Combination engineered cell therapy

Abstract

The present disclosure provides a combination engineered cell therapy comprising pluripotent stem cell-derived cells engineered to express an inert cell surface protein and regulatory T cells engineered to express a chimeric antigen receptor specific for said cell surface protein. Further provided are methods of treating a disease, such as type 1 diabetes, by administering said combination engineered cell therapy.

Description

[0001] DESCRIPTION

[0002] COMBINATION ENGINEERED CELL THERAPY

[0003] PRIORITY CLAIM

[0004] This application claims benefit of priority to U.S. Provisional Application Serial No. 63 / 583,706, filed September 19, 2023, the entire contents of which are hereby incorporated by reference.

[0005] STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

[0006] This invention was made with U.S. Government support under grant no. RO1 DK120444 and TR001451, awarded by the National Institutes of Health. The government has certain rights in the invention.

[0007] BACKGROUND

[0008] This invention was created whole or in part from funding received under Award No. 3- APF-2024-1492-A-N from Breakthrough T1D.

[0009] 1. Field

[0010] The disclosure relates generally to the field of molecular biology. More particularly, it concerns cell therapy comprising regulatory T cells and stem cell-derived cells, such as for the treatment of Type 1 diabetes (T1D).

[0011] 2. Related Art

[0012] Type 1 diabetes (T1D) is a devastating disease caused by autoimmune attack of the insulinproducing pancreatic beta cells. Between five and ten percent of all diabetes cases are type 1 , which imposes an annual economic burden of more than $16 billion on the U.S. healthcare system (1). Although type 1 diabetes (T1D) can be managed with diet and medication, including 2-4 insulin injections per day, a cure remains elusive, and it cannot be prevented or reversed in humans. Transplanting pancreatic islets obtained from cadaveric donors results in insulin independence for approximately 3 years in patients (2). Yet, insufficient donor pancreas availability, donor histoincompatibility, and the recipient’s alloimmunity and autoimmunity against the transplanted cadaveric islets greatly limit the widespread use of this approach (3).

[0013] Protocols have been developed to derive human pancreatic beta-like cells from an inexhaustible source: human pluripotent stem cells (hPSCs) (4). To begin to address immune rejection of transplanted hPSC-derived beta-like cells (sBCs), CRISPR / Cas9 genome editing was used in hPSCs to delete B2M, whose protein product is required for HLA class I surface expression and hence recognition by cytotoxic CD8+ T cells, and to integrate PD-L1, whose protein product is a potent suppressor of T cell function. The resulting differentiated genome-edited sBCs were significantly protected from human diabetogenic CD8+ T cells (5). Nevertheless, the autoimmune response in type 1 diabetes involves multiple cell types, including natural killer (NK) cells, which kill precisely cells lacking HLA surface expression. Moreover, complete ablation of HLA expression in transplanted cells poses the risk of creating tissue that cannot be effectively surveilled by the immune system in case of viral infection or cancer formation. Hence, it would be more desirable to rather induce immune tolerance, i.e., unresponsiveness specifically towards transplanted islets.

[0014] Pancreas and pancreatic islet transplantation are the only curative treatments for type 1 diabetes (T1D), an autoimmune disease where the patient’s immune system destroys the insulinproducing pancreatic beta cells. The severe shortage of pancreas and islet donors has fueled interest in alternative islet sources, chiefly human pluripotent stem cells (hPSC), which would represent an inexhaustible source of beta cells (Sneddon et al., 2018). However, allogeneic (caused by genetic mismatches between islet donor and recipient) and autoimmune (caused by the recipient’ s immune system attack towards islets) immune rejection signify that patients have to subject themselves to life-long immunosuppressant drug regimens, with their associated toxicities and comorbidities (Burrack et al., 2017).

[0015] Regulatory T cells (Tregs), a subset of CD4+T cells, are extremely powerful inhibitory cells, harnessing a multitude of mechanisms to provide peripheral immune suppression (Ferreira el al., 2019). While harnessing these cells holds great promise to create therapies to suppress unwanted immunity, such as autoimmunity and organ transplant rejection, the efficacy of current Treg therapy approaches is low due to generally low abundance of antigen specific Tregs. To address this issue, Tregs can be redirected using artificial immune receptors, such as a chimeric antigen receptor (CAR), to specific cells and tissues to protect them from immune attack (Muller etal., 2021 ). However, one of the biggest challenges in CAR design is identifying a target molecule uniquely expressed by the desired target cells.

[0016] SUMMARY

[0017] In certain embodiments, the present disclosure provides a cell therapy composition comprising pluripotent stem cell (PSC) derived cells engineered to express a cell surface protein or fragment thereof and regulatory T cells (Tregs) engineered to express a chimeric antigen receptor (CAR) to said cell surface protein or fragment thereof.

[0018] In some aspects, the PSCs are induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs). In certain aspects, the PSC are human PSCs. In some aspects, the PSC-derived cells contain a cell surface protein or fragment thereof knocked-in at the AAVS1, CCR5, hRosa26, Rogil (GHS1), Rogi2 (GSH2), GSH7, GSH8, GSH31, TRAC, HSH323, HSH325, HSH289, HSH227, HSH229, HSH253, HSH255, HSH257, HSH259, HSH261, HSH263, HSH317,

[0019] HSH3O3, HSH331, HSH283, HSH231, HSH315, HSH327, HSH305, HSH307, HSH309,

[0020] HSH285, HSH233, HSH311, HSH299, HSH301, HSH293, HSH319, HSH333, HSH295,

[0021] HSH329, HSH297, HSH291, HSH313, HSH321 or GAPDH safe harbor locus of the PSC.

[0022] In certain aspects, the Tregs are human Tregs. In some aspects, the Tregs are autologous. In some aspects, the Tregsare allogeneic. In some aspects, the human TiegSare CD4+CD25+CD127‘ cells activated with anti-CD3 / CD28-coated magnetic beads and interleukin-2 (IL-2).

[0023] In some aspects, the CAR construct comprises a cell surface protein or fragment thereof selected from the group consisting of F(ab’)2, Fab’, Fab, Fv, and scFv. In specific aspects, the cell surface protein is biologically inert. In some aspects, the cell surface protein is a cell surface receptor, ligand, glycoprotein, cell adhesion molecule, antigen, integrin or cluster of differentiation (CD). In specific aspects, the cell surface protein is epidermal growth factor receptor (EGFR), an erbB-2 receptor tyro sine-protein kinase (errb2, HER2), an erbB-3 receptor tyrosine-protein kinase, an erbB-4 receptor tyrosine-protein kinase, a hepatocyte growth factor receptor (HGFR / c-MET), an insulin-like growth factor receptor-1 (IGF-1 R), EpCAM, VEGFR, integrin (e.g., integrins avP3, a4, allbp3, a4p7, a5P 1, avP3, av), a member of the TNF receptor superfamily (e.g., TRAIL- Rl, TRAIE-R2), a member of the epidermal growth factor receptor family, PDGF Receptor, interferon receptor, folate receptor, GPNMB, ICAM-1, HLA-DR, CEA, CA-125, MUC1, TAG- 72, IL-6 receptor, 5T4, GD2, GD3, prostate-specific membrane antigen (PSMA), or clusters of differentiation (e.g., CD2, CD3, CD4, CD5, CD11, CDl la / LFA-1, CD15, CD18 / ITGB2, CD19, CD20, CD22, CD23 / IgE Receptor, CD25, CD28, CD30, CD33, CD38, CD40, CD41 , CD44, CD51, CD52, CD62L, CD74, CD80, CD125, CD147 / basigin, CD152 / CTLA-4, CD154 / CD40L, CD195 / CCR5 or CD319 / SLAMF7.

[0024] In some aspects, the cell surface protein is epidermal growth factor receptor (EGFR). In particular aspects, the EGFR is a truncated EGFR (EGFRt). In some aspects, the EGFRt comprises SEQ ID NO:1. In certain aspects, the CAR contains an anti-EGFR cetuximab single chain fragment variable (scFv).

[0025] In some aspects, the CAR comprises a CD28-CD3S, intracellular domain. In certain aspects, the CAR further comprises a reporter protein. In some aspects, the reporter protein is linked to the cell surface protein or fragment thereof by a peptide linker. In certain aspects, the peptide linker is a 2A peptide linker.

[0026] In certain aspects, the PSC-derived cells comprise one or more cell types. In some aspects, the PSC-derived cells form a tissue or organ. In specific aspects, the PSC-derived cells are insulinproducing beta cells. In some aspects, the PSC-derived cells are further engineered to comprise a deletion of P? microglobulin. In certain aspects, the PSC-derived cells are further engineered to express an immune checkpoint protein. In some aspects, the immune checkpoint protein is CD39, CD47, CD73, CTLA-4, HLA-G, HLA-E, PD-1, PD-L1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3, KIR, or A2aR. In particular aspects, the immune checkpoint protein is PD-L1.

[0027] A further embodiment provides a pharmaceutical composition comprising the composition of the present embodiments and aspects thereof (e.g., a cell therapy composition comprising pluripotent stem cell (PSC) derived cells engineered to express a cell surface protein or fragment thereof and regulatory T cells (Tregs) engineered to express a chimeric antigen receptor (CAR) to said cell surface protein or fragment thereof) and a pharmaceutical carrier.

[0028] Another embodiment provides a composition comprising an effective amount of the composition of the present embodiments and aspects thereof for use in the treatment of a disease in a subject. In some aspects, the disease is an autoimmune disease. In certain aspects, the autoimmune disease is type I diabetes.

[0029] In yet another embodiment, a method of treating a disease in a subject comprising administering a cell therapy composition comprising PSC-derived cells engineered to express a cell surface protein or fragment thereof and Tregs engineered to express a CAR to said cell surface protein or fragment thereof is provided.

[0030] In some aspects, the PSCs are induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs). In certain aspects, the PSC are human PSCs. In some aspects, the PSC-derived cells were obtained by knocking in the cell surface protein or fragment thereof at the AAVS1, CCR5, hRosa26, Rogil (GHS1), Rogi2 (GSH2), GSH7, GSH8, GSH31, TRAC, HSH323, HSH325,

[0031] HSH289, HSH227, HSH229, HSH253, HSH255, HSH257, HSH259, HSH261, HSH263,

[0032] HSH317, HSH3O3, HSH331, HSH283, HSH231, HSH315, HSH327, HSH305, HSH307,

[0033] HSH309, HSH285, HSH233, HSH311, HSH299, HSH301, HSH293, HSH319, HSH333,

[0034] HSH295, HSH329, HSH297, HSH291, HSH313, HSH321 or GAPDH safe harbor locus of the PSC.

[0035] In certain aspects, the Tregs are human Tregs. In some aspects, the Tregs are autologous. In some aspects, the Tregs are allogeneic. In some aspects, the human Tregs were isolated from human peripheral blood by sorting for CD4+CD25+CD127‘ cells and activating them with anti- CD3 / CD28-coated magnetic beads and interleukin-2 (IL-2).

[0036] In some aspects, the CAR construct comprises a cell surface protein or fragment thereof selected from the group consisting of F(ab’)2, Fab’, Fab, Fv, and scFv. In specific aspects, the cell surface protein is biologically inert. In some aspects, the cell surface protein is a cell surface receptor, ligand, glycoprotein, cell adhesion molecule, antigen, integrin or cluster of differentiation (CD). In specific aspects, the cell surface protein is epidermal growth factor receptor (EGFR), an erbB-2 receptor tyro sine-protein kinase (errb2, HER2), an erbB-3 receptor tyro sine-protein kinase, an erbB-4 receptor tyrosine-protein kinase, a hepatocyte growth factor receptor (HGFR / c-MET), an insulin-like growth factor receptor-1 (IGF-1 R), EpCAM, VEGFR, integrin (e.g., integrins avP3, a4, allbp3, a4p7, a5pi, avP3, av), a member of the TNF receptor superfamily (e.g., TRAIL- Rl, TRAIL-R2), a member of the epidermal growth factor receptor family, PDGF Receptor, interferon receptor, folate receptor, GPNMB, ICAM-1, HLA-DR, CEA, CA-125, MUC1, TAG- 72, IL-6 receptor, 5T4, GD2, GD3, prostate-specific membrane antigen (PSMA), or clusters of differentiation (e.g., CD2, CD3, CD4, CD5, CD11, CDl la / LFA-1, CD15, CD18 / ITGB2, CD19, CD20, CD22, CD23 / IgE Receptor, CD25, CD28, CD30, CD33, CD38, CD40, CD41, CD44, CD51 , CD52, CD62L, CD74, CD80, CD125, CD147 / basigin, CD152 / CTLA-4, CD154 / CD40L, CD195 / CCR5 or CD319 / SLAMF7.

[0037] In some aspects, the cell surface protein is epidermal growth factor receptor (EGFR). In particular aspects, the EGFR is a truncated EGFR (EGFRt). In some aspects, the EGFRt comprises SEQ ID NO:1. In certain aspects, the CAR contains an anti-EGFR cetuximab single chain fragment variable (scFv).

[0038] In some aspects, the CAR comprises a CD28-CD3^ intracellular domain. In certain aspects, the CAR further comprises a reporter protein. In some aspects, the reporter protein is linked to the cell surface protein or fragment thereof by a peptide linker. In certain aspects, the peptide linker is a 2A peptide linker.

[0039] In certain aspects, the PSC-derived cells comprise one or more cell types. In some aspects, the PSC-derived cells form a tissue or organ. In specific aspects, the PSC-derived cells are insulinproducing beta cells. In some aspects, the PSC-derived cells are further engineered to comprise a deletion of P2 microglobulin. In certain aspects, the PSC-derived cells are further engineered to express an immune checkpoint protein. In some aspects, the immune checkpoint protein is CD39, CD47, CD73, CTLA-4, HLA-G, HLA-E, PD-1, PD-L1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3, KIR, or A2aR. In particular aspects, the immune checkpoint protein is PD-L1.

[0040] In some aspects, the cell therapy composition is administered intravenously, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, percutaneously, subcutaneously, regionally, or by direct injection or perfusion. In some aspects, the cell therapy composition is delivered intravenously or subcutaneously.

[0041] In additional embodiment, the method further comprises administering at least one additional therapy to the subject. In specific aspects, the CAR Tregs and PSC-derived cells are administered more than once.

[0042] A further embodiment provides a method of preventing allogeneic immune rejection upon transplantation of PSC-derived cells by administering a cell therapy composition of the present embodiments or aspects thereof.

[0043] In some aspects, the PSCs are induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs). In certain aspects, the PSC are human PSCs. In some aspects, the PSC-derived cells were obtained by knocking in the cell surface protein or fragment thereof at the AAVS1, CCR5, hRosa26, Rogil (GHS1), Rogi2 (GSH2), GSH7, GSH8, GSH31, TRAC, HSH323, HSH325, HSH289, HSH227, HSH229, HSH253, HSH255, HSH257, HSH259, HSH261 , HSH263,

[0044] HSH317, HSH3O3, HSH331, HSH283, HSH231, HSH315, HSH327, HSH305, HSH307,

[0045] HSH309, HSH285, HSH233, HSH311, HSH299, HSH301, HSH293, HSH319, HSH333,

[0046] HSH295, HSH329, HSH297, HSH291, HSH313, HSH321 or GAPDH safe harbor locus of the

[0047] PSC.

[0048] In certain aspects, the Tregs are human Tregs. In some aspects, the Tregsarc autologous. In some aspects, the Tregsare allogeneic. In some aspects, the human Tregs were isolated from human peripheral blood by sorting for CD4+CD25+CD127‘ cells and activating with anti-CD3 / CD28- coated magnetic beads and interleukin-2 (IL-2).

[0049] In some aspects, the CAR construct comprises a cell surface protein or fragment thereof selected from the group consisting of F(ab’)2, Fab’, Fab, Fv, and scFv. In specific aspects, the cell surface protein is biologically inert. In some aspects, the cell surface protein is epidermal growth factor receptor (EGFR). In particular aspects, the EGFR is a truncated EGFR (EGFRt). In some aspects, the EGFRt comprises SEQ ID NO:1. In certain aspects, the CAR contains an anti-EGFR cetuximab single chain fragment variable (scFv).

[0050] In some aspects, the CAR comprises a CD28-CD3S, intracellular domain. In certain aspects, the CAR further comprises a reporter protein. In some aspects, the reporter protein is linked to the cell surface protein or fragment thereof by a peptide linker. In certain aspects, the peptide linker is a 2A peptide linker.

[0051] In certain aspects, the PSC-derived cells comprise one or more cell types. In some aspects, the PSC-derived cells form a tissue or organ. In specific aspects, the PSC-derived cells are insulinproducing beta cells. In some aspects, the PSC-derived cells are further engineered to comprise a deletion of 02 microglobulin (B2M). In certain aspects, the PSC-derived cells are further engineered to express an immune checkpoint protein. In some aspects, the immune checkpoint protein is CD39, CD47, CD73, CTLA-4, HLA-G, HLA-E, PD-1, PD-L1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3, KIR, or A2aR. In particular aspects, the immune checkpoint protein is PD- Ll.

[0052] It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein. For example, a compound synthesized by one method may be used in the preparation of a final compound according to a different method.

[0053] Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

[0054] BRIEF DESCRIPTION OF THE DRAWINGS

[0055] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0056] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0057] FIG. 1: Co-engineering human pluripotent stem cells and regulatory T cells to provide localized immune protection to stem cell-derived cells and tissues. Human pluripotent stem cells were modified to express a unique CAR antigen, a truncated form of epidermal growth factor receptor (EGFRt) and differentiated into beta cells (Et-sBC). In parallel, human regulatory T cells (Tregs) were engineered to express a chimeric antigen receptor (CAR) recognizing EGFRt. Activation of EGFRt CAR Tregs by Et-sBC resulted in inhibition of effector immune cells, such as cytotoxic CD8+T cells and helper CD4+T cells, which recognize cells via interaction of their T cell receptor (TCR) with peptide-bound to human leukocyte antigen (HLA) on the surface of the target cell, as well as natural killer (NK) cells and macrophages. Tregs use multiple mechanisms to suppress immune responses, including secretion of the anti-inflammatory cytokines interleukin- 10 (IL- 10) and transforming growth factor beta (TGF-P).

[0058] FIGS. 2A-2N: Genetically engineered EGFRt-hPSC efficiently differentiate into stem cell-derived beta-like cells. FIG. 2A) Schematic for TALEN-mediated genome recombination for insertion of constitutive luciferase reporter and EGFRt constructs into MelllNS-GFP hPSC line. FIG. 2B) Immunofluorescence staining for EGFR / EGFRt, OCT3 / 4, NANOG, and DAPI in WT or EGFRt-hPSCs at 20x magnification. FIG. 2C) Representative triplicate luciferase assay and quantification of relative light units (RLUs) in WT and EGFRt-hPSC. FIG. 2D) Flow cytometry of C-peptide expression on day 23 of differentiation in WT and EGFRt-sBC. FIG. 2E) Representative live brightfield (BF) images of clusters on day 23 as well as the GFP insulin reporter (pINS.GFP) for WT and EGFRt-expressing cells. FIG. 2F) Immunofluorescence staining of day 23 WT and EGFRt-sBC for Insulin, PDX1, and NKX6.1 merged with DAPI. FIG. 2G) Flow cytometry quantification of EGFR surface expression (MFI, mean fluorescence intensity) in WT and EGFRt-sBC on day 23. FIGS. 2H-M) Gene expression analysis of hormones INS, GCG, and SST and transcription factors PDX1, NKX6.1, and NEUR0D1 within WT and EGFRt sBC on day 23. FIG. 2N) Dynamic glucose stimulated insulin secretion assay on day 30 WT and EGFRt sBC normalized to insulin content. Data arc representative of at least 3 independent experiments. Data analyzed by unpaired t-test. ns = not significant; ** p<0.01.

[0059] FIGS. 3A-3R: Generation of EGFRt-hPSC line and characterization of EGFRt sBC. FIG. 3A) Biallelic knock-in strategy using TALEN-mediated recombination at the AAVS1 locus to integrate EGFRt and firefly luciferase along with antibiotic resistance genes for selection. FIG. 3B) EGFRt surface expression detected by Row cytometry. FIG. 3C) Bright field images from early stages of sBC differentiation of WT and EGFRt hPSC lines. FIG. 3D) Flow cytometry of pluripotency markers TRA-1-60 and SOX2 on day 0 of differentiation. FIG. 3E) Flow cytometry of definitive endoderm markers SOX17 and FOXA2 on day 3 of differentiation. FIG. 3F) Flow cytometry of PDX1 expression on day 23 of differentiation. FIG. 3G) Flow cytometry of NKX6.1 expression within PDX1+ cells on day 23 of differentiation. FIG. 3H) Flow cytometry of NKX6.1 expression within the C-peptide+ cells on day 23 of differentiation. FIG. 31) Flow cytometry of EGFR expression on day 23 of differentiation within EGFRt sBC. Grey histogram is staining control. FIGS. 3J-R) Gene expression analysis of NKX2.2 (J), NEUROG3 (K), CHGA (L), IAPP (M), ISL1 (N), GC (O), KCNJ11 (P), SLC18A (Q), SLC30A8 (R).

[0060] FIG. 4: Workflow for the generation and testing of human CAR Tregs. Human Tregs are isolated from human peripheral blood, activated, transduced with CAR lentivirus (e.g. EGFR CAR), expanded, and tested in immune assays. The peak of Treg activation is two days post stimulation with anti-CD3 / CD28 beads and IL-2 and the cells take 9-12 days to return to a resting state, at which time they are activated again via either the endogenous TCR / CD28 or the CAR in immune assays.

[0061] FIG. 5: Gene map of EGFR CAR construct. An EF1A strong promoter was used to drive expression of an EGFR CAR consisting of a signal peptide (SP), a Myc-tag, an anti-EGFR cetuximab single chain fragment variable (scFv), a CD8 hinge domain (H), a CD28 transmembrane domain (TM), a CD28 intracellular domain, and a CD3zeta intracellular domain. CAR expression was linked to mCherry expression by a 2A peptide.

[0062] FIGS. 6A-6F: EGFR CAR-Tregs are stable after transduction and respond to EGFRt-expressing target cells. FIG. 6A) EGFR CAR construct and schematic for lentiviral CAR transduction approach into regulatory human T cells. FIG. 6B) Representative flow cytometry plot and quantification graph of transduction efficiency employing CAR mCherry reporter expression in untransduccd (UT) and CAR transduced regulatory T cells (Trcgs). FIG. 6C) Flow cytometry and quantification of Treg identity markers HELIOS and FOXP3 expression in untransduced (UT) effector T cells (Teff), UT Tregs, CAR-Teff cells, or CAR-Tregs. FIG. 6D) Flow cytometry analysis of activation markers CD25 and CD71 expression on CAR-Tregs cultured with control (WT) or EGFRt-expressing K562 cells. FIG. 6E) Quantification of flow cytometry data for FOXP3 and HELIOS expression 9 days after stimulation with anti-CD3 / CD28 beads or EGFRt-K562 cells. FIG. 6F) Flow cytometry analysis of FOXP3 and HELIOS expression in CAR-Tregs during repeated stimulation with either anti-CD3 / CD28 beads or EGFRt-K562 cells. Data are representative of at least 2 independent experiments with triplicate wells. Data analyzed by two- way ANOVA with multiple comparisons or t-test where appropriate, ns = not significant; * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001.

[0063] FIG. 7: EGFR CAR Tregs are specifically activated in the presence of EGFRt- expressing human pluripotent stem cells (Et-hPSCs) in vitro. Histograms with surface expression levels of CD71 in either UT Tregs or EGFR CAR Tregs alone or co-incubated with Et- hPSCs or anti-CD3 / CD28 beads for 48h.

[0064] FIG. 8: EGFR CAR Tregs are specifically activated in the presence of EGFRt- expressing human pluripotent stem cell-derived beta cells (Et-sBCs) in vitro. EGFR CAR Tregs are marked by mCherry expression (mCherry, red), while Et-sBCs are marked by insulin expression (GFP, green). The circles in the merged imaged indicate the interaction between EGFR CAR-expressing Tregs and insulin-expressing Et-sBCs. In the bottom right, histograms with surface expression levels of the early activation marker CD69 in either UT Tregs or EGFR CAR Tregs alone or co-incubated with Et-sBCs or anti-CD3 / CD28 beads for 24h.

[0065] FIGS. 9A-9L: CAR-Tregs cultured with EGFRt-sBC reduce dendritic cell responses and effector T cell proliferation. FIGS. 9A-B) Flow quantification of activation markers CD71 and CD69 expression in Tregs after 48-hour co-culture with EGFRt-sBC. FIGS. 9C-G) Quantification of IL-10 (C), IL-13 (D), MCP-1 (E) protein levels in supernatants from co-cultures. FIG. 9F) Flow quantification of cleaved caspase 3 frequency in sBC after co-culture. FIG. 9G) Schematic for CAR-Treg suppression of dendritic cells (moDCs) in co-culture. FIG. 9H) Flow cytometry analysis of CD80 and CD86 expression within CD83+ moDCs. FIG. 9I&J) Quantification of IL-12p40 (I), and IL-15 (J) protein levels in supernatants from co-cultures with moDCs. FIG. 9K) Schematic for CAR-Treg-mediated suppression of T responder (Tresp) cell proliferation after being stimulated by EGFRt-sBC. FIG. 9L) Quantification of suppression of CAR CD4+ (white boxes) or CAR CD8+ (black boxes) Tresp cells compared to Tresp cells cultured alone at different Treg to Tresp cell ratios. Data are representative of at least 3 independent experiments. Data analyzed by two-way ANOVA with multiple comparisons, ns = not significant; * p<0.05; ** p<0.01; *** p<0.001; **** pcO.OOOl.

[0066] FIGS. 10A-10G: CAR-Tregs are activated by EGFRt-hPSC. FIG. 10A) Flow cytometry analysis of CD71 surface expression after co-culture of untransduced (UT) or CAR- Tregs with EGFRt-hPSC. FIG. 10B) Flow cytometry analysis CD80 and CD86 expression within CD83+ moDCs after co-culture with UT or CAR-Tregs activated by EGFRt-hPSC. FIG. IOC) Flow cytometry analysis CD80 and CD86 expression within CD83+ moDCs after co-culture with UT or CAR-Tregs activated by beads or EGFRt sBC. FIG. 10D) Representative flow plots of FOXP3 and HELIOS expression in Tregs after repeated stimulation with beads or EGFRt K562 cells. FIGS. 10E-G) Quantification of IFNg (E), IL-17A (F), IL-17F (G) protein levels in supernatants from co-cultures in D). Data analyzed by two-way ANOVA corrected for multiple comparisons, ns = not significant; * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001.

[0067] FIG. 11: Experimental strategy to demonstrate EGFRt-sBC protection by EGFR CAR Treg in vivo. Luciferase-labeled EGFRt-expressing stem cell-derived beta cells (sBCs) were transplanted into the kidney capsule of immunodeficient NSG mice. Three weeks later, after the EGFRt-sBCs have engrafted, mice were challenged intravenously with either EGFR chimeric antigen receptor (CAR) effector T (Teff) cells alone or with EGFR CAR regulatory T cells (Tregs) at a 1:2 Teff:Treg ratio. Mice were imaged for luciferase activity three times per week to monitor EGFRt-sBC graft survival. Four weeks after immune challenge, mice were euthanized and the spleen and grafted kidney collected for analysis by histology and flow cytometry.

[0068] FIGS. 12A-12H: CAR-Tregs traffic to EGFRt-sBC grafts and suppress CAR-T effector cell-mediated graft destruction. FIG. 12A) Schematic for transplantation study using EGFRt-sBC grafts and infusion of CAR-Teff cells + / - CAR-Tregs. FIG. 12B) In vivo luciferase imaging at different days post-T-cell infusion. FIGS. 12C&D) Quantification of luciferase bioluminescence as a percentage of luciferase intensity before T-cell infusion and area under the curve (AUC) analysis of the data. FIG. 12B-D) Data from two independent cohorts with N = 13 total mice. FIG. 12E) Immunofluorescence staining of EGFRt sBC grafts after T-cell infusion for insulin, CD3, F0XP3, and DAPI. Scale bar is 100 pm. FIG. 12F) Immunofluorescence staining of EGFRt sBC grafts after T-cell infusion for insulin, EGFR, HNA, and DAPI. Scale bar is 100 pm. FIGS. 12G&H) Flow cytometry analysis for FOXP3+ cell frequencies of total CD4+ cells in the spleen or in grafts of transplanted mice 4 weeks post T-cell infusion. Data analyzed by unpaired t- test, ns = not significant; * p<0.05; ** p<0.01; **** p<0.0001.

[0069] FIG. 13: FOXP3+EGFR CAR Tregs localize to sBC grafts transplanted in the kidney capsule of NSG mice. Human CD4+mCherry+T cells were found in the kidney capsule and spleen one month post injection. The kidney of EGFR CAR Treg-treated mice contained FOXP3+ Tregs, as detected by flow cytometry. FIG. 14: CAR is required for Treg protection of and localization to sBC grafts.

[0070] EGFRt-sBC graft survival, as indicated by luciferase activity, was only maintained in the presence of EGFR CAR Tregs, not with EGFR CAR Teff alone or with EGFR CAR Teff and untransduced (UT) Tregs at a 1:2 ratio of Teff:Treg. EGFR CAR Tregs, but not UT Tregs, could be found in the grafted kidney four weeks after intravenous infusion, as assessed by the presence of FOXP3+cells.

[0071] DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0072] The present studies provided in vitro and in vivo proof-of-concept that human pluripotent stem cells (hPSCs) and regulatory T cells (Tregs) can be co-engineered to protect target cells from immune rejection upon transplantation. Such strategies can allow for directed protection of a multitude of stem cell-derived tissues without off-target suppression of other immune responses, having implications for multiple fields of study. This novel dual engineering approach can be used to synergize with or be a safer alternative to hypoimmunogenic cells for cell replacement therapies. In the diabetes realm, this strategy provides a solution for both beta cell replacement and immune tolerance re-establishment to treat type 1 diabetes (T1D). Thus, in certain embodiments, the present disclosure provides a method to specifically protect stem cell-derived cells, tissues, and organs from immune rejection by the recipient without requiring global immune suppression.

[0073] To overcome this obstacle, the present inventors engineered a unique, biologically inert surface bait on hPSCs, that can be specifically recognized by a chimeric antigen receptor (CAR). In parallel, human Tregs were modified with a CAR recognizing this unique surface bait. This innovative combinatorial engineering strategy has the potential for providing effective, localized immune suppression for any stem cell-derived tissue, including stem cell derived beta-like cells (sBC) (Castro-Gutierrez et al., 2021).

[0074] Thus, in certain embodiments, the present disclosure provides hPSCs engineered to express a unique artificial surface marker which can be transplanted with CAR Tregs recognizing said marker. Specifically, a gene coding for an inert cell surface protein may be knocked-in in a safe harbor locus in hPSCs which may be differentiated to a specific cell type, such as insulin-producing beta cells. Further, Tregs may be isolated from human peripheral blood and engineered with a CAR targeting said inert cell surface protein in them using lentivirus. These engineered beta cellspecific CAR Tregs can then be co-infused with the engineered beta cells to protect them from immune attack. Therefore, hPSCs and Tregs can be co-engineered to protect differentiated target cells from allogeneic immune rejection upon transplantation.

[0075] The present engineered beta cells with engineered CAR Tregs that specifically protect the engineered beta cells hold the potential to re-establish insulin independence and avoid global immunosuppression. In some aspects, hPSC lines also express additional immune modulating molecules, e.g., PD-L1. The present combination engineered cell therapy can be used to address both beta cell replacement and immune tolerance rc-cstablishmcnt to treat T1D. Simply transplanting beta cells into T1D patients will result in their rejection from both allogeneic rejection and autoimmunity. Combining such transplants with lifelong immunosuppression will leave the patients susceptible to infections and cancer for life, a particularly nefarious price to pay since T1D patients are often children and adolescents. Transplanting beta cells engineered to be opaque to the immune system by deleting their major histocompatibility complex (MHC) expression and / or overexpressing immune checkpoint inhibitor proteins may prolong their life, but if these transplanted cells become cancerous or the target of a virus infection, the immune system will be incapable to clear them, endangering the patients. Immune therapy to quell autoimmunity alone is unlikely to help most cases of T1D, as by the time of T1D diagnosis a significant portion of the beta cell mass has already been lost and beta cells in adults have next to non-existent replicative potential (<1%).

[0076] I. Definitions

[0077] As used herein, “essentially free,” in terms of a specified component, is used herein to mean that none of the specified component has been purposefully formulated into a composition and / or is present only as a contaminant or in trace amounts. The total amount of the specified component resulting from any unintended contamination of a composition is therefore well below 0.05%, preferably below 0.01%. Most preferred is a composition in which no amount of the specified component can be detected with standard analytical methods.

[0078] As used herein the specification, “a” or “an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising,” the words “a” or “an” may mean one or more than one.

[0079] The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or.” As used herein “another” may mean at least a second or more.

[0080] The term “about” means, in general, within a standard deviation of the stated value as determined using a standard analytical technique for measuring the stated value. The terms can also be used by referring to plus or minus 5% of the stated value.

[0081] The phrase “effective amount” or “therapeutically effective” means a dosage of a drug or agent sufficient to produce a desired result. The desired result can be subjective or objective improvement in the recipient of the dosage, increased lung growth, increased lung repair, reduced tissue edema, increased DNA repair, decreased apoptosis, a decrease in tumor size, a decrease in the rate of growth of cancer cells, a decrease in metastasis, or any combination of the above.

[0082] As used herein, the term “antibody” refers to an immunoglobulin, derivatives thereof which maintain specific binding ability, and proteins having a binding domain which is homologous or largely homologous to an immunoglobulin binding domain. These proteins may be derived from natural sources, or partly or wholly synthetically produced. An antibody may be monoclonal or polyclonal. The antibody may be a member of any immunoglobulin class, including any of the human classes: IgG, IgM, IgA, IgD, and IgE. The antibody may be a bi-specific antibody. In exemplary embodiments, antibodies used with the methods and compositions described herein are derivatives of the IgG class. The term antibody also refers to antigen-binding antibody fragments. Examples of such antibody fragments include, but are not limited to, Fab, Faby, F(aby)2, scFv, Fv, dsFv diabody, and Fd fragments. Antibody fragments may be produced by any means. For instance, the antibody fragment may be enzymatically or chemically produced by fragmentation of an intact antibody, it may be recombinantly produced from a gene encoding the partial antibody sequence, or it may be wholly or partially synthetically produced. The antibody fragment may optionally be a single chain antibody fragment. Alternatively, the fragment may comprise multiple chains which are linked together, for instance, by disulfide linkages. The fragment may also optionally be a multimolecular complex. A functional antibody fragment will typically comprise at least about 10 amino acids and more typically will comprise at least about 200 amino acids.

[0083] “Subject” and “patient” refer to either a human or non-human, such as primates, mammals, and vertebrates. In particular embodiments, the subject is a human.

[0084] As used herein, the terms "treat," "treatment," "treating," or "amelioration" when used in reference to a disease, disorder or medical condition, refer to therapeutic treatments for a condition, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a symptom or condition. The term "treating" includes reducing or alleviating at least one adverse effect or symptom of a condition. Treatment is generally "effective" if one or more symptoms or clinical markers are reduced. Alternatively, treatment is "effective" if the progression of a condition is reduced or halted. That is, "treatment" includes not just the improvement of symptoms or markers, but also a cessation or at least slowing of progress or worsening of symptoms that would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of the deficit, stabilized (z.e., not worsening) state of a tumor or malignancy, delay or slowing of tumor growth and / or metastasis, and an increased lifespan as compared to that expected in the absence of treatment.

[0085] The term “T cell” refers to T lymphocytes as defined in the art and is intended to include thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. The T cells can be CD4+T cells, CD8+T cells, CD4+CD8+T cells, or CD4-CD8“ cells. The T cells can also be T helper cells, such as T helper 1 (TH1), or T helper 2 (TH2) cells, or TH17 cells, as well as cytotoxic T cells, regulatory T cells, natural killer T cells, naive T cells, memory T cells, or gamma delta T cells (Wilson et al., 2009; Wynn, 2005; Ladi et al., 2006). T cells that differ from each other by at least one marker, such as CD4, are referred to herein as “subsets” of T cells.

[0086] “CD4+T cells” refers to a subset of T cells that express CD4 on their surface and are associated with cell-mediated immune response. They are characterized by the secretion profiles following stimulation, which may include secretion of cytokines such as IFN-gamma, TNF-alpha, IL-2, IL-4 and IL- 10. “CD4” are 55-kD glycoproteins originally defined as differentiation antigens on T-lymphocytes, but also found on other cells including monocytes / macrophages. CD4 antigens are members of the immunoglobulin supergene family and are implicated as associative recognition elements in MHC (major histocompatibility complex) class Il-restricted immune responses. On T-lymphocytes they define the helper / inducer subset.

[0087] “CD8+T cells” refers to a subset of T cells which express CD8 on their surface, are MHC class I-restricted, and function as cytotoxic T cells. “CD8” molecules are differentiation antigens found on thymocytes and on cytotoxic and suppressor T-lymphocytes. CD8 antigens are members of the immunoglobulin supergene family and are associative recognition elements in major histocompatibility complex class I-rcstrictcd interactions.

[0088] “Regulatory T cells” refer to a subset of T cells which act to suppress immune responses, thereby maintaining homeostasis and self-tolerance. Self-tolerance refers to a state of immune unresponsiveness towards self-antigens, important to avoid the development of autoimmune disease.

[0089] The term “chimeric antigen receptors (CARs),” as used herein, may refer to artificial T cell receptors, chimeric T cell receptors, or chimeric immunoreceptors, for example, and encompass engineered receptors that graft an artificial specificity onto a particular immune effector cell. CARs may be employed to impart the specificity of a monoclonal antibody onto a T cell, thereby allowing a large number of specific T cells to be generated, for example, for use in adoptive cell therapy. In specific embodiments, CARs direct specificity of the cell to a tumor associated antigen, for example. In some embodiments, CARs comprise an intracellular activation domain, a transmembrane domain, and an extracellular domain comprising a tumor associated antigen binding region. In particular aspects, CARs comprise fusions of single-chain variable fragments (scFv) derived from monoclonal antibodies, fused to CD3-zeta a transmembrane domain and endodomain. The specificity of other CAR designs may be derived from ligands of receptors (e.g. , peptides) or from pattern-recognition receptors, such as Dectins. In certain cases, the spacing of the antigen-recognition domain can be modified to reduce activation-induced cell death. In certain cases, CARs comprise domains for additional co-stimulatory signaling, such as CD3(j, FcR, CD27, CD28, CD137, DAP10, and / or 0X40. In some cases, molecules can be co-expressed with the CAR, including co-stimulatory molecules, reporter genes for imaging (e.g., for positron emission tomography), gene products that conditionally ablate the T cells upon addition of a pro-drug, homing receptors, chemokines, chemokine receptors, cytokines, and cytokine receptors.

[0090] The term “antigen presenting cells (APCs)” refers to a class of cells capable of presenting one or more antigens in the form of peptide-MHC complex recognizable by specific effector cells of the immune system, and thereby inducing an effective cellular’ immune response against the antigen or antigens being presented. APCs can be intact whole cells such as macrophages, B cells, endothelial cells, activated T cells, and dendritic cells; or other molecules, naturally occurring or synthetic, such as purified MHC Class I molecules complexed to p2-microglobulin. While many types of cells may be capable of presenting antigens on their cell surface for T cell recognition, only dendritic cells have the capacity to present antigens in an efficient amount to activate naive T cells for cytotoxic T lymphocyte (CTL) responses.

[0091] The term biologically “inert” refers to a molecule, such as a cell surface protein, that is biologically inert to the cell expressing it or modified to become biologically inert to the cell expressing it and / or is not recognized as foreign by the recipient’s immune system and has central tolerance. For example, the inert molecule may be a partial (i.e., truncated) or full-length human cell surface molecule on the surface of hPSCs and hPSC-derived cells, tissues or organs, and any immune receptor, natural or artificial, that recognizes said molecule on hPSCs and hPSC-derived cells, tissues or organs.

[0092] The term “safe harbor locus” refers to a place in the genome which allows for expression of an inserted transgene do not cause alterations of the host genome posing a risk (i.e., without disrupting the function of endogenous genes) to the host cell or organism.

[0093] IL Pluripotent Stem Cell-Derived Cells

[0094] In some embodiments, the present disclosure provides pluripotent stem cells (PSCs) engineered to express a cell surface protein or fragment thereof. PSCs may comprise embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) which have the ability to differentiate into several cell types that can be used in drug testing and also in the study and treatment of diseases. The term "differentiated cell" as used herein can refer to a precursor cell that has developed from an unspecialized phenotype to a specialized phenotype. iPSCs are artificial stem cells produced from somatic cells through co-expression of defined pluripotency-associated factors. Like embryonic stem cells (ESCs), they can typically proliferate and self-renew indefinitely in vitro and differentiate into derivatives of all three primary germ layers (i.e., ectoderm, mesoderm, and endoderm) as well as germ cells that give rise to the gametes. The basic paradigm in the use of PSCs for cell therapy purposes is that they are first differentiated into the desired cell types of interest, and the resulting specialized tissue- specific cells are then transplanted as cell suspensions or more complex tissue constructs into patients.

[0095] In 2006, Takahashi and Yamanaka discovered that mouse embryonic and adult fibroblasts could be reprogrammed to cells with the characteristics of ESCs by overexpression of a defined set of ESC-enriched transcription factors (Oct4, Sox2, Klf4, and c-Myc). The resulting cells, termed iPSCs, display infinite self-renewal ability (sternness) and can differentiate into all three

[0096] 19

[0097] SUBSTITUTE SHEET (RULE 26) embryonic germ layers (pluripotency). Human iPSCs are a promising prospect for cell therapy in a wide range of diseases for which there are currently no cures or effective therapies, such as neurodegenerative diseases of the central nervous system, heart infarction, diabetes mellitus, and diseases of the liver, lung, and kidney. Given that iPSCs can be produced in a patient- specific manner, they may be used in autologous transplantation.

[0098] In some aspects, PSCs may be genetically engineered to knock-out and / or knock-in a gene. This may be performed by various genome-editing approaches, including CRISPR technology, zinc finger nuclease (ZFN) and transcription activator-like effector nuclease (TALEN) technologies. The AAVS1 (adeno-associated virus integration site 1) locus is a "safe harbor" and has an open chromatin structure that allows insertion and stable expression of transgene. In some aspects, the cell surface protein is integrated at an AAVS1 targeting site, such as by TALENs.

[0099] The PSCs or PSC-derived cells may be engineered to express an inert cell surface protein. In particular aspects, the inert cell surface protein is truncated EGFR, such as comprising a sequence 90%, 95%, or 100% similar to SEQ ID NO:1:

[0100] EGFRt (SEQ ID NO: 1)

[0101] MLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSI SGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLH AFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCY ANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRD CVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGR GPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPN CTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM

[0102] PSCs may be differentiated into any of the 216 cell types found in an adult organism, such as neurons, cardiomyocytes, smooth muscle cells, osteocytes, hepatocytes, keratinocytes, insulinproducing cells, hematopoietic cells, and endothelial cells. In particular aspects, the present PSC- derived cells may comprise one or more of the 216 cell types. In some aspects, the transplanted engineered stem cell-derived tissue or organ can be made up of multiple cell types. The major cell types used from the endoderm include hepatic cells and insulin-producing cells. The mesodermal progenitors obtained from ESCs and iPSCs includes cardiomyocytes, endothelial cells, and hematopoietic cells. These cell types could be used for treatment of ischemic heart disease, repair of ischemic tissue, and to obtain all types of blood cells, respectively. The cells differentiated into 20

[0103] SUBSTITUTE SHEET (RULE 26) the ectoderm lineage include cells of the epidermis, external sense organs, and central and peripheral nervous system, such as functional neurons that can be used for the treatment of neurodegenerative diseases, such as acute spinal cord injury.

[0104] The induction of ESC and iPSC differentiation to produce different cell types requires complex differentiation steps with specific culture medium and growth factors, addition of cytokines, and supplements. The high differentiation potential of ESCs into specific cell lineages through in vitro systems, EB formation, or co-culture with stromal cells represents a source of cells that can be used for testing new drugs and also for cell therapy clinical trials.

[0105] In particular embodiments, the PSCs are differentiated to insulin-producing beta cells. For example, human pluripotent stem (hPSC) MelllNS-GFP reporter cells may be maintained on hES qualified Matrigel in mTeSR+ media. Differentiation to stem cell derived beta like cells (sBC) can be carried out in suspension-based, bioreactor magnetic stirring system as follows. Confluent hPSC cultures may be dissociated into single-cell suspension by incubation with TrypLE for 8 min at 37 °C. Detached cells can then be quenched with mTESR-i- media. Cells are then counted, followed by seeding 0.5 x 106cells / ml in mTeSR+ media supplemented with lOuM ROCK inhibitor (Y-27632, R&D Systems #1254-50). Bioreactors may be placed on a magnetic stirring system set at 60rpm in a cell culture incubator with 5% CO2 to induce sphere formation for 48 hours. To induce definitive endoderm differentiation, spheres may be collected in a 50ml Falcon tube, allowed to settle by gravity, washed once with RPMI + 0.2% FBS, and re-suspended in dl media [RPMI containing 0.2% FBS, 1:5,000 ITS (Gibco #41400-045), lOOng / ml Activin A (R&D Systems #338-AC-01M), and 3uM CHIR99021 (STEMCELL Technologies #72054)]. Differentiation media may be changed daily by letting spheres settle by gravity for 3-10min. Most supernatant may be removed by aspiration; fresh media added, and bioreactors placed back on stirrer system. sBC differentiation media may comprise: day 2-3: RPMI containing 0.2% FBS, 1:2,000 ITS, and lOOng / ml Activin A; d4-5: RPMI containing 2% FBS, 1:1,000 ITS, and 50ng / ml KGF (Prepotech #100-19-lMG); d6: DMEM with 4.5g / L D-glucose (Gibco #11960-044) containing 1:100 SMI (STEMCELL Technologies #5711), 1:100 NEAA (Gibco #11140-050), ImM Sodium Pyruvate (Gibco #11360-070), 1:100 GlutaMAX (Gibco #35050-061), 3nM TTNPB, (R&D Systems #0761), 250nM Sant-1 (R&D Systems #1974), 250nM LDN (STEMCELL Technologies #72149), 30nM PMA (Sigma Aldrich #P1585-1MG), 50pg / ml 2-phospho-L-ascorbic acid

[0106] 21

[0107] SUBSTITUTE SHEET (RULE 26) trisodium salt (VitC) (Sigma #49752- 10G); d7: DMEM containing 1 :100 SM I , 1 :100 NEAA, ImM Sodium Pyruvate, 1:100 GlutaMAX, 3nM TTNPB, and 50pg / ml VitC; d8-9: DMEM containing 1:100 SMI, 1:100 NEAA, ImM Sodium Pyruvate, 1:100 GlutaMAX, lOOng / ml EGF (R&D Systems #236-EG-01M), 50ng / ml KGF, and 50ug / ml VitC; dl0-16: DMEM containing 2% fraction V BSA, 1:100 NEAA, ImM Sodium Pyruvate, 1:100 GlutaMAX, 1:100 ITS, IO g / ml Heparin (Sigma #H3149-250KU), 2mM N-Acetyl-L-cysteine (Cysteine) (Sigma #A9165-25G), lO M Zinc sulfate heptahydrate (Zinc) (Sigma #Z0251-100g), lx BME, lOpM Alk5i II RepSox (R&D Systems #3742 / 50), I M 3,3’,5-Triiodo-L-thyronine sodium salt (T3) (Sigma #T6397), 0.5pM LDN, IpM Gamma Secretase Inhibitor XX (XXi) (AsisChem #ASIS-0149) and 1:250 IM NaOH to adjust pH to ~7.4; dl7-23: CMRL (Gibco #11530-037) containing 1% BSA, 1:100 NEAA, ImM Sodium Pyruvate, 1:100 GlutaMAX, lOug / ml Heparin, 2mM Cysteine, lOuM Zinc, lx BME, lOpM Alk5i II RepSox, luM T3, 50ug / ml VitC, and 1:250 NaOH to adjust pH to ~7.4. All media may also contain lx PenStrep.

[0108] III. CAR-Modified Regulatory T Cells

[0109] In certain embodiments, the present disclosure provides Tregs engineered to express a CAR vector. The CAR Tregs may be used to treat a disease or disorder, such as an autoimmune disease.

[0110] Certain embodiments of the present disclosure concern obtaining a starting population of Tregs, modifying the Tregs, and administering the modified Tregs to a subject as protection against immune rejection with transplantation. In particular, the Tregs express CAR.

[0111] In some embodiments, the starting population of Tregs are derived from the blood, cord blood, bone marrow, lymph, or lymphoid organs, such as the thymus. In some aspects, the cells are human cells. The cells typically are primary cells, such as those isolated directly from a subject and / or isolated from a subject and frozen. With reference to the subject to be treated, the cells may be allogeneic and / or autologous. In some embodiments, the methods include isolating cells from the subject, preparing, processing, culturing, and / or engineering them, as described herein, and reintroducing them into the same patient, before or after cryopreservation.

[0112] In some embodiments, one or more of the T cell populations is enriched for or depleted of cells that arc positive for a specific marker, such as surface markers, or that are negative for a specific marker. In some cases, such markers are those that are absent or expressed at relatively low levels on certain populations of T cells (e.g., regulatory T cells) but are present or expressed at relatively higher levels on certain other populations of T cells (e.g., regulatory T cells).

[0113] In some embodiments, regulatory T cells are separated from a peripheral blood mononuclear cell (PBMC) sample by negative selection of markers expressed on non-T cells, such as B cells, monocytes, or other white blood cells, such as CD 14. In some aspects, CD4+CD25+CD127’ Tregs are purified from human peripheral blood.

[0114] In some embodiments, the T cells are autologous T cells. In this method, tumor samples are obtained from patients and a single cell suspension is obtained. The single cell suspension can be obtained in any suitable manner, e.g., mechanically (disaggregating the tumor using, e.g., a gentleMACS™ Dissociator, Miltenyi Biotec, Auburn, Calif.) or enzymatically (e.g., collagenase or DNase). Single-cell suspensions of tumor enzymatic digests are cultured in interleukin-2 (IL- 2). The cells are cultured until confluence (e.g., about 2xl06lymphocytes), e.g., from about 5 to about 21 days, preferably from about 10 to about 14 days. For example, the cells may be cultured from 5 days, 5.5 days, or 5.8 days to 21 days, 21.5 days, or 21.8 days, such as from 10 days, 10.5 days, or 10.8 days to 14 days, 14.5 days, or 14.8 days.

[0115] The cultured T cells can be pooled and rapidly expanded. Rapid expansion provides an increase in the number of antigen- specific T-cells of at least about 50-fold (e.g., 50-, 60-, 70-, 80- , 90-, or 100-fold, or greater) over a period of about 10 to about 14 days. More specifically, rapid expansion provides an increase of at least about 200-fold (e.g., 200-, 300-, 400-, 500-, 600-, 700-, 800-, 900-, or greater) over a period of about 10 to about 14 days. Expansion can be accomplished by any of a number of methods as are known in the art. For example, T cells can be rapidly expanded using non-specific T-cell receptor stimulation in the presence of feeder lymphocytes and either interleukin-2 (IL-2) or interleukin- 15 (IL-15). The non-specific T-cell receptor (TCR) stimulus can include around 30 ng / ml of OKT3, a mouse monoclonal anti-CD3 antibody (available from Ortho-McNeil®, Raritan, N.J.). Alternatively, T cells can be rapidly expanded by stimulation of peripheral blood mononuclear cells (PBMC) in vitro with one or more antigens (including antigenic portions thereof, such as epitope(s), or a cell) of the cancer, which can be optionally expressed from a vector, such as a human leukocyte antigen A2 (HLA-A2) binding peptide, in the presence of a T-cell growth factor, such as 300 lU / ml IL-2. The in vi / ra-induccd T-cells are rapidly expanded by re- stimulation with the same antigen(s) of the cancer pulsed onto HLA-A2- expressing antigen-presenting cells. Alternatively, the T-cells can be re-stimulated with irradiated, autologous lymphocytes or with irradiated HLA-A2+allogeneic lymphocytes and IL-2, for example.

[0116] The autologous T-cells can be modified to express a T-cell growth factor that promotes the growth and activation of the autologous T-cells. Suitable T-cell growth factors include, for example, interleukin (IL)-2, IL-7, IL- 15, and IL- 12. Suitable methods of modification are known in the art. See, for instance, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rded., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994. In particular aspects, modified autologous T-cells express the T-cell growth factor at high levels. T- cell growth factor coding sequences, such as that of IL- 12, are readily available in the art, as are promoters, the operable linkage of which to a T-cell growth factor coding sequence promote high- level expression.

[0117] One of skill in the art would be well-equipped to construct a vector through standard recombinant techniques (see, for example, Sambrook et al., 2001 and Ausubel et al., 1996, both incorporated herein by reference) for the expression of the antigen receptors of the present disclosure. Vectors include but are not limited to, plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs), such as retroviral vectors (e.g. derived from Moloney murine leukemia virus vectors (MoMLV), MSCV, SFFV, MPSV, SNV etc), lentiviral vectors (e.g. derived from HIV-1, HIV-2, SIV, BIV, FIV etc.), adenoviral (Ad) vectors including replication competent, replication deficient and gutless forms thereof, adeno-associated viral (AAV) vectors, simian vims 40 (SV-40) vectors, bovine papilloma vims vectors, Epstein-Barr vims vectors, herpes virus vectors, vaccinia virus vectors, Harvey murine sarcoma vims vectors, murine mammary tumor virus vectors, Rous sarcoma virus vectors, parvovirus vectors, polio virus vectors, vesicular stomatitis virus vectors, maraba virus vectors and group B adenovims enadenotucirev vectors.

[0118] In some embodiments, the CAR contains an extracellular antigen-recognition domain that specifically binds to an antigen. In some embodiments, the antigen is a protein expressed on the surface of cells. In some embodiments, the CAR is a TCR-like CAR and the antigen is a processed peptide antigen, such as a peptide antigen of an intracellular protein, which, like a TCR, is recognized on the cell surface in the context of a major histocompatibility complex (MHC) molecule.

[0119] Exemplary antigen receptors, including CARs and recombinant TCRs, as well as methods for engineering and introducing the receptors into cells, include those described, for example, in international patent application publication numbers W0200014257, WO2013126726, WO2012 / 129514, WO2014031687, WO2013 / 166321, W02013 / 071154, WO2013 / 123061, WO2023019144A1, U.S. patent application publication numbers US2002131960, US2013287748, US20130149337, U.S. Patent Nos.: 6,451,995, 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118, and European patent application number EP2537416, and / or those described by Sadelain et al., Cancer Discov. 2013 April; 3(4): 388-398; Davila et al. (2013) PLoS ONE 8(4): e61338; Turtle et al., Curr. Opin. Immunol., 2012 October; 24(5): 633-39; Wu et al., Cancer, 2012 March 18(2): 160-75. In some aspects, the genetically engineered antigen receptors include a CAR as described in U.S. Patent No.: 7,446,190, and those described in International Patent Application Publication No.: WO / 2014055668 Al.

[0120] In some embodiments, the CAR comprises: a) an intracellular signaling domain, b) a transmembrane domain, and c) an extracellular domain comprising an antigen binding region.

[0121] In some embodiments, the engineered antigen receptors include CARs, including activating or stimulatory CARs, costimulatory CARs (see WO2014 / 055668), and / or inhibitory CARs (iCARs, see Fedorov et al., 2013). The CARs generally include an extracellular antigen (or ligand) binding domain linked to one or more intracellular signaling components, in some aspects via linkers and / or transmembrane domain(s). Such molecules typically mimic or approximate a signal through a natural antigen receptor, a signal through such a receptor in combination with a costimulatory receptor, and / or a signal through a costimulatory receptor alone.

[0122] Certain embodiments of the present disclosure concern the use of nucleic acids, including nucleic acids encoding an antigen- specific CAR polypeptide, including a CAR that has been humanized to reduce immunogenicity (hCAR), comprising an intracellular signaling domain, a transmembrane domain, and an extracellular domain comprising one or more signaling motifs. In certain embodiments, the CAR may recognize an epitope comprising the shared space between one or more antigens. In certain embodiments, the binding region can comprise complementary determining regions of a monoclonal antibody, variable regions of a monoclonal antibody, and / or antigen binding fragments thereof. In another embodiment, that specificity is derived from a peptide (e.g., cytokine) that binds to a receptor.

[0123] It is contemplated that the human CAR nucleic acids may be human genes used to enhance cellular immunotherapy for human patients. In a specific embodiment, the invention includes a full-length CAR cDNA or coding region. The antigen binding regions or domain can comprise a fragment of the VH and VL chains of a single-chain variable fragment (scFv) derived from a particular human monoclonal antibody, such as those described in U.S. Patent 7,109,304, incorporated herein by reference. The fragment can also be any number of different antigen binding domains of a human antigen- specific antibody. In a more specific embodiment, the fragment is an antigen- specific scFv encoded by a sequence that is optimized for human codon usage for expression in human cells.

[0124] The arrangement could be multimeric, such as a diabody or multimers. The multimers are most likely formed by cross pairing of the variable portion of the light and heavy chains into a diabody. The hinge portion of the construct can have multiple alternatives from being totally deleted, to having the first cysteine maintained, to a proline rather than a serine substitution, to being truncated up to the first cysteine. The Fc portion can be deleted. Any protein that is stable and / or dimerizes can serve this purpose. One could use just one of the Fc domains, e.g., either the CH2 or CH3 domain from human immunoglobulin. One could also use the hinge, CH2 and CH3 region of a human immunoglobulin that has been modified to improve dimerization. One could also use just the hinge portion of an immunoglobulin. One could also use portions of CD8alpha.

[0125] In some embodiments, the CAR nucleic acid comprises a sequence encoding other costimulatory receptors, such as a transmembrane domain and a modified CD28 intracellular signaling domain. Other costimulatory receptors include, but are not limited to one or more of CD28, CD27, OX-40 (CD134), DAP10, and 4-1BB (CD137). In addition to a primary signal initiated by CD3^, an additional signal provided by a human costimulatory receptor inserted in a human CAR is important for full activation of NK cells and could help improve in vivo persistence and the therapeutic success of the adoptive immunotherapy.

[0126] In some embodiments, CAR is constructed with a specificity for a particular antigen (or marker or ligand), such as an antigen expressed in a particular cell type to be targeted by adoptive therapy, e.g., a cancer marker, and / or an antigen intended to induce a dampening response, such as an antigen expressed on a normal or non-diseased cell type. Thus, the CAR typically includes in its extracellular portion one or more antigen binding molecules, such as one or more antigenbinding fragment, domain, or portion, or one or more antibody variable domains, and / or antibody molecules. In some embodiments, the CAR includes an antigen-binding portion or portions of an antibody molecule, such as a single-chain antibody fragment (scFv) derived from the variable heavy (VH) and variable light (VL) chains of a monoclonal antibody (mAb).

[0127] In certain embodiments of the chimeric antigen receptor, the antigen- specific portion of the receptor (which may be referred to as an extracellular domain comprising an antigen binding region) comprises a cell surface protein, particularly an inert cell surface protein. Antigens include carbohydrate antigens recognized by pattern-recognition receptors, such as Dectin- 1.

[0128] The sequence of the open reading frame encoding the chimeric receptor can be obtained from a genomic DNA source, a cDNA source, or can be synthesized (e.g., via PCR), or combinations thereof. Depending upon the size of the genomic DNA and the number of introns, it may be desirable to use cDNA or a combination thereof as it is found that introns stabilize the mRNA. Also, it may be further advantageous to use endogenous or exogenous non-coding regions to stabilize the mRNA.

[0129] It is contemplated that the chimeric construct can be introduced into immune cells as naked DNA or in a suitable vector. Methods of stably transfecting cells by electroporation using naked DNA are known in the art. See, e.g., U.S. Pat. No. 6,410,319. Naked DNA generally refers to the DNA encoding a chimeric receptor contained in a plasmid expression vector in proper orientation for expression.

[0130] Alternatively, a viral vector (e.g., a retroviral vector, adenoviral vector, adeno-associated viral vector, or lentiviral vector) can be used to introduce the chimeric construct into immune cells. Suitable vectors for use in accordance with the method of the present disclosure are non-replicating in the immune cells. A large number of vectors are known that are based on viruses, where the copy number of the virus maintained in the cell is low enough to maintain the viability of the cell, such as, for example, vectors based on HIV, SV40, EBV, HSV, or BPV.

[0131] In some aspects, the antigen- specific binding, or recognition component is linked to one or more transmembrane and intracellular signaling domains. In some embodiments, the CAR includes a transmembrane domain fused to the extracellular domain of the CAR. In one embodiment, the transmembrane domain that naturally is associated with one of the domains in the CAR is used. In some instances, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.

[0132] The transmembrane domain in some embodiments is derived either from a natural or from a synthetic source. Where the source is natural, the domain in some aspects is derived from any membrane-bound or transmembrane protein. Transmembrane regions include those derived from (i.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T- cell receptor, CD28, CD3 zeta, CD3 epsilon, CD3 gamma, CD3 delta, CD45, CD4, CD5, CD8, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD137, CD154, ICOS / CD278, GITR / CD357, NKG2D, and DAP molecules. Alternatively, the transmembrane domain in some embodiments is synthetic. In some aspects, the synthetic transmembrane domain comprises predominantly hydrophobic residues such as leucine and valine. In some aspects, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain.

[0133] In certain embodiments, the platform technologies disclosed herein to genetically modify immune cells, such as NK cells, comprise (i) non-viral gene transfer using an electroporation device (e.g., a nucleo lector), (ii) CARs that signal through endodomains (e.g., CD28 / CD3-(^, CD137 / CD3-(^, or other combinations), (iii) CARs with variable lengths of extracellular domains connecting the antigen-recognition domain to the cell surface, and, in some cases, (iv) artificial antigen presenting cells (aAPC) derived from K562 to be able to robustly and numerically expand CAR+immune cells (Singh et al., 2008; Singh et al., 2011).

[0134] Among the target proteins of antigens targeted by the present CARs arc those expressed in the context of a disease, condition, or cell type to be targeted via the CAR. Among the diseases and conditions are proliferative, neoplastic, and malignant diseases and disorders, including cancers and tumors, including hematologic cancers, cancers of the immune system, such as lymphomas, leukemias, and / or myelomas, such as B, T, and myeloid leukemias, lymphomas, and multiple myelomas. In some embodiments, the antigen is selectively expressed or overexpressed on cells of the disease or condition, e.g., the tumor or pathogenic cells, as compared to normal or non-targeted cells or tissues. In other embodiments, the antigen is expressed on normal cells and / or is expressed on the engineered cells. Any suitable antigen may find use in the present methods. Exemplary antigens include, but are not limited to, antigenic molecules from infectious agents, auto- / sclf- antigens, tumor- / canccr-associatcd antigens, and tumor neoantigens.

[0135] The terms “tumor-associated antigen,” “tumor antigen” and “cancer cell antigen” are used interchangeably herein. In each case, the terms refer to proteins, glycoproteins or carbohydrates that are specifically or preferentially expressed by cancer cells.

[0136] Exemplary embodiments of antigens include, but are not limited to, CD 19, CD20, carcinoembryonic antigen, alphafetoprotein, CA-125, MUC-1, CD56, EGFR, EGFRt, c-Met, AKT, Her2, Her3, epithelial tumor antigen, melanoma-associated antigen, mutated p53, mutated ras, and so forth. In particular aspects, the antigens include NY-ESO, EGFRvIII, Muc-1, Her2, CA-125, WT-1, Mage-A3, Mage-A4, Mage-AlO, TRAIL / DR4, and CEA. In particular aspects, the antigens for the two or more antigen receptors include, but are not limited to, CD 19, EBNA, WT1, CD123, NY-ESO, EGFRvIII, MUC1, HER2, CA-125, WT1, Mage-A3, Mage-A4, Mage- A10, TRAIL / DR4, and / or CEA. The sequences for these antigens are known in the art, for example, CD19 (Accession No. NG_007275.1), EBNA (Accession No. NG_002392.2), WT1 (Accession No. NG_009272.1), CD123 (Accession No. NC_000023.11), NY-ESO (Accession No. NC_000023.11), EGFRvIII (Accession No. NG_007726.3), MUC1 (Accession No. NG_029383.1), HER2 (Accession No. NG_007503.1), CA-125 (Accession No. NG_055257.1), WT1 (Accession No. NG_009272.1), Mage- A3 (Accession No. NG_013244.1), Mage-A4 (Accession No. NG_013245.1), Mage-AlO (Accession No. NC_000023.11), TRAIL / DR4 (Accession No. NC_000003.12), and / or CEA (Accession No. NC_000019.10).

[0137] Other tumor associated antigens include Plu-1, HASH-1, HasH-2, Cripto and Criptin. Additionally, a tumor antigen may be a self-peptide hormone, such as whole length gonadotrophin hormone releasing hormone (GnRH), a short 10 amino acid long peptide, useful in the treatment of many cancers.

[0138] Tumor antigens include tumor antigens derived from cancers that are characterized by tumor-associated antigen expression, such as HER-2 / neu expression. Tumor-associated antigens of interest include lineage- specific tumor antigens such as the melanocyte-melanoma lineage antigens MART-l / Melan-A, gplOO, gp75, mda-7, tyrosinase and tyrosinase-related protein. Illustrative tumor-associated antigens include, but are not limited to, tumor antigens derived from or comprising any one or more of, p53, Ras, c-Myc, cytoplasmic serine / threonine kinases (e.g., A- Raf, B-Raf, and C-Raf, cyclin-dependent kinases), MAGE-A1, MAGE-A2, MAGE-A3, MAGE- A4, MAGE-A6, MAGE- A 10, MAGE-A12, MART-1 , BAGE, DAM-6, -10, GAGE-1 , -2, -8, GAGE-3, -4, -5, -6, -7B, NA88-A, MART-1, MC1R, GplOO, PSA, PSM, Tyrosinase, TRP-1, TRP-2, ART-4, CAMEL, CEA, Cyp-B, hTERT, hTRT, iCE, MUC1, MUC2, Phosphoinositide 3- kinases (PI3Ks), TRK receptors, PRAME, P15, RU1, RU2, SART-1, SART-3, Wilms' tumor antigen (WT1), AFP, -catenin / m, Caspase-8 / m, CEA, CDK-4 / m, ELF2M, GnT-V, G250, HSP70- 2M, HST-2, KIAA0205, MUM-1, MUM-2, MUM-3, Myosin / m, RAGE, SART-2, TRP-2 / INT2, 707-AP, Annexin II, CDC27 / m, TPI / mbcr-abl, BCR-ABL, interferon regulatory factor 4 (IRF4), ETV6 / AML, LDLR / FUT, Pml / RAR, Tumor-associated calcium signal transducer 1 (TACSTD1) TACSTD2, receptor tyrosine kinases (e.g., Epidermal Growth Factor receptor (EGFR) (in particular, EGFRvIII), platelet derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR)), cytoplasmic tyrosine kinases (e.g., sre-family, syk-ZAP70 family), integrin-linked kinase (ILK), signal transducers and activators of transcription STAT3, STATS, and STATE, hypoxia inducible factors e.g., HIF-1 and HIF-2), Nuclear Factor- Kappa B (NF-B), Notch receptors (e.g., Notchl-4), c-Met, mammalian targets of rapamycin (mTOR), WNT, extracellular signal-regulated kinases (ERKs), and their regulatory subunits, PMSA, PR-3, MDM2, Mesothelin, renal cell carcinoma-5T4, SM22-alpha, carbonic anhydrases I (CAI) and IX (CAIX) (also known as G250), STEAD, TEL / AML1, GD2, proteinase3, hTERT, sarcoma translocation breakpoints, EphA2, ML-IAP, EpCAM, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, ALK, androgen receptor, cyclin Bl, polysialic acid, MYCN, RhoC, GD3, fucosyl GM1, mesothelin, PSCA, sLe, PLAC1, GM3, BORIS, Tn, GLoboH, NY-BR-1, RGsS, SART3, STn, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, legumain, TIE2, Page4, MAD-CT-1, FAP, MAD-CT-2, fos related antigen 1, CBX2, CLDN6, SPANX, TPTE, ACTL8, ANKRD30A, CDKN2A, MAD2L1, CTAG1B, SUNCI, LRRN1 and idiotype.

[0139] Antigens may include epitopic regions or epitopic peptides derived from genes mutated in tumor cells or from genes transcribed at different levels in tumor cells compared to normal cells, such as telomerase enzyme, survivin, mesothelin, mutated ras, bcr / abl rearrangement, Her2 / neu, mutated or wild-type p53, cytochrome P450 IB 1, and abnormally expressed intron sequences such as N-acetylglucosaminyltransferase-V; clonal rearrangements of immunoglobulin genes generating unique idiotypes in myeloma and B-cell lymphomas; tumor antigens that include epitopic regions or epitopic peptides derived from oncoviral processes, such as human papilloma virus proteins E6 and E7; Epstein bar virus protein LMP2; nonmutated oncofetal proteins with a tumor- selective expression, such as carcinocmbryonic antigen and alpha-fetoprotein.

[0140] Antigen-presenting cells (APCs), which include macrophages, B lymphocytes, and dendritic cells, are distinguished by their expression of a particular MHC molecule. APCs internalize antigen and re-express a part of that antigen, together with the MHC molecule on their outer cell membrane. The MHC is a large genetic complex with multiple loci. The MHC loci encode two major classes of MHC membrane molecules, referred to as class I and class II MHCs. T helper lymphocytes generally recognize antigen associated with MHC class II molecules, and T cytotoxic lymphocytes recognize antigen associated with MHC class I molecules. In humans the MHC is referred to as the HLA complex and in mice the H-2 complex.

[0141] In some cases, artificial APCs (aAPCs) arc useful in preparing therapeutic compositions and cell therapy products of the embodiments. For general guidance regarding the preparation and use of antigen-presenting systems, see, e.g., U.S. Patent Nos. 6,225,042, 6,355,479, 6,362,001 and 6,790,662. aAPC systems may comprise at least one exogenous assisting molecule. Any suitable number and combination of assisting molecules may be employed. The assisting molecule may be selected from assisting molecules such as co- stimulatory molecules and adhesion molecules. Exemplary co-stimulatory molecules include CD86, CD64 (FcyRI), 41BB ligand, and IL-21. Adhesion molecules may include carbohydrate-binding glycoproteins such as selectins, transmembrane binding glycoproteins such as integrins, calcium-dependent proteins such as cadherins, and single-pass transmembrane immunoglobulin (Ig) superfamily proteins, such as intercellular adhesion molecules (ICAMs), which promote, for example, cell-to-cell or cell-to- matrix contact. Exemplary adhesion molecules include LFA-3 and ICAMs, such as ICAM-1. Techniques, methods, and reagents useful for selection, cloning, preparation, and expression of exemplary assisting molecules, including co-stimulatory molecules and adhesion molecules, are exemplified in, e.g., U.S. Patent Nos. 6,225,042, 6,355,479, and 6,362,001.

[0142] IV. Formulation and Administration

[0143] The present disclosure provides pharmaceutical compositions comprising PSC-derived cells and CAR Trcgs. Such compositions comprise a prophylactically or therapeutically effective amount of PSC-derived cells, Tregs and a pharmaceutically acceptable carrier. In a specific embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a particular carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Other suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.

[0144] The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical agents are described in “Remington's Pharmaceutical Sciences.” Such compositions will contain a prophylactically or therapeutically effective amount of the antibody or fragment thereof, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration, which can be oral, intravenous, intraarterial, intrabuccal, intranasal, nebulized, bronchial inhalation, or delivered by mechanical ventilation.

[0145] Generally, the ingredients of compositions of the disclosure are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration. The compositions of the disclosure can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

[0146] A. Autoimmune Diseases

[0147] In one embodiment, the subject has an inflammatory condition or autoimmune disease. Non-limiting examples of inflammatory diseases include: alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune diseases of the adrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac spate-dermatitis, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatrical pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, glomerulonephritis, Graves' disease, Guillain- Barre, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), IgA neuropathy, juvenile arthritis, lichen planus, lupus erythematosus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, type 1 or immune-mediated diabetes mellitus, myasthenia gravis, nephrotic syndrome (such as minimal change disease, focal glomerulosclerosis, or membranous nephropathy), pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomenon, Reiter's syndrome, Rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, stiff-man syndrome, systemic lupus erythematosus, lupus erythematosus, ulcerative colitis, uveitis, vasculitides (such as polyarteritis nodosa, takayasu arteritis, temporal arteritis / giant cell arteritis, or dermatitis herpetiformis vasculitis), vitiligo, and Wegener's granulomatosis. Thus, some examples of an autoimmune disease that can be treated using the methods disclosed herein include, but are not limited to, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosis, type I diabetes mellitus, Crohn's disease; ulcerative colitis, myasthenia gravis, glomerulonephritis, ankylosing spondylitis, vasculitis, myocarditis, or psoriasis. Tn other aspects, the disease is a disease that results from the loss of specialized cell function.

[0148] B. Methods of Treatment

[0149] In particular compositions that may be used in treating an autoimmune disease in a subject (e.g., a human subject) are disclosed herein. The compositions described above are preferably administered to a mammal (e.g., rodent, human, non-human primates, canine, bovine, ovine, equine, feline, etc.) in an effective amount, that is, an amount capable of producing a desirable result in a treated subject (e.g., causing apoptosis of cancerous cells or killing bacterial cells). Toxicity and therapeutic efficacy of the compositions utilized in methods of the disclosure can be determined by standard pharmaceutical procedures. As is well known in the medical and veterinary arts, dosage for any one animal depends on many factors, including the subject's size, body surface area, body weight, age, the particular composition to be administered, time and route of administration, general health, the clinical symptoms of the infection or cancer and other drugs being administered concurrently.

[0150] In some embodiments, the present disclosure provides methods for treating a disease and / or preventing immune rejection comprising administering an effective amount of the PSC-derived cells and Tregsof the present disclosure. In one embodiment, a medical disease or disorder is treated by transfer of an immune cell population that elicits an immune response.

[0151] To determine the suitability of cells provided herein for therapeutic applications, the cells can first be tested in a suitable animal model. At one level, cells are assessed for their ability to survive and maintain their phenotype in vivo. Cells provided herein are administered to immunodeficient animals (such as NOG mice, or animals rendered immunodeficient chemically or by irradiation) at a site amenable for further observation, such as under the kidney capsule, into the spleen, into a liver lobule, or into the bone marrow. Tissues are harvested after a period of a few days to several weeks or more, and assessed as to whether starting cell types such as erythrocytes are still present. This can be performed by providing the administered cells with a detectable label (such as green fluorescent protein, or 0-galactosidase); or by measuring a constitutive marker specific for the administered human cells. Where cells provided herein are being tested in a rodent model, the presence and phenotype of the administered cells can be assessed by immunohistochemistry or ELISA using human- specific antibody, or by RT-PCR analysis using primers and hybridization conditions that cause amplification to be specific for human polynucleotide sequences. Suitable markers for assessing gene expression at the mRNA or protein level are provided elsewhere in this disclosure.

[0152] Tregs and PSC-derived cells provided by methods of the present disclosure may be tested in various animal models for their ability to treat disorders. For example, a sickle cell anemia mouse model or the T / B cell-deficient Rag-2 knockout mouse may be particularly useful animal models for testing the myeloid and lymphoid cells disclosed herein.

[0153] Tregs and PSC-derived cells provided in certain aspects of the present disclosure that demonstrate desirable functional characteristics or efficacy in animal models, may also be suitable for direct administration to human subjects in need thereof. For purposes of hemostasis, the cells can be administered at any site that has adequate access to the circulation. Hematopoietic cells or precursors thereof may also be delivered at a site of injury or disease.

[0154] The Tregs and PSC-derived cells provided in certain aspects of this present disclosure can be used for therapy of any subject in need thereof. Human conditions that may be appropriate for such therapy include the various anemias and hemoglobinopathies, as well as diseases characterized by decreased numbers of hematopoietic cells (such as, for example, myelodysplastic syndrome, myelofibrosis, neutropenia, agranulocytosis, Glanzmann’s thrombasthenia, thrombocytopenia, and acquired immune deficiency syndrome). For human therapy, the dose is generally between about 109and 1012cells, and typically between about 5xl09and 5xlO10cells, making adjustments for the body weight of the subject, nature and severity of the affliction, and the replicative capacity of the administered cells. The ultimate responsibility for determining the mode of treatment and the appropriate dose lies with the managing clinician.

[0155] Therapeutically effective amounts of Tregs and PSC-derived cells can be administered by a number of routes, including parenteral administration, for example, intravenous, intraperitoneal, intramuscular, intrastemal, or intraarticular injection, or infusion.

[0156] The therapeutically effective amount of Tregs f and PSC-derived cells or use in adoptive cell therapy is that amount that achieves a desired effect in a subject being treated. For instance, this can be the amount of Tregs necessary to inhibit advancement, or to cause regression of an autoimmune or alloimmune disease, or which is capable of relieving symptoms caused by an autoimmune disease, such as pain and inflammation. It can be the amount necessary to relieve symptoms associated with inflammation, such as pain, edema and elevated temperature. It can also be the amount necessary to diminish or prevent rejection of a transplanted organ.

[0157] The Tregs and PSC-derived cell population can be administered in treatment regimens consistent with the disease, for example a single or a few doses over one to several days to ameliorate a disease state or periodic doses over an extended time to inhibit disease progression and prevent disease recurrence. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. The therapeutically effective amount of Tregs and PSC-derived cells will be dependent on the subject being treated, the severity and type of the affliction, and the manner of administration. In some embodiments, doses that could be used in the treatment of human subjects range from at least 3.8xl04, at least 3.8xl05, at least 3.8xl06, at least 3.8xl07, at least 3.8xl08, at least 3.8xl09, or at least 3.8xlO10immune cells / m2. In a certain embodiment, the dose used in the treatment of human subjects ranges from about 3.8xl09to about 3.8xlOloimmune cells / m2. In additional embodiments, a therapeutically effective amount of immune cells can vary from about 5xl06cells per kg body weight to about 7.5xl08cells per kg body weight, such as about 2xl07cells to about 5xl08cells per kg body weight, or about 5x IO7cells to about 2x IO8cells per kg body weight. The exact amount of immune cells is readily determined by one of skill in the art based on the age, weight, sex, and physiological condition of the subject. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0158] The Tregs and PSC-derived cells may be administered in combination with one or more other therapeutic agents for the treatment of the immune- mediated disorder. Combination therapies can include, but are not limited to, one or more anti-microbial agents (for example, antibiotics, anti-viral agents and anti-fungal agents), anti-tumor agents (for example, fluorouracil, methotrexate, paclitaxel, fludarabine, etoposide, doxorubicin, or vincristine), immune-depleting agents (for example, fludarabine, etoposide, doxorubicin, or vincristine), immunosuppressive agents (for example, azathioprine, or glucocorticoids, such as dexamethasone or prednisone), antiinflammatory agents (for example, glucocorticoids such as hydrocortisone, dexamethasone or prednisone, or non-steroidal anti-inflammatory agents such as acetylsalicylic acid, ibuprofen or naproxen sodium), cytokines (for example, interleukin- 10 or transforming growth factor-beta), hormones (for example, estrogen), or a vaccine. In addition, immunosuppressive or tolerogenic agents including but not limited to calcineurin inhibitors (e.g., cyclosporin and tacrolimus); mTOR inhibitors (e.g., Rapamycin); mycophcnolatc mofctil, antibodies (e.g., recognizing CD3, CD4, CD40, CD154, CD45, IVIG, or B cells); chemotherapeutic agents (e.g., Methotrexate, Treosulfan, Busulfan); irradiation; or chemokines, interleukins or their inhibitors (e.g., BAFF, IL-2, anti-IL- 2R, IL-4, JAK kinase inhibitors) can be administered. Such additional pharmaceutical agents can be administered before, during, or after administration of the immune cells, depending on the desired effect. This administration of the cells and the agent can be by the same route or by different routes, and either at the same site or at a different site.

[0159] The therapeutic methods of the disclosure (which include prophylactic treatment) in general include administration of a therapeutically effective amount of the compositions described herein to a subject in need thereof, including a mammal, particularly a human. Such treatment will be suitably administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk for a disease, disorder, or symptom thereof. Determination of those subjects "at risk" can be made by any objective or subjective determination by a diagnostic test or opinion of a subject or health care provider (e.g., genetic test, enzyme or protein marker, marker (as defined herein), family history, and the like).

[0160] In one embodiment, the disclosure provides a method of monitoring treatment progress. The method includes the step of determining a level of changes in hematological parameters and / or cancer stem cell (CSC) analysis with cell surface proteins as diagnostic markers or diagnostic measurement (e.g., screen, assay) in a subject suffering from or susceptible to a disorder or symptoms thereof in which the subject has been administered a therapeutic amount of a composition as described herein. The level of marker determined in the method can be compared to known levels of marker either in healthy normal controls or in other afflicted patients to establish the subject's disease status. In preferred embodiments, a second level of marker in the subject is determined at a time point later than the determination of the first level, and the two levels are compared to monitor the course of disease or the efficacy of the therapy. In certain preferred embodiments, a pre-treatment level of marker in the subject is determined prior to beginning treatment according to the methods described herein; this pre-treatment level of marker can then be compared to the level of marker in the subject after the treatment commences, to determine the efficacy of the treatment. C. Additional Therapy

[0161] In certain embodiments, the compositions and methods of the present embodiments involve Tregs and PSC-derived cells, in combination with a second or additional therapy.

[0162] In certain embodiments, the compositions and methods of the present embodiments involve Tregs and PSC-derived cells in combination with at least one additional therapy. The additional therapy may be therapies for preventing immune rejection.

[0163] The methods and compositions, including combination therapies, enhance the therapeutic or protective effect, and / or increase the therapeutic effect of another immune therapy. Therapeutic and prophylactic methods and compositions can be provided in a combined amount effective to achieve the desired effect. This process may involve contacting the cells with both an antibody or antibody fragment and a second therapy. A tissue, tumor, or cell can be contacted with one or more compositions or pharmacological formulation(s) comprising one or more of the agents, or by contacting the tissue, tumor, and / or cell with two or more distinct compositions or formulations.

[0164] The terms “contacted” and “exposed,” when applied to a cell, are used herein to describe the process by which a therapeutic construct and a chemotherapeutic or radiotherapeutic agent are delivered to a target cell or are placed in direct juxtaposition with the target cell. To achieve cell killing, for example, both agents are delivered to a cell in a combined amount effective to kill the cell or prevent it from dividing.

[0165] In certain embodiments, a course of treatment will last 1-90 days or more (this such range includes intervening days). It is contemplated that one agent may be given on any day of day 1 to day 90 (this such range includes intervening days) or any combination thereof, and another agent is given on any day of day 1 to day 90 (this such range includes intervening days) or any combination thereof. Within a single day (24-hour period), the patient may be given one or multiple administrations of the agent(s). Moreover, after a course of treatment, it is contemplated that there is a period of time at which no anti-cancer treatment is administered. This time period may last 1-7 days, and / or 1-5 weeks, and / or 1-12 months or more (this such range includes intervening days), depending on the condition of the patient, such as their prognosis, strength, health, etc. It is expected that the treatment cycles would be repeated as necessary. V. Kits

[0166] In various aspects of the embodiments, a kit is envisioned containing therapeutic agents and / or other therapeutic and delivery agents. In some embodiments, the present embodiments contemplate a kit for preparing and / or administering a T cell and PSC-derived cell composition of the embodiments. The kit may comprise one or more sealed vials containing any of the pharmaceutical compositions of the present embodiments. The kit may include, for example, T cells and PSC-derived cells as well as reagents to prepare, formulate, and / or administer the components of the embodiments or perform one or more steps of the inventive methods. In some embodiments, the kit may also comprise a suitable container, which is a container that will not react with components of the kit, such as an Eppendorf tube, an assay plate, a syringe, a bottle, or a tube. The container may be made from sterilizable materials such as plastic or glass.

[0167] The kit may further include an instruction sheet that outlines the procedural steps of the methods set forth herein, and will follow substantially the same procedures as described herein or are known to those of ordinary skill in the art. The instruction information may be in a computer readable media containing machine-readable instructions that, when executed using a computer, cause the display of a real or virtual procedure of delivering a pharmaceutically effective amount of a therapeutic agent.

[0168] VI. Examples

[0169] The following examples are included to demonstrate preferred embodiments of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure.

[0170] Example 1 - Combination Engineered Cell Therapy For Type 1 Diabetes hPSCs constitutively expressing a truncated form of epidermal growth factor receptor (EGFRt) on their surface were generated, as well as a luciferase reporter gene for in vivo imaging (Et-hPSCs). In parallel, human Tregs were modified with a CAR recognizing EGFR, with the goal to provide localized protection to transplanted Et-hPSC-derived sBCs (Et-sBCs) against recipient’s effector immune cells, such as cytotoxic CD8+T cells and helper CD4+T cells (FIG. 1). Specifically, an inert artificial gene, a truncated human EGF receptor (EGFRt), was knocked into the AAVS1 safe harbor locus in hPSCs (FIG. 2, FIG. 3), as previously described (5). EGFRt is a version of EGFR that is devoid of extracellular N-terminal ligand binding domains and intracellular receptor tyrosine kinase activity yet retains cell surface localization and a binding epitope for the anti-EGFR monoclonal antibody cetuximab (Erbitux) (6). Then, these cells were differentiated into sBCs and quality-controlled for EGFRt surface expression, beta cell phenotype, and function (FIG. 2, FIG. 3).

[0171] To generate EGFR CAR Tregs, human peripheral blood cells were obtained in the form of a leukopak, isolated total CD4+T cells using negative magnetic selection, sorted CD4+CD25+CD127’ Tregs using fluorescence-assisted cell sorting (FACS) and activated with anti-CD3 / CD28-coated magnetic beads and 1,000 lU / ml interleukin-2 (IL-2). Two days later, activated Tregs were transduced with lentivirus encoding EGFR CAR and expanded the cells in vitro in the presence of 1,000 lU / ml IL-2. In parallel, CD4+CD25‘CD127+effector T (Tefl cells and CD8+T cells were purified, activated, transduced when applicable, and expanded. Transduction efficiency was assessed, and EGFR CAR Tregs were evaluated using in vitro and in vivo immune assays (FIG. 4).

[0172] The CAR construct utilized consisted of an EGFR CAR gene linked to an mCherry reporter gene by a 2A peptide, ensuring equimolar production of EGFR CAR and mCherry proteins (FIG.

[0173] 5). Of note, the antigen-binding domain (single chain variable fragment - scFv) in the EGFR CAR used in the experiments was derived from cetuximab and can thus recognize both full-length human EGFR and EGFRt. Transduction with EGFR CAR lentivirus resulted in 80% transduction efficiency of human Tregs across multiple blood donors, as assessed by mCherry expression (FIG.

[0174] 6).

[0175] Engineering the CAR into human Tregs did not negatively impact Treg identity, as measured by simultaneous expression of the Treg lineage transcription factors FOXP3 and HELIOS, compared to polyclonally activated untransduced (UT) Tregs (FIG. 6).

[0176] Next, the inventors sought to test the ability for the unique target protein, EGFRt, to activate EGFR CAR Tregs (FIG. 6, FIG. 7, FIG. 8). First, EGFRt CAR Tregs were co-cultured with irradiated EGFRt-expressing K562 cells. These target cells have the advantage of lacking expression of HLA and co-stimulatory molecules, such as CD80 and CD86. Hence, only activation via the CAR, not via TCR or CD28, can occur when Trcgs arc co-incubatcd with these cells. Irradiation of K562 cells prevents their division while preserving cell surface molecule expression. It was found that EGFR CAR Tregs, but not UT Tregs, were activated specifically in the presence of EGFRt-expressing K562 cells, to levels comparable to those achieved by polyclonal Treg activation with anti-CD3 / CD28-coated beads (FIG. 6).

[0177] Similarly, EGFR CAR Tregs, but not UT Tregs, were activated in the presence of Et-hPSCs (FIG. 7). Importantly, EGFRt-expressing human pluripotent stem cell-derived beta cells (Et- SBCs) also activated EGFR CAR Tregs. The presence of an insulin GFP reporter gene in the Et- hPSCs further allowed confirmation that co-incubation of Et-sBCs with EGFR CAR Tregs in vitro did not inhibit insulin expression by the Et-sBCs (FIG. 8).

[0178] Overall, an upregulation of activation markers was observed on EGFR CAR Tregs when co-cultured with EGFRt-expressing target cells, demonstrating the specificity of this interaction. Importantly, EGFR CAR Tregs maintained a Treg phenotype following activation via the EGFR CAR and expansion in vitro (FIG. 6, FIG. 9, FIG. 10).

[0179] Moreover, EGFR CAR Tregs displayed suppressive function once activated, suppressing effector T cell proliferation as well as downregulating co-stimulatory receptor expression on dendritic cells (DCs) in vitro. To assess inhibition of T cell proliferation by EGFR CAR Tregs, EGFR CAR Tregs were activated with irradiated EGFRt-K562 and activated CellTrace Violet (CTV) labeled CD8+T cells with anti-CD3 / CD28 beads overnight in parallel. The following day, activated CD8+T cells were debeaded and co-incubated with the activated EGFR CAR Tregs for three days, after which cells were analyzed by flow cytometry to quantify CTV dilution as a proxy for CD8 T cell proliferation (FIG. 9, FIG. 10).

[0180] In addition to inhibiting T cell proliferation, Tregs suppress immune responses also by directly modulating antigen-presenting cells (APCs), such as DCs. More specifically, Tregs express high levels of CTLA4, which competes with the costimulatory receptor CD28 on T cells for binding to CD80 and CD86 costimulatory molecules on the surface of DCs. The strong binding of CTLA4 to CD80 and CD86 removes these molecules from the surface of DCs, rendering them incapable of inducing pro -inflammatory T cell responses. Here, EGFR CAR Tregs were activated and their capacity to downregulate CD80 and CD86 expression in human DCs was evaluated (FIG. 9, FIG. 10). In light of the observations that EGFR CAR Tregs can be generated with high efficiency, retain a Trcg phenotype, arc activated specifically by EGFRt-cxprcssing cells, and suppress T cells and DCs in vitro, the suppressive function of EGFR CAR Tregs in vivo. Et-sBCs were generated from Et-hPSCs and transplanted into the kidney capsule of immunodeficient nonobese diabetic (NOD) severe combined immunodeficiency (SCID) common gamma chain mutant (NSG) mice. In parallel, EGFR CAR Tregs and EGFR CAR T cells were generated. Ten days after the sBC transplant, sBC engraftment as measured by bioluminescence (BLI), as the sBCs express a luciferase gene, the successfully transplanted mice were divided into groups, and infused intravenously (i.v.). with EGFR CAR T cells alone or with EGFR CAR Tregs at a 1:2 T cell:Treg ratio (FIG. 11, FIG. 12). The mice were then monitored for the presence of sBCs in the NSG mice over time using BLI (FIG. 11, FIG. 12).

[0181] Mice that received EGFR CAR Tregs protected sBCs from immune rejection by EGFR CAR T cells for at least 10 days (FIG. 12), demonstrating the suppressive capacity of EGFR CAR Tregs in vivo in response to EGFRt-expressing sBCs. Moreover, FOXP3+Tregs could be found in the grafted kidney of mice that received EGFR CAR Tregs, but not in those mice that received CAR Teff cells alone (FIG. 12, FIG. 13) or together with untransduced (no CAR) Tregs (FIG. 14). These data suggest that local activation of EGFRt CAR Tregs by EGFRt-expressing target cells results in effective suppression of different immune cells, including T cell-mediated immune rejection.

[0182] In summary, the present studies provide in vitro and in vivo proof-of-concept that hPSCs and Tregs can be co-engineered to protect target cells from immune rejection upon transplantation. Such strategies can allow for directed protection of a multitude of stem cell-derived tissues without off-target suppression of other immune responses, having implications for multiple fields of study. This novel dual engineering approach may be able to synergize with or be a safer alternative to hypoimmunogenic cells for cell replacement therapies.

[0183] * * * * * * * * *

[0184] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which arc both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

[0185] REFERENCES

[0186] The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

[0187] 1. Peterson M. Economic Costs of Diabetes in the U.S. in 2017. Diabetes Care. 2018;41: 917-928. doi:10.2337 / dcil8-0007

[0188] 2. Ryan EA, Lakey JR, Paty BW, Imes S, Korbutt GS, Kneteman NM, et al. Successful Islet

[0189] Transplantation: Continued Insulin Reserve Provides Long-Term Glycemic Control. Diabetes. 2002; 51:2148-57. doi: 10.2337 / diabetes.51.7.2148

[0190] 3. Brusko TM, Russ HA, Stabler CL. Strategies for Durable P Cell Replacement in Type 1

[0191] Diabetes. Science. 2021;373:516-522. doi: 10.1126 / science.abhl657

[0192] 4. Nair GG, Liu JS, Russ HA, Tran S, Saxton MS, Chen R, et al. Recapitulating Endocrine Cell

[0193] Clustering in Culture Promotes Maturation of Human Stem-Cell-Derived P Cells. Nat Cell Biol. 2019;21:263-274. doi:10.1038 / s41556-018-0271-4

[0194] 5. Castro-Gutierrez R, Alkanani A, Mathews, CE, Michels A, Russ HA. Protecting Stem Cell

[0195] Derived Pancreatic Beta-Like Cells from Diabetogenic T Cell Recognition. Front Endocrinol. 2021;12:707881. doi: 10.3389 / fendo.2021.707881

[0196] 6. Ferreira LMR, Muller YD, Bluestone JA, Tang Q. Next- Generation Regulatory T Cell Therapy.

[0197] Nat Rev Drug Discov. 2019;18:749-774.

[0198] 7. Bluestone JA, Buckner JH, Fitch M, Gitelman SE, Gupta S, Hellerstein MK, et al. Type 1

[0199] Diabetes Immunotherapy Using Polyclonal Regulatory T Cells. Sci Transl Med. 2015 ;7 :315ra 189. doi: 10.1126 / scitranslmed. aad4134

[0200] 8. Tang Q, Henriksen JK, Bi M, Finger EB, Szot G, Ye J, et al. In vitro-Expanded Antigen- Specific

[0201] Regulatory T Cells Suppress Autoimmune Diabetes. J Exp Med. 2004;199:1455-1466. doi: 10.1084 / jem.20040139

[0202] 9. Muller YD, Ferreira LMR, Ronin E, Ho P, Nguyen V, Faleo G, et al. Precision Engineering of an Anti-HLA-A2 Chimeric Antigen Receptor in Regulatory T Cells for Transplant Immune Tolerance. Front Immunol. 2021;12:686439. doi:10.3389 / fimmu.2021.686439

[0203] 10. Wang X, Chang WC, Wong CW, Colcher D, Sherman M, Ostberg JR, et al. A Transgene-

[0204] Encoded Cell Surface Polypeptide for Selection, in vivo Tracking, and Ablation of Engineered Cells. Blood. 2011;118:1255-1263. doi: 10.1182 / blood-2011-02-337360 11 . Zhou Y, Drummond DC, Zou H, Hayes ME, Adams GP, Kirpotin DP, et al. Impact of Single Chain scFv Antibody Fragment Affinity on Nanoparticlc Targeting of Epidermal Growth Factor Receptor Expressing Tumor Cells. J Mol Biol. 2007; 371:934-947

[0205] Burrack, A.L., Martinov, T., and Fife, B.T. (2017). T Cell-Mediated Beta Cell Destruction: Autoimmunity and Alloimmunity in the Context of Type 1 Diabetes. Frontiers in Endocrinology 8, 343. 10.3389 / fendo.2017.00343.

[0206] Castro-Gutierrez, R., Alkanani, A., Mathews, C.E., Michels, A., and Russ, H.A. (2021). Protecting Stem Cell Derived Pancreatic Beta-Eike Cells From Diabetogenic T Cell Recognition. Front Endocrinol (Eausanne) 12, 707881. 10.3389 / fendo.2021.707881.

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[0209] Sneddon, J.B., Tang, Q., Stock, P., Bluestone, J. A., Roy, S., Desai, T., and Hebrok, M. (2018). Stem Cell Therapies for Treating Diabetes: Progress and Remaining Challenges. Cell Stem Cell 22, 810-823. 10.1016 / j.stem.2018.05.016.

Claims

WHAT IS CLAIMED:

1. A cell therapy composition comprising pluripotent stem cell (PSC) derived cells engineered to express a cell surface protein or fragment thereof and regulatory T cells (Tregs) engineered to express a chimeric antigen receptor (CAR) to said cell surface protein or fragment thereof.

2. The composition of claim 1, wherein PSCs are induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs).

3. The composition of claim 1 or 2, wherein the PSC are human PSCs.

4. The composition of any of claims 1-3, wherein the PSC-dcrivcd cells comprise said cell surface protein or fragment thereof knocked-in at the AAVS1, CCR5, hRosa26, Rogil (GHS1), Rogi2 (GSH2), GSH7, GSH8, GSH31, TRAC, HSH323, HSH325, HSH289, HSH227, HSH229, HSH253, HSH255, HSH257, HSH259, HSH261, HSH263, HSH317, HSH303, HSH331, HSH283, HSH231, HSH315, HSH327, HSH305, HSH307, HSH309, HSH285, HSH233, HSH311, HSH299, HSH301, HSH293, HSH319, HSH333, HSH295, HSH329, HSH297, HSH291, HSH313, HSH321 or GAPDH safe harbor locus of said PSC- derived cells.

5. The composition of any of claims 1-4, wherein the Tregs are human Tregs.

6. The composition of any of claims 1-5, wherein the Tregsare autologous.

7. The composition of any of claims 1-5, wherein the Tregsare allogeneic.

8. The composition of any of claims 5-7, wherein the human TregscompriseCD4+CD25+CD127‘ cells activated with anti-CD3 / CD28-coated magnetic beads and interleukin-2 (IL-2).

9. The composition of any of claims 1-8, wherein the CAR construct comprises a cell surface protein or fragment thereof selected from the group consisting of F(ab’)2, Fab’, Fab, Fv, and scFv.

10. The composition of any of claim 1-9, wherein the cell surface protein is biologically inert.

11. The composition of any of claims 1-9, wherein the cell surface protein is epidermal growth factor receptor (EGFR).

12. The composition of any of claims 1-11 , wherein the cell surface protein a cell surface receptor, ligand, glycoprotein, cell adhesion molecule, antigen, integrin or cluster of differentiation (CD).

13. The composition of any of claims 1-12, wherein the cell surface protein is epidermal growth factor receptor (EGFR), an erbB-2 receptor tyro sine-protein kinase (errb2, HER2), an erbB-3 receptor tyrosine-protein kinase, an erbB-4 receptor tyrosine-protein kinase, a hepatocyte growth factor receptor (HGFR / c-MET), an insulin-like growth factor receptor- 1 (IGF-1 R), EpCAM, VEGFR, integrin (e.g., integrins avP3, a4, allbp3, a4p7, a5pi, avp3, av), a member of the TNF receptor superfamily (e.g., TRAIL-R1, TRAIL-R2), a member of the epidermal growth factor receptor family, PDGF Receptor, interferon receptor, folate receptor, GPNMB, ICAM-1, HLA-DR, CEA, CA-125, MUC1, TAG-72, IL-6 receptor, 5T4, GD2, GD3, prostate-specific membrane antigen (PSMA), or clusters of differentiation (e.g., CD2, CD3, CD4, CD5, CD11, CDl la / LFA-1, CD15, CD18 / ITGB2, CD19, CD20, CD22, CD23 / IgE Receptor, CD25, CD28, CD30, CD33, CD38, CD40, CD41, CD44, CD51, CD52, CD62L, CD74, CD80, CD125, CD147 / basigin, CD152 / CTLA-4, CD154 / CD40L, CD195 / CCR5 or CD319 / SLAMF7.

14. The composition of claim 11, wherein the EGFR is a truncated EGFR (EGFRt).

15. The composition of claim 14, wherein the EGFRt comprises SEQ ID NO: 1.

16. The composition of claim 14, wherein the CAR contains an anti-EGFR cetuximab single chain fragment variable (scFv).

17. The composition of any of claims 1-16, wherein the CAR comprises a CD28- CD c, intracellular domain.

18. The composition of any of claims 1-17, wherein the CAR further comprises a reporter protein.

19. The composition of claim 18, wherein the reporter protein is linked to the cell surface protein or fragment thereof by a peptide linker.

20. The composition of claim 19, wherein the peptide linker is a 2A peptide linker.

21. The composition of any of claims 1-20, wherein the PSC-derived cells comprise one or more cell types.

22. The composition of claim 21, wherein the PSC-derived cells form a tissue or organ.

23. The composition of any of claims 1-20, wherein the PSC-derived cells are insulinproducing beta cells.

24. The composition of any of claims 1-23, wherein the PSC-derived cells are further engineered to comprise a deletion of P? microglobulin.

25. The composition of any of claims 1-24, wherein the PSC-derived cells are further engineered to express an immune checkpoint protein.

26. The composition of claim 25, wherein the immune checkpoint protein is CD39, CD47, CD73, CTLA-4, HLA-G, HLA-E, PD-1, PD-L1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3, KIR, or A2aR.

27. The composition of claim 25, wherein the immune checkpoint protein is PD-L1.

28. A pharmaceutical composition comprising the composition of any of claims 1-27 and a pharmaceutical carrier.

29. A composition comprising an effective amount of the composition of claim 28 for use in the treatment of a disease in a subject.

30. The composition of claim 29, wherein the disease is an autoimmune disease.

31. The composition of claim 30, wherein the autoimmune disease is type I diabetes.

32. A method of treating a disease in a subject comprising administering a cell therapy composition comprising PSC-derived cells engineered to express a cell surface protein or fragment thereof and Tregs engineered to express a CAR to said cell surface protein or fragment thereof.

33. The method of claim 32, wherein PSCs are induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs).

34. The method of claim 32 or 33, wherein the PSC are human PSCs.

35. The method of any of claims 32-34, wherein the PSC-derived cells were obtained by knocking in the cell surface protein or fragment thereof at the AAVS1, CCR5, hRosa26, Rogil (GHS1), Rogi2 (GSH2), GSH7, GSH8, GSH31, TRAC, HSH323, HSH325, HSH289, HSH227, HSH229, HSH253, HSH255, HSH257, HSH259, HSH261, HSH263, HSH317, HSH303, HSH331, HSH283, HSH231, HSH315, HSH327, HSH305, HSH307, HSH309, HSH285, HSH233, HSH311, HSH299, HSH301, HSH293, HSH319, HSH333, HSH295, HSH329, HSH297, HSH291, HSH313, HSH321 or GAPDH safe harbor locus of the PSC.

36. The method of any of claims 32-35, wherein the Tregs arc human Tregs.

37. The method of any of claims 32-36, wherein the Tregs are autologous.

38. The method of any of claims 32-36, wherein the Tregs are allogeneic.

39. The method of any of claims 36-38, wherein the human Tregswere isolated from human peripheral blood by sorting for CD4+CD25+CD127‘ cells and activating with anti- CD3 / CD28-coated magnetic beads and interleukin-2 (IL-2).

40. The method of any of claims 32-39, wherein the CAR construct comprises a cell surface protein or fragment thereof selected from the group consisting of F(ab’)2, Fab’, Fab, Fv, and scFv.

41. The method of any of claims 32-40, wherein the cell surface protein is biologically inert.

42. The method of any of claims 32-41, wherein the cell surface protein a cell surface receptor, ligand, glycoprotein, cell adhesion molecule, antigen, integrin or cluster of differentiation (CD).

43. The method of any of claims 32-41, wherein the cell surface protein is epidermal growth factor receptor (EGFR), an erbB-2 receptor tyrosine-protein kinase (errb2, HER2), an erbB-3 receptor tyrosine-protein kinase, an erbB-4 receptor tyrosine-protein kinase, a hepatocyte growth factor receptor (HGFR / c-MET), an insulin-like growth factor receptor- 1 (IGF-1 R), EpCAM, VEGFR, integrin (e.g., integrins avP3, a4, allbp3, a4p7, a5pi, avP3, av), a member of the TNF receptor superfamily (e.g., TRAIL-R1, TRAIL-R2), a member of the epidermal growth factor receptor family, PDGF Receptor, interferon receptor, folate receptor, GPNMB, ICAM-1, HLA-DR, CEA, CA-125, MUC1, TAG-72,IL-6 receptor, 5T4, GD2, GD3, prostate-specific membrane antigen (PSMA), or clusters of differentiation (c.g., CD2, CD3, CD4, CD5, CD11, CDl la / LFA-1, CD15, CD18 / ITGB2, CD19, CD20, CD22, CD23 / IgE Receptor, CD25, CD28, CD30, CD33, CD38, CD40, CD41, CD44, CD51, CD52, CD62L, CD74, CD80, CD125, CD147 / basigin, CD152 / CTLA-4, CD154 / CD40L, CD195 / CCR5 or CD319 / SLAMF7.

44. The method of any of claims 32-40, wherein the cell surface protein is epidermal growth factor receptor (EGFR).

45. The method of claim 44, wherein the EGFR is a truncated EGFR (EGFRt).

46. The method of claim 45, wherein the CAR contains an anti-EGFR cetuximab single chain fragment variable (scFv).

47. The method of any of claims 32-46, wherein the CAR comprises a CD28- CD3^ intracellular domain.

48. The method of any of claims 32-47, wherein the CAR further comprises a reporter protein.

49. The method of claim 48, wherein the reporter protein is linked to the cell surface protein or fragment thereof by a peptide linker.

50. The method of claim 49, wherein the peptide linker is a 2A peptide linker.

51. The method of any of claims 32-50, wherein the PSC-derived cells are insulin-producing beta cells.

52. The method of any of claims 32-51, wherein the PSC-derived cells are further engineered to comprise a deletion of P? microglobulin.

53. The method of any of claims 32-52, wherein the PSC-derived cells are further engineered to express an immune checkpoint protein.

54. The method of claim 53, wherein the immune checkpoint protein is CD39, CD47, CD73, CTLA-4, HLA-G, HLA-E, PD-1, PD-L1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3, KIR, or A2aR.

55. The method of claim 53, wherein the immune checkpoint protein is PD-L1.

56. The method of any of claims 32-55, wherein the disease is an autoimmune disease.

57. The method of claim 56, wherein the autoimmune disease is type I diabetes.

58. The method of any of claims, wherein the cell therapy composition is administered intravenously, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, percutaneously, subcutaneously, regionally, or by direct injection or perfusion.

59. The method of any of claims 32-58, wherein the cell therapy composition is delivered intravenously or subcutaneously.

60. The method of any of claims 32-59, further comprising administering at least one additional therapy to the subject.

61. The method of any of claims 32-59, wherein the CAR Tregs and PSC-derived cells are administered more than once.

62. A method of preventing allogeneic immune rejection upon transplantation of PSC-derived cells by administering a cell therapy composition of any of claims 1-27.