KI, CD58 KO, and ICAM3 KO provided a greater than additive reduction inNK clearance, compared to the sum of the CD58 KO and ICAM3 KO groups (Fig. 20B, "Whole" versus "Sum of Parts"). While adhesion molecule KOs (i.e., CD58 KO and ICAM3 KO) in the absence of HLA-E KI were not very protective, the combination of HLA-E KI with CD58 KO and ICAM3 KO provided greater protection than the sum of their parts (Fig. 20B), suggesting synergy.

[0203] Next, the function of iPSC-derived TCR-T cells with and without allogeneic edits was assessed in the presence of NK and tumor cells. A 5-day three-way co-culture assay was used to examine iPSC-CD8+ TCR-T cell proliferation and cytotoxicity at varying iPSC-CD8+ TCR-T cell to tumor cell or iPSC-CD8+ TCR-T cell to NK cell ratios. All iPSC-CD8+ cell lines, including P2M knockout cells, were able to control tumor growth under the tested conditions. Cell proliferation was greatly reduced in the P2M knockout-only cells (Fig. 21A, 21B). The expression of an HLA-E SCT and knockout of CD58 provided protection to iPSC- derived CD8+ cells in the presence of NK cells, recovering proliferation to the same level as observed in unmodified cells (Fig. 21A, 21B).

[0204] A similar assay was performed with unmodified, P2M knockout and P2M / CD58 knockout and HLA-E SCT-transduced iPSC-derived CD8+ CAR-T cells in the presence of NK cells and Raji target cells (B- cell lymphoma target cells expressing CD19). Cells were co-cultured for 7 days with rechallenge with additional Raji target cells on days 2 and 4. Expression of an HLA-E SCT and knockout of CD58 recovered the loss of function due to P2M knockout in the presence of NK cells across two tumor rechallenges (Fig. 22A). The HLA-E+ and P2M- / CD58- / - line also recovered the loss of proliferation caused by HLA- dependent and HLA-independent clearance in the P2M knockout line (Fig. 22B).

[0205] Next, modified iPSC-derived CD8+ CAR-T cells were generated to assess the combined effects of HLA-E SCT expression and P2M, CD58 and ICAM3 knockout. P2M KO iPSC-derived CD8+ CAR-T cells were activated with CD3 / CD28 Dynabeads™ and transduced with the NT363 HLA-E SCT L\Z\Z construct. Cells were then expanded for two days and electroporated with Cas9 RNP targeting CD58 and / or ICAM3, reactivated with CD3 / CD28 Dynabeads™ for three days and further expanded for an additional two days before assessment. A CD58 blocking antibody was included in CD58 KO co-cultures to reduce uncleared CD58 expression. The LW -driven expression of the NT363 HLA-E SCT was approximately 40,000 molecules per cell (Fig. 23A), well above the desired range of expression identified from the primary T cell study (Fig. 1). This enabled observation of HLA-E mediated mitigation of NKG2A-skewed NK clearance in the co-culture assay. The CD58 / ICAM3 double KO editing was 40% efficient (Fig. 23B), with higher efficiency of CD58 single KO editing at approximately 70-75% (Fig. 23B). In the NK clearance cell assay, HLA-E expression alone, while still protective, did not completely bridge the gap between WT andP2M KO cells (Fig. 24). The combination of HLA-E expression, CD58 KO and ICAM3 KO provided robust protection against HLA-dependent NK clearance and completely bridged the gap between WT and P2M KO cells (Fig. 24).

[0206] The function of the modified iPSC-derived CD8+ CAR-T cells was assessed in an in vitro assay with Raji target cells. No differences in tumor growth inhibition (TGI) or specificity against CD19 target cells were observed between groups (Fig. 25A). Further, on-target proliferation and pre- versus postassay phenotype were similar with and without KO of CD58 or CD58 and ICAM3 (Fig. 25B, 25C), suggesting that the KOs did not impact function of the iPSC-derived CAR-T cells.EXAMPLE 3: Generation and Function of SSI Allogeneic iPSC-T Cells

[0207] Clonal iPSC cell lines were generated using MAD7 or Cas9 Ribonucleoprotein (RNP) (Liu et al., 2020; Prochazka et al., 2024). Cell lines included an EBV TCR, a CD19 CAR, and one or more of HLA-SCT insertion, P2M KO, CIITA KO, CD58 KO and ICAM3 KO (Table 6). Knockout of CD58 was incomplete in the PCE490 line. Insertion of the HLA-E SCT NT363 construct resulted in 10 extra tandem copies of the HLA-E SCT at the GAPDH locus.

[0208] Engineered CD8+ T cells were generated by staged differentiation of iPSC-derived hematopoietic stem / progenitor cells, generated from the iPSC lines described above, to progenitor T cells and CD8+ cells on an engineered Notch ligand support (Carpenedo et al., 2024). Briefly, iPSCs were differentiated to CD34+ cells (Trotman-Grant et al., 2021) and 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 2.70xl07beads / mL of microbeads modified with DL4 and VCAM-1 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); 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. 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).

[0209] The differentiated iPSC-derived CD8+ CAR-TCR lines had similar expansion and viability postactivation with CD3 / CD28 Dynabeads™ (data not shown). While the GAPDH HLA-E CD58 single KO clonal line cleanly lacked CD58 expression, the CD58 / ICAM3 double KO clonal line displayed some residual CD58 expression, with no expression of ICAM3 (Fig. 26A, 26B). Except for the CIITA TCR KI line (NTX4J26- PCE332), the edited lines retained HLA class II expression (Fig. 26C). After activation and expansion, cells modified with the HLA-E SCT construct had consistent HLA-E expression (Fig. 26D).

[0210] The clearance of these modified cells by NK cells from an NKG2A-prominent donor (having a relative NKG2A toNKG2C expression ratio of greater than 10) and NKG2C-prominent donors (having a relative NKG2A to NKG2C expression ratio of less than 3) were evaluated. A CD58 blocking antibody was included in the CD58 and ICAM3 KO group to reduce uncleared CD58. Layering the CD58 and ICAM3 KOs onto the GAPDH HLA-E-modified cells resulted in the greatest decrease in comprehensive NK clearance (Fig- 27).

[0211] An additional set of iPSC lines were generated with HLA-E SCT KI and CD58 or CD58 and ICAM3 KO (Table 7). All cell lines included a CD19 CAR and an EBV TCR.

[0212] These cells were differentiated to CD8+ T cells via staged presentation of an immobilized Notch ligand as described above (Carpenedo et al., 2024). Normal growth kinetics and cell phenotype was observed, with greater than 80% CD5+CD7+ cells, less than 40% CD4+ cells, greater than 45% CD8a+CD8P+ and less than 15% CD56+ cells observed for all cell lines at the end of differentiation (data not shown). Minimal expression of CD58 was observed in CD58 KO lines (Fig. 28A) and no expression of ICAM3 was observed in CD58 and ICAM3 double knockout lines (Fig. 28B). Lower expression and MFI of HLA-E was observed with insertion at the CIITA site compared to the GAPDH site (Fig. 28C).

[0213] The in vitro function and proliferation of these cells were evaluated in a 5-day single-stim assay with CD19+ Raji target cells ("WT") or CD19 KO Raji cells ("KO"). Raji target cell growth inhibition (TGI) by the iPSC-derived CD8+ T cells, Raji target cell proliferation and iPSC-derived CD8+ T cell proliferation was assessed after a 5-day co-culture of iPSC-derived CD8+ T cells expressing a CD19 CAR and Raji CD19+ tumor target cells at multiple effector to target cell ratios (E:Ts). TGI was determined by lncucyte®-based monitoring of Raji target cell fluorescence, and Raji target cell proliferation and iPSC-derived CD8+ T cell proliferation was quantified by normalizing manual counts at the end of the co-culture with % Raji cells and % iPSC-derived CD8+ T cells as determined by flow cytometry. HLA-E KI at the GAPDH locus with additional CD58 / ICAM3 KOs did not impact the CAR function or proliferation of iPSC-derived T cells in a single stimulation assay (Fig. 29). All cell lines exhibited similar cytotoxicity against CD19+ Raji target cells (Fig. 29A) and similar reduction in proliferation of the Raji target cells was also observed across cell lines (Fig. 29B). Proliferation of the iPSC-derived CD8+ T cells was antigen-dependent, and similar across lines (Fig. 29C).

[0214] Further, three iPSC cell lines were generated with the insertion of the HLA-E SCT at the GAPDH site, in addition to KO of P2M, CIITA, CD58 and ICAM3. Following differentiation to hematopoietic stem / progenitor cells, these cells continued to demonstrate HLA-E expression and minimal CD58 expression, with expected expression of hematopoietic markers CD34 and CD43 (Table 8).

[0215] Additional analysis of NK cell clearance was performed on the following iPSC-derived CD8+ T cell lines: NTX4J26 and NTX4H27 (P2M-intact and P2M-deficient controls, respectively); P2M-deficient cells with insertion of an HLA-E SCT (NT363) at the GAPDH locus alone (NTX4I36-PCE575) and P2M-deficient cells with insertion of an HLA-E SCT at the GAPDH locus and additional layering of a CD58 KO (NTX4K36- PCE496), or CD58 and ICAM3 KO (NTX4M36-PCE488). Clearance at a 1:1 NK to iPSC-derived CD8+ T cell ratio was evaluated by tracking iPSC-derived CD8+ T cell viability daily in the presence and absence of NKcells for up to 96 h by flow cytometry and analyzing the difference in viability as % reduction in the area under the curve (AUC). The combination of HLA-E KI, CD58 KO, and ICAM3 KO resulted in increased protection from NK cell clearance (Fig. 30A). A significant reduction in NK cell clearance (n=10 unique donors) was observed with the combination of HLA-SCT KI, CD58 KO and ICAM3 KO (fifth column) compared to HLA-SCT KI alone (third column) or HLA-SCT KI and CD58 KO (fourth column) (Fig. 30A, oneway ANOVA with Holm-Sidak correction). The ratio of % NKG2A+NKG2C+ expression over % NKG2A- NKG2C+ expression on CD3-CD56+ NK cells used in the NK clearance assay was also analyzed. The expression of NKG2A and NKG2C on the donor NK cells used in this assay comprised a wide range of NKG2A / NKG2C ratios (Fig. 30B).

[0216] Figure 31 provides a schematic summarizing a proposed engineering approach to mitigate allogeneic cell clearance. Allogeneic cell clearance is known to limit the persistence of iPSC-derived CD8 T-cell therapies. For example, iPSC-derived CD8 T cells can be recognized and eliminated by patient CD8 and CD4 T-cells through human leukocyte adhesion (HLA) class I and class II molecules. HLA class I molecules form heterodimers with the P2 microglobulin protein that is encoded by the / 32M gene. CIITA is a transcriptional coactivator essential for all HLA class II expression. Deletion of / 32M and CIITA genes leads to the formation of cells that can completely mitigate patient T-cell mediated clearance. However, patient NK cells eliminate cells that do not express HLA class I receptors on their surface, since it is an important inhibitory ligand for NK cell receptors. To address NK clearance, a unique layered approach was designed to knock-in HLA-E and knock-out adhesion ligands CD58 and ICAM3 to prevent the formation of a stable immune synapse between product cells and patient NK cells. As demonstrated in the Examples, the layered approach results in reduced cell clearance compared to HLA-E KI alone in a NKG2C+ human NK donor pool and has the potential to support immune evasion and product persistence in a broader population of patients than previous approaches.

[0217] As described above in Example 2, expanded iPSC-derived CD8+ T cells were generated with clonal P2M and / or CIITA knockouts (KOs). HLA-ABC (Class I) and HLA-DR / DP / DQ (Class II) expression on the expanded iPSC-derived CD8+ T cells was measured by flow cytometry. P2M and CIITA double KO cells do not express any HLA class I and II, in contrast to single KO cells (Fig. 32A, 32B). Further, the mixed lymphocyte reaction of CellTrace™ Violet (CTV, ThermoFisher Scientific)-labeled allogeneic primary T cells to iPSC-derived CD8+ T cells was examined by co-culture of primary T cells with iPSC- derived CD8+ T cells containing single or double P2M and CIITA Kos at a 3:1 E:T ratio for 6 days. The proliferation of allogeneic CD4+ and CD8+ T cells was tracked by % CTV dilution as a metric ofproliferation, and statistical differences between groups were assessed by one-way ANOVA with Dunnett's test. Allogeneic CD4+ T cells proliferated when co-cultured with P2M single KO iPSC-derived T cells that had intact HLA class II expression but not with CIITA single KO iPSC-derived T cells (Fig. 32C), whereas allogeneic CD8+ T cells proliferated when co-cultured with CIITA single KO iPSC-derived T cells that had intact HLA class I expression but not with P2M single KO iPSC-derived T cells (Fig. 32D). The resulting lysis of iPSC-derived T cells after co-culture with allogeneic CD4+ and CD8+ T cells was also analyzed and statistical differences between groups were assessed by one-way ANOVA with Dunnett's test. While P2M and CIITA single KO iPSC-derived T cells were lysed after co-culture, P2M and CIITA double KO cells were protected (Fig. 32E).

[0218] As described above in Example 1, iPSCs were generated with targeted integration of HLA-E at GAPDH, 62M, and CIITA loci, differentiated to CD8+ T cells and expanded (n = 6 clones for GAPDH, n = 1 clone for P2M and CIITA). The percent HLA-E expression on iPSCs, CD34 hematopoietic progenitor cells (HPCs), progenitor T cells (ProTs), unexpanded CD8 single-positive T cells (CD8SPs) and expanded CD8SPs (Post-Exp) was measured by flow cytometry. HLA-E expression at GAPDH and 62M loci was maintained throughout differentiation, whereas expression at CIITA locus was silenced at the end of differentiation but upregulated post-expansion (Fig. 33A). The correlation of NKG2A-NKG2C+ phenotype on CD3-CD56+ NK cells from n = 7 donors with NK lysis of expanded P2M KO iPSC-derived T cells containing targeted integration of HLA-E at GAPDH locus was investigated. NK lysis correlated positively with NKG2C expression, suggesting, as described above, that further engineering on top of HLA-E knock- in such as KO of adhesion ligands may be needed to comprehensively mitigate NK clearance from different donors (Fig. 33B).

[0219] Since NK lysis of HLA-E KI P2M KO iPSC-derived T cells correlated with expression of activating receptor NKG2C on NK cells, it was further explored, as described above, if additional layering of synapse disruption by knockout of adhesion molecules such as CD58 and ICAM3 (CD50) could lead to best-in-class NK cell protection against NKG2C+ NK donors. As described above, two iPSC clonal cell lines were derived from an iPSC cell line with targeted integration of HLA-E at the GAPDH locus. Each clonal line featured bi-allelic knockouts, targeting either CD58 alone or both CD58 and ICAM3. The percentage of HLA-E, CD58 and ICAM3 expression on iPSCs, CD34+ hematopoietic progenitor cells (HPCs), progenitor T cells (ProTs), unexpanded and expanded T cells in the combination HLA-E KI, CD58 KO and ICAM3 KO cell line was measured by flow cytometry (n = 2 clones). CD58 and ICAM3 KOs resulted in disruption of protein expression through all stages of differentiation (Fig. 34A). Representative flow cytometry plots of key phenotypic attributes of iPSC-derived T cells (CD5 and CD7expression, CD8a and CD8P expression, CD7 expression and lack of CD56 expression, CAR (CD19 CAR as described above) and CD3 expression, HLA-E expression and lack of ICAM3 and CD58 expression) are shown in Fig. 34B. CD58 and ICAM3 KOs did not impact the differentiation of iPSC-derived T cells.

[0220] The percentage of Raji cells and iPSC-derived T cell proliferation was assessed in a co-culture after a 7-day persistence assay of Raji CD19+ target cells and iPSC-derived CD8+ T cells, modified with HLA-E KI at the GAPDH locus and CD58 and ICAM3 double KO. iPSC-derived T cells were co-cultured with Raji cells at a 4:1 E:T for 3 days and split 1:4 onto fresh Raji target cells every 2 days thereafter for 7 days total (3 stimulations). At the end of each stimulation, the percentage of Raji cells and iPSC-derived T cell proliferation were determined by manual counting and flow cytometry to distinguish the two cell types. HLA-E KI at the GAPDH locus with additional CD58 / ICAM3 KOs did not impact the CAR function and proliferation of iPSC-derived T cells in a 7-day persistence assay (Fig. 35A, 35B).

[0221] Next, Raji target cell TGI and iPSC-derived CD8+ T cell proliferation was assessed after a 5-day co-culture with allogeneic (primary donor) NK and CD8+T cells at a 1:1:0.5:1 NK:CD8T:iPSC:Raji ratio. TGI and iPSC-derived CD8+ T cell proliferation were determined by manual counting and flow cytometry to distinguish the different cell types. HLA-E KI at the GAPDH locus and additional CD58 / ICAM3 KOs resulted in improved tumor control and proliferation of P2M KO iPSC-derived T cells in the presence of allogeneic NK and CD8+ T cells compared to HLA-E KI alone and was comparable to wild-type counterparts (Fig. 36A, 36B).

[0222] As shown in these Examples, HLA-E knock-in combined with allogeneic synapse disruption provided comprehensive protection of P2M KO allogeneic iPSC-derived T-cells against a broad and diverse population of donor NK cells. The knockout of adhesion ligands that disrupt the allogeneic synapse, CD58 and ICAM3, did not decrease the potency of iPSC-derived CAR-T cells. The novel combination of allogeneic edits improved the performance of iPSC-derived CAR-T cells in the presence of co-cultured allogeneic T and NK cells in vitro. This work may support the development of scalable, potent, and persistent off-the-shelf allogeneic CAR-T cell therapies for oncology and autoimmune patients.

[0223] 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 forthin 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. Arnold, C. Autoimmune disease is the next frontier for CAR T cell therapy. Nat. Med. 1-4 (2024).2. Baulu, E., et al. 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Cell 181, 46-62 (2020).SEQ ID NO:1HLA-SCT NT363 (DNA)ATGGTGGTCATGGCCCCTAGAACACTGTTCCTGCTGCTGTCTGGCGCCCTGACACTGACAGAGACATGGGCCGTGATGGCCCCCAGAACCCTGATCCTGGGCGGCGGTGGTTCAGGCGGAGGAGGTTCAGGAGGAGGGGGTAGTGGAGGTGGTGGTTCTATCCAGCGGACCCCTAAGATCCAGGTGTACAGCAGACACCCCGCCGAGAACGGCAAGAGCAACTTCCTGAACTGCTACGTGTCCGGCTTTCACCCCAGCGACATTGAGGTGGACCTGCTGAAGAACGGCGAGCGGATCGAGAAGGTGGAACACAGCGATCTGAGCTTCAGCAAGGACTGGTCCTTCTACCTGCTGTACTACACCGAGTTCACCCCTACCGAGAAGGACGAGTACGCCTGCAGAGTGAACCACGTGACACTGAGCCAGCCTAAGATCGTGAAGTGGGATCGCGATATGGGCGGAGGCGGATCTGGTGGCGGAGGAAGTGGCGGCGGAGGATCTGGCTCCCACTCCTTGAAGTATTTCCACACTTCCGTGTCCCGGCCCGGCCGCGGGGAGCCCCGCTTCATCTCTGTGGGCTACGTGGACGACACCCAGTTCGTGCGCTTCGACAACGACGCCGCGAGTCCGAGGATGGTGCCGCGGGCGCCGTGGATGGAGCAGGAGGGGTCAGAGTATTGGGACCGGGAGACACGGAGCGCCAGGGACACCGCACAGATTTTCCGAGTGAATCTGCGGACGCTGCGCGGCTACTACAATCAGAGCGAGGCCGGGTCTCACACCCTGCAGTGGATGCATGGCTGCGAGCTGGGGCCCGACGGGCGCTTCCTCCGCGGGTATGAACAGTTCGCCTACGACGGCAAGGATTATCTCACCCTGAATGAGGACCTGCGCTCCTGGACCGCGGTGGACACGGCGGCTCAGATCTCCGAGCAAAAGTCAAATGATGCCTCTGAGGCGGAGCACCAGAGAGCCTACCTGGAAGACACATGCGTGGAGTGGCTCCACAAATACCTGGAGAAGGGGAAGGAGACGCTGCTTCACCTGGAGCCCCCAAAGACACACGTGACTCACCACCCCATCTCTGACCATGAGGCCACCCTGAGGTGCTGGGCCCTGGGCTTCTACCCTGCGGAGATCACACTGACCTGGCAGCAGGATGGGGAGGGCCATACCCAGGACACGGAGCTCGTGGAGACCAGGCCTGCAGGGGATGGAACCTTCCAGAAGTGGGCAGCTGTGGTGGTGCCTTCTGGAGAGGAGCAGAGATACACGTGCCATGTGCAGCATGAGGGGCTACCCGAGCCCGTCACCCTGAGATGGAAGCCGGCTTCCCAGCCCACCATCCCCATCGTGGGCATCATTGCTGGCCTGGTTCTCCTTGGATCTGTGGTCTCTGGAGCTGTGGTTGCTGCTGTGATATGGAGGAAGAAGAGCTCAGGTGGAAAAGGAGGGAGCTACTCTAAGGCTGAGTGGAGCGACAGTGCCCAGGGGTCTGAGTCTCACAGCTTGSEQ ID NO: 2HLA-SCT NT363 (Protein)MVVMAPRTLFLLLSGALTLTETWAVMAPRTLILGGGGSGGGGSGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGSHSLKYFHTSVSRPGRGEPRFISVGYVDDTQFVRFDNDAASPRMVPRAPWMEQEGSEYWDRETRSARDTAQIFRVNLRTLRGYYNQSEAGSHTLQWMHGCELGPDGRFLRGYEQFAYDGKDYLTLNEDLRSWTAVDTAAQISEQKSNDASEAEHQRAYLEDTCVEWLHKYLEKGKETLLHLEPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQQDGEGHTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPVTLRWKPASQPTIPIVGIIAGLVLLGSWSGAWAAVIWRKKSSGGKGGSYSKAEWSDSAQGSESHSLSEQ ID NO:3HLA-SCT NT568 (DNA)ATGGTGGTCATGGCCCCTAGAACACTGTTCCTGCTGCTGTCTGGCGCCCTGACACTGACAGAGACATGGGCCGTGATGCCTCCCAGAACCCTGCTCCTGGGCGGCGGTGGTTCAGGCGGAGGAGGTTCAGGAGGAGGGGGTAGTGGAGGTGGTGGTTCTATCCAGCGGACCCCTAAGATCCAGGTGTACAGCAGACACCCCGCCGAGAACGGCAAGAGCAACTTCCTGAACTGCTACGTGTCCGGCTTTCACCCCAGCGACATTGAGGTGGACCTGCTGAAGAACGGCGAGCGGATCGAGAAGGTGGAACACAGCGATCTGAGCTTCAGCAAGGACTGGTCCTTCTACCTGCTGTACTACACCGAGTTCACCCCTACCGAGAAGGACGAGTACGCCTGCAGAGTGAACCACGTGACACTGAGCCAGCCTAAGATCGTGAAGTGGGATCGCGATATGGGCGGAGGCGGATCTGGTGGCGGAGGAAGTGGCGGCGGAGGATCTGGCTCCCACTCCTTGAAGTATTTCCACACTTCCGTGTCCCGGCCCGGCCGCGGGGAGCCCCGCTTCATCTCTGTGGGCTACGTGGACGACACCCAGTTCGTGCGCTTCGACAACGACGCCGCGAGTCCGAGGATGGTGCCGCGGGCGCCGTGGATGGAGCAGGAGGGGTCAGAGTATTGGGACCGGGAGACACGGAGCGCCAGGGACACCGCACAGATTTTCCGAGTGAATCTGCGGACGCTGCGCGGCTACTACAATCAGAGCGAGGCCGGGTCTCACACCCTGCAGTGGATGCATGGCTGCGAGCTGGGGCCCGACAGACGCTTCCTCCGCGGGTATGAACAGTTCGCCTACGACGGCAAGGATTATCTCACCCTGAATGAGGACCTGCGCTCCTGGACCGCGGTGGACACGGCGGCTCAGATCTCCGAGCAAAAGTCAAATGATGCCTCTGAGGCGGAGCACCAGAGAGCCTACCTGGAAGACACATGCGTGGAGTGGCTCCACAAATACCTGGAGAAGGGGAAGGAGACGCTGCTTCACCTGGAGCCCCCAAAGACACACGTGACTCACCACCCCATCTCTGACCATGAGGCCACCCTGAGGTGCTGGGCCCTGGGCTTCTACCCTGCGGAGATCACACTGACCTGGCAGCAGGATGGGGAGGGCCATACCCAGGACACGGAGCTCGTGGAGACCAGGCCTGCAGGGGATGGAACCTTCCAGAAGTGGGCAGCTGTGGTGGTGCCTTCTGGAGAGGAGCAGAGATACACGTGCCATGTGCAGCATGAGGGGCTACCCGAGCCCGTCACCCTGAGATGGAAGCCGGCTTCCCAGCCCACCATCCCCATCGTGGGCATCATTGCTGGCCTGGTTCTCCTTGGATCTGTGGTCTCTGGAGCTGTGGTTGCTGCTGTGATATGGAGGAAGAAGAGCTCAGGTGGAAAAGGAGGGAGCTACTCTAAGGCTGAGTGGAGCGACAGTGCCCAGGGGTCTGAGTCTCACAGCTTGSEQ ID NO: 4HLA-SCT NT568 (Protein)MVVMAPRTLFLLLSGALTLTETWAVMPPRTLLLGGGGSGGGGSGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGSHSLKYFHTSVSRPGRGEPRFISVGYVDDTQFVRFDNDAASPRMVPRAPWMEQEGSEYWDRETRSARDTAQIFRVNLRTLRGYYNQSEAGSHTLQWMHGCELGPDRRFLRGYEQFAYDGKDYLTLNEDLRSWTAVDTAAQISEQKSNDASEAEHQRAYLEDTCVEWLHKYLEKGKETLLHLEPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQQDGEGHTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPVTLRWKPASQPTIPIVGIIAGLVLLGSVVSGAVVAAVIWRKKSSGGKGGSYSKAEWSDSAQGSESHSL

Claims

1. CLAIMSWe claim:

1. An engineered cell, comprising an exogenous HLA single-chain trimer construct (HLA-SCT), a knockout of the endogenous gene encoding CD58, a knockout of the endogenous gene encoding ICAM3, and a knockout of at least one gene encoding a protein or regulator of HLA class I or HLA class II complex.

2. The engineered cell of claim 1, wherein the knockout of the at least one gene encoding a protein or regulator of the HLA class I complex is a knockout of the gene encoding P2M.

3. The engineered cell of claim 1 or claim 2, wherein the knockout of the at least one gene encoding a protein or regulator of the HLA class II complex is a knockout of the gene encoding CIITA.

4. The engineered cell of claim 1, wherein the knockout of at least one gene encoding a protein or regulator of the HLA class I or HLA class II complex comprises a knockout of a gene encoding P2M and a knockout of a gene encoding CIITA.

5. The engineered cell of any one of claims 1-4, wherein the HLA-SCT encodes a non-classical HLA molecule.

6. The engineered cell of any one of claims 1-5, wherein the HLA-SCT encodes a molecule comprising HLA-E.

7. The engineered cell of any one of claims 1-6, wherein the HLA-SCT nucleic acid sequence is SEQ ID NO:1 or SEQ ID NO:3.

8. The engineered cell of any one of claims 1-6, wherein the HLA-SCT encodes a protein with the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4.

9. The engineered cell of any one of claims 1-8, wherein the HLA-SCT is expressed at the GAPDH locus under the control of the endogenous GAPDH promoter.

10. The engineered cell of any one of claims 1-8, wherein the HLA-SCT is expressed at the CIITA locus under the control of a CAG promoter.

11. The engineered cell of any one of claims 1-10, wherein insertion of the HLA-SCT into the cell is monoallelic or biallelic.

12. The engineered cell of any one of claims 1-11, wherein the cell is an induced pluripotent stem cell (iPSC).

13. The engineered cell of any one of claims 1-12, wherein the cell is derived from an iPSC.

14. The engineered cell of claim 13, wherein the cell derived from the iPSC is a T cell.

15. The engineered cell of any one of claims 1-14, 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).

16. The engineered cell of any one of claims 1-15, wherein the cell exhibits increased viability in the presence of natural killer (NK) cells compared to a non-engineered cell.

17. The engineered cell of any one of claims 1-15, wherein the cell exhibits increased viability in the presence of natural killer (NK) cells compared to a cell lacking one or more of the exogenous HLA- SCT, the knockout of the endogenous gene encoding CD58, and the knockout of the endogenous gene encoding ICAM3.

18. A population of cells comprising the engineered cells of any one of claims 1-17.

19. A use of the engineered cell of any one of claims 1-17 in the treatment of a disease or a condition in a subject.

20. A pharmaceutical composition comprising the engineered cell of any one of claims 1-17 and a pharmaceutically acceptable carrier.

21. An engineered cell, comprising an exogenous HLA single-chain trimer construct (HLA-SCT), a knockout or knockdown of the endogenous gene encoding CD58, a knockout or knockdown of the endogenous gene encoding ICAM3, and a knockout or knockdown of at least one gene encoding a protein or regulator of HLA class I or HLA class II complex.

22. The engineered cell of claim 21, wherein the knockout or knockdown of the at least one gene encoding a protein or regulator of the HLA class I complex is a knockout of the gene encoding P2M.

23. The engineered cell of claim 21 or claim 22, wherein the knockout or knockdown of the at least one gene encoding a protein or regulator of the HLA class II complex is a knockout of the gene encoding CIITA.

24. The engineered cell of claim 21, wherein the knockout or knockdown of at least one gene encoding a protein or regulator of HLA class I or HLA class II complex comprises a knockout or knockdown of a gene encoding P2M and a knockout or knockdown of a gene encoding CIITA.

25. The engineered cell of any one of claims 21-24, wherein the HLA-SCT encodes a non-classical HLA molecule.

26. The engineered cell of any one of claims 21-25, wherein the HLA-SCT encodes a molecule comprising HLA-E.

27. The engineered cell of any one of claims 21-26, wherein the HLA-SCT nucleic acid sequence is SEQ ID NO:1 or SEQ ID NO:3.

28. The engineered cell of any one of claims 21-26, wherein the HLA-SCT encodes a protein with the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4.

29. The engineered cell of any one of claims 21-28, wherein the HLA-SCT is expressed at the GAPDH locus under the control of the endogenous GAPDH promoter.

30. The engineered cell of any one of claims 21-28, wherein the HLA-SCT is expressed at the CIITA locus under the control of a CAG promoter.

31. The engineered cell of any one of claims 21-30, wherein insertion of the HLA-SCT into the cell is monoallelic or biallelic.

32. The engineered cell of any one of claims 21-31, wherein the cell is an induced pluripotent stem cell (iPSC).

33. The engineered cell of any one of claims 21-32, wherein the cell is derived from an iPSC.

34. The engineered cell of claim 33, wherein the cell derived from the iPSC is a T cell.

35. The engineered cell of any one of claims 21-34, 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).

36. The engineered cell of any one of claims 21-35, wherein the cell exhibits increased viability in the presence of natural killer (NK) cells compared to a non-engineered cell.

37. The engineered cell of any one of claims 21-35, wherein the cell exhibits increased viability in the presence of natural killer (NK) cells compared to a cell lacking one or more of the exogenous HLA- SCT, the knockout or knockdown of the endogenous gene encoding CD58, the knockout or knockdown of the endogenous gene encoding ICAM3.

38. A population of cells comprising the engineered cells of any one of claims 21-37.

39. A use of the engineered cell of any one of claims 21-37 in the treatment of a disease or a condition in a subject.

40. A pharmaceutical composition comprising the engineered cell of any one of claims 21-37 and a pharmaceutically acceptable carrier.

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