Pluripotent stem cell-engineered immune cells for off-the-shelf cell therapy

Abstract

Embodiments of the disclosure include compositions and methods related to engineered monoclonal TCR-Armed Gene-Engineered T (TARGET) cells for off-the-shelf use for clinical therapy. In particular embodiments, the TARGET cells are produced from a pluripotent stem cell line and therefore can be of unlimited supply. The TARGET cell products can be conventional ab T cells, gd T cells, and unconventional T cells such as invariant natural killer T (iNKT) cells, non-invariant nature killer T (NKT) cells, mucosal-associated invariant T (MAIT) cells, and others. The TARGET cell products can also be designed to be the CD8-positive or CD4-positive T cell subtypes, and the CD4-positive TARGET cells can be further designed to be various TH-polarized subtypes.

Description

PLURIPOTENT STEM CELL-ENGINEERED IMMUNE CELLS FOR OFF-THE-SHELF CELL THERAPYCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. Section 119(e) of copending and commonly-assigned U.S. Provisional Patent Application No. 63 / 485,700, filed February 17, 2023, entitled “PLURIPOTENT STEM CELL-ENGINEERED IMMUNE CELLS FOR OFF-THE-SHELF CELL THERAPY”, which application is incorporated by reference herein.TECHNICAL FIELD

[0002] Embodiments of the disclosure concern at least the fields of immunology, cell biology, molecular biology, and medicine, including at least cancer medicine.BACKGROUND

[0003] Cancer affects tens of millions of people worldwide and is a leading threat to public health in the United States. In 2024, over 2 million new cancer cases and over 600,000 cancer deaths are projected to occur in the United States. Despite the existing therapies, cancer patients still suffer from the ineffectiveness of these treatments, their toxicities, and the risk of relapse. Novel therapies for cancer are therefore in desperately needed. Over the past decade, immunotherapy has become the new-generation cancer medicine. In particular, cell-based cellular therapies have shown great promise. An outstanding example is the chimeric antigen receptor (CAR)-engineered adoptive T cells therapy, which targets certain blood cancers at impressive efficacy.

[0004] However, most of the current protocols for treatment consist of autologous adoptive cell transfer, wherein immune cells collected from a patient are manufactured and used to treat this single patient. Such an approach is costly, manufacture labor intensive, and difficult to broadly deliver to all patients in need. Allogenic immunecellular products that can be manufactured at a large-scale and can be readily distributed to treat a higher number of patients therefore are in great demand.

[0005] Despite existing therapies, cancer patients still suffer from the ineffectiveness of these treatments, their toxicities, and the risk of relapse. Novel therapies for diseases, such as cancer and autoimmune diseases, are therefore in desperate demand. The present disclosure provides solutions to a long-felt need for these therapies, but also therapies that can be delivered or distributed more widely.BRIEF SUMMARY

[0006] Embodiments are provided to address the need for new therapies, more particularly, the need for cellular therapies that are not hampered by the challenges posed for individualizing therapy using autologous cells. Embodiments of the invention include methods designed to use pluripotent stem cells (PSCs) cells to generate populations of selected immune cells for “off-the-shelf’ uses. The ability to manufacture a therapeutic cell population or a cell population that can be used to create a therapeutic cell population “off-the-shelf’ increases the availability and usefulness of new cellular therapies.

[0007] In this context, embodiments of the invention include methods of using PSCs to make monoclonal TCR-Armed Gene-Engineered T (TARGET) cells, immune cells which are useful in a wide variety of therapeutic contexts. The methods of the invention can include introducing into TARGET cells a selected monoclonal T cell receptor (TCR) gene as an endogenous TCR gene when a T cell -reprogrammed induced PSC (T-iPSC) line is used to make the TARGET cells, or introducing into TARGET cells a selected monoclonal TCR gene as an exogeneous TCR transgene when a non-T-iPSC PSC line is used to make the TARGET cells. A TCR transgene can comprise nucleic acid molecule encoding a TCR selected from but not limited to: an a.p TCR (a conventional CD4 a|3 TCR, a conventional CD8 a|3 TCR, or an unconventional a|3 TCR); a y5 TCR (a Vy9V52 TCR, a V51 TCR, or other y5 TCRs); an invariant NKT TCR (iNKT TCR); a non-invariant NKT TCR; and / or a mucosal associated invariantTCR (MAIT TCR). Embodiments of the invention include monoclonal TCR-Armed Gene-Engineered T (TARGET) cells made by the methods disclosed herein.

[0008] In certain embodiments of the invention, the TARGET cell comprises a gene expression profile characterized as being at least one of: monoclonal TCR-positive CD3-postive; HLA-I-low / negative; HLA-II-low / negative; expression of immune modulatory and / or suicide / marker transgene(s); and / or disrupted expression of endogenous immune modulatory gene(s). In certain embodiments of the invention, the transgene(s) delivered into the TARGET cells can encode any of the following: immune targeting molecules (e.g., chimeric antigen receptors, CARs; T-cell receptors, TCRs; native or synthetic receptor / ligands, and others), immune regulatory molecules (e.g., IL-2, IL-4, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, IFN-y, TNF-a, TL1A, CD27, CD28, 4-1BB, 0X40, ICOS, DAP10, Bell lb, Batf3, ThPOK, FOXP3, Runx3, and others), immune allorejection resistance molecules (e.g., HLA-C, HLA-E, HLA-G, CD47, and others), and / or suicide control and imaging marker molecules (e.g., sr39TK, iCasp9, CD20, and others). In certain embodiments of the invention, the endogenous gene(s) disrupted in the TARGET cells can encode any of the following: immune checkpoint molecules (e.g, PD-1, PD-L1, CTLA-4, LAG-3, TIM-3, TIGIT, B7-H3 / B7- H4, BTLA, VISTA, NKG2A, A2aR, PVRIG, IDO, CD73, CD39, CD96, CD161, and others), immune regulatory molecules (e.g, TET2, PI3K5 / y, DGK, DNMT3a, Suv39hl, and others), or / and immune allorejection molecules (e.g, HLA-I / II, B2M, CIITA, and others).

[0009] In the methods of the invention, a single transgene or multiple transgenes can be incorporated into a TARGET cell product via any of a wide variety of gene delivery vectors (e.g, lentivector, retrovector, adenovector, AAV, and others) and / or vector-free systems (e.g, CRISPR, TALEN, Zinc-Finger, and others), while a single endogenous gene or multiple endogenous genes of a TARGET cell product can be disrupted from expression via any of a wide variety of gene editing tools (e.g, CRISPR, TALEN, Zinc- Finger, and others). In some embodiments of the invention, an All-in-One engineering (AO-Engineering) strategy can be employed when all the desired gene modificationsintended for a designated TARGET cell product are integrated in a master PSC line. In some embodiments of the invention, an Assembly-Line engineering (AL- Engineering) strategy can be employed when all the desired gene modifications intended for a designated TARGET cell product occur stepwise on a master PSC line as well as its progeny hematopoietic stem and progenitor cells (HSPCs or HSCs; both terms are alternatively used in this invention document).

[0010] In the methods of the invention, PSCs are cultured ex vivo to produce TARGET cells. In certain embodiments of the invention, the ex vivo culture can be divided into three to four stages: Stage 0 (PSC master cell bank generation and maintenance), Stage 1 (ex vivo PSC-HSPC differentiation), Stage 2 (ex vivo HSPC- TARGET cell differentiation), and Stage 3 (ex vivo TARGET cell expansion). In certain embodiments of the invention, an additional “CD4-Induction Step” can be added between the Stage 2 and Stage 3 cultures to enable the generation of CD4+ TARGET cells, and another additional “Tn-Polarization Step” can be further added in Stage 3 culture to enable the generation of Tn-polarized CD4+ TARGET cells. In certain embodiments of the invention, all three culture Stages can be feeder-free and / or serum-free, while in other embodiments of the invention, the Stage 3 culture can contain feeder cells (e.g., artificial antigen presenting cells; APCs). In certain embodiments of the invention, the cell culture media can comprise a base medium supplemented with one or more factors selected to facilitate the PSC-derived TARGET cell differentiation, expansion, and sublineage commitment (see the Detailed Description Section of this invention document). In certain embodiments of the invention, all three stages (Stages 1, 2, and 3) of ex vivo culture can achieve high purity, eliminating the need for in- process purification steps. In certain embodiments of the invention, the PSC-derived HSPC and / or HSPC-derived TARGET intermediate cell product(s) can be cultured freshly or cryopreserved and then thawed for continued culture. In certain embodiments of the invention, an All-in-One engineered (AO-Engineered) master PSC line is cultured ex vivo to produce a designated TARGET cell product, without the need for additional gene engineering steps; in other embodiments, an Assembly-Line engineered(AL-Engineered) master PSC line is cultured ex vivo to make a designated TARGET cell product, requiring additional gene engineering step(s) on the PSC-derived HSPCs and / or other TARGET cell progenitors.

[0011] In some embodiments, a TARGET cell product produced by the methods described herein can be cryopreserved. In some embodiments, the cryo-recovered cell product can be stable at room temperature for at least one hour. In some embodiments, the cryo-recovered cell product is stable at room temperature for at least 1, 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, 24, 30, or 48 hours (or any derivable range therein). In certain embodiments, a cell product contains a solution comprising dextrose, one or more electrolytes, albumin, dextran, and / or DMSO. In further embodiments, a cell product is in a solution that is sterile, nonpyogenic, and isotonic.

[0012] Methods of treating patients with a TARGET cell product are also provided. In certain embodiments, the patient has a cancer. In other embodiments, the patient has a viral, bacterial, fungal or parasitic infection. In some embodiments, the patient has a disease or condition involving inflammation, which, in some embodiments, excludes cancer. In specific embodiments, the patient has an autoimmune disease or condition. In some embodiments, the TARGET cell product is allogeneic with respect to the patient. In additional embodiments, the patient does not exhibit signs of rejection or depletion of the TARGET cells. Some therapeutic methods further include administering to the patient a stimulatory reagent that activates TARGET cells, or a reagent that triggers the suicide gene kill-switch.

[0013] The foregoing has outlined rather broadly the features and technical advantages of the present disclosure in order that the detailed description that follows may be better understood. Additional features and advantages will be described hereinafter which form the subject of the claims herein. It should be appreciated by those skilled in the art that the conception and specific embodiments disclosed may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present designs. It should also be realized by those skilled in the art that such equivalent constructions do not depart from the spirit and scope as setforth in the appended claims. The novel features which are believed to be characteristic of the designs disclosed herein, both as to the organization and method of operation, together with further objects and advantages will be better understood from the following description when considered in connection with the accompanying figures. It is to be expressly understood, however, that each of the figures is provided for the purpose of illustration and description only and is not intended as a definition of the limits of the present disclosure.BRIEF DESCRIPTION OF THE DRAWINGS

[0014] For a more complete understanding of the present disclosure, reference is now made to the following descriptions taken in conjunction with the accompanying drawings.Figure 1. Overview of the PSC-T invention. Schematics showing the generation of “Off-The-Shelf PSC-derived T cell products from pluripotent stem cells (PSCs). (a) PSCs can originate from commercially available or proprietary (homemade) sources. These sources encompass embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) derived from CD34+ hematopoietic stem and progenitor cells (HSPC-iPSCs or HSC-iPSCs), iPSCs derived from mature T cells (T -iPSCs), as well as iPSCs derived from other non-T hematopoietic or non-hematopoietic cells (non- T iPSCs). The examples of T-iPSCs include conventional alpha beta T cell derived iPSCs (Tc-iPSCs), mucosal-associated invariant T cell derived iPSCs (MAIT -iPSCs), natural killer T cell derived iPSCs (NKT-iPSCs), and gamma delta T cell derived iPSCs (yST-iPSCs). (b) Gene engineering strategy & toolbox includes: (bl) “All-in-One" Engineering (AO-Engineering) strategy: all gene engineering operations occur at the PSC stage to generate designated PSC master cell lines that can be banked and used to produce designated T cell products via a streamlined PSC-T differentiation culture without further gene engineering needs. (b2) "Assembly-Line" Engineering (AL- Engineering) strategy: genetic engineering operations can occur at the PSC stage as well as at the PSC-derived CD34+ HSPC stage in a step-wise manner, allowing flexible"plug-in" of various genetic manipulations to generate an array of designated T cell products. (b3) The gene engineering toolbox include gene engineering methods like viral and non-viral vectors (e.g., Lentivector) and gene-editing tools (e.g., CRISPR KO / KI), and genes of interest (e.g., TCR, CAR, immune modulatory genes), (c) Scalable Ex Vivo PSC-T Cell Culture Method: a chemically defined, feeder-free and serum-free culture method that can differentiate selected PSCs into mature T cells, and compatible with all kinds of PSC sources and engineering strategies of choice, (d) "Off-The-Shelf PSC-derived T cell products: the final products are various types of PSC-derived T (PSCT) cells, such as conventional T (pscTc), natural killer T (PSCNKT), mucosal-associated invariant T (PSCMAIT), gamma-delta T (pscy5T), and otherPSCT cells, characterized by high yield, high purity, robustness, and monoclonal TCR, ready for therapeutic applications.Figure 2. Generation and characterization of MAIT reprogrammed iPSC (MAIT-iPSC) lines. (A) Schematics showing the experimental design to generate MAIT-iPSC lines. MAIT cells were isolated from healthy donor peripheral blood mononuclear cells (PBMCs) via MR1-5-0P-RU tetramer staining followed by magnetic activated cell sorting (MACS). The isolated PBMC-derived MAIT (PBMC- MAIT) cells were enriched from ~5% to over 90%. The isolated PBMC-MAIT cells were then subjected to reprogramming using a CytoTune™-iPS 2.0 Sendai Reprogramming Kit (ThermoFisher Scientific, A16517). Following successful reprogramming, individual MAIT-iPSC clones were selected for further verification and characterization, including assessments of iPSC pluripotency, confirmation of MAIT TCR rearrangement, evaluation of tri-lineage differentiation potential (ectoderm, mesoderm, and endoderm), elimination of Sendai viral vectors, mycoplasma testing, and karyotyping analysis. (B) Fluorescence-activated cell sorting (FACS) plots showing the enrichment of PBMC-MAIT cells (gated as CD3+ Va7.2+) after MR1-5- OP-RU tetramer-mediated MACS sorting. (C) Microscopic image showing the morphology of a representative MAIT-iPSC line. Three previously established PSC lines were included as controls: Hl ESC (WiCell, WA01) is a human embryonic stemcell line, iPS21 (ALSTEM) is a human fibroblast-reprogrammed iPSC line, and T-iPSC (UCLA) is a human T cell -reprogrammed iPSC line. (D) FACS plots showing the pluripotency markers (i.e., EpCAM, SSEA-4, TRA-1-81, TRA-1-60) expressed on a representative MAIT-iPSC line. (E) DNA gel image showing the detection of recombined MAIT TCR (Va7.2 chain) PCR products from nine reprogrammed single MAIT-iPSC clones. L: DNA ladder; Samples #1 and #2: PBMC-MAIT cells as positive controls; Sample #3: Hl ESC as a negative control; Samples #4-#12: MAIT-iPSC single clones. The expected PCR product size is 312 bp.Figure 3. Differentiation of PSCs into CD34+ HSPCs in a scalable Feeder- Free / Serum-Free Ex Vivo culture (without gene engineering). (A) Schematics showing the generation of PSC-derived T cells in a scalable Feeder-free / Serum-Free Ex Vivo Culture, highlighting the initial step to differentiate PSCs into CD34+ HSPCs. PSCs tested include Hl ESC (WiCell, WA01; a human embryonic stem cell line), iPS21 (ALSTEM; a human fibroblast-reprogrammed iPSC line), T-iPSC (UCLA; a human T cell-reprogrammed iPSC line), and MAIT-iPSC (home-made; a human MAIT cell- reprogrammed iPSC line). (B) Microscopic images showing the formation of embryoid bodies (EBs; left) on day 5 and the emergence of hematopoietic stem and progenitor cells (HSPCs; right) on day 13 of the Ex Vivo HSPC Differentiation Culture. Data generated using Hl ESCs are presented. (C) FACS plots showing the detection of HSPC markers (i.e., CD34, CD31, CD43, CD44, CD45, CD144, and CD235a) on cells differentiated from PSCs for 13 days. Data generated using Hl ESCs are presented. The objective of HSPC differentiation is to yield CD34+ HSPCs that have undergone definitive hematopoiesis, signifying hematopoietic commitment and enhanced lymphoid lineage potential. Throughout the differentiation process from PSCs, hemogenic endothelial cells (HECs) initially express CD34 and CD31, followed by upregulation of CD43 upon commitment to hematopoietic fate. CD44 serves as a marker for endothelial-to-hematopoietic transition (EHT), crucial for generating definitive HSPCs from PSCs. CD144 denotes endothelial cells, while CD235a, an erythrocyte marker, indicates primitive hematopoiesis. Additionally, CD45 is utilizedto confirm the identity of our HSPCs since it is the hematopoietic lineage-restricted antigen that is expressed on all hematopoietic cells. (D) Quantification of (C) showing the percentage (left) and yield (right) of CD34+CD45+ cells on day 13 of the Ex Vivo HSPC Differentiation Culture (n = 3; n indicates biological replicates). The sources of the starting PSCs are indicated. Data are presented as the mean ± SEM.Figure 4. Differentiation of PSC-derived CD34+ HSPCs into T cells in a scalable Ex Vivo Feeder-Free / Serum-Free Culture (without gene engineering). (A) Schematics showing the generation of PSC-derived T cells in a scalable Feeder- free / S erum -Free Ex Vivo Culture, highlighting the second step to differentiate PSC- derived CD34+ HSPCs into T cells. Note the resulting T cells are expected to express a pair of successfully rearranged endogenous TCR, that is either generated during the differentiation culture when a non-T-iPSC is used, or inherited from the pre-rearranged endogenous TCR when a T-iPSC is used. (B) FACS analysis of T cell differentiation in the Ex Vivo T Cell Differentiation Culture over 5 weeks. Data generated using Hl ESC-derived CD34+ HSPCs are presented. Note both alpha-beta and gamma-delta T cells are generated.Figure 5. Characterization of PSC-derived T cells (without gene engineering). (A) Schematics showing the generation of PSC-derived T cells in a scalable Feeder-free / S erum -Free Ex Vivo Culture, highlighting the characterization of the resultingPSCT cell products. Three cell products were characterized: a Hl ESC- derived conventional T cell (pscTc) product, a T-iPSC-derived conventional T cell (pscTc) product, and a M AIT -iP SC -derived MAIT cell (PSCMAIT) product. (B and C) The PSC-derived T cell products were stimulated with 1 pg / ml anti-CD3 / CD28 (forpscTc) or 1 pM MAIT-activating ligand, 5-(2-oxopropylideneamino)-6-D- ribitylaminouracil (5-OP-RU; forPSCMAIT) in the presence of irradiated healthy donor PBMCs as antigen-presenting cells (APCs) for 14 days, followed by analyses of the antigen response of these PSC-derived T cell products. (B) FACS plots showing the expression of TCR (conventional aPTCR or MAIT TCR) and co-receptors (dominantly CD4'CD8aP+) on the indicatedPSCT cell products. (C) Quantification of the antigen-stimulated expansion of the indicatedPSCT cell products (n = 3-5; n indicates biological replicates). All the testedPSCT cell products mounted significant expansion in response to TCR antigen stimulation, supporting the functionality of these PSC-derived T cells. Data are presented as the mean ± SEM. **P < 0.01, ****P < 0.0001, by Student’ s t test.Figure 6. Generation of PSC-derived MAIT cells and their CAR / LL-15- armed derivatives (AO-Engineering strategy). (A) Schematics showing the experimental design to generate PSC-derived MAIT cells and their CAR / IL- 15 -armed derivatives using an “All-in-One” Engineering (AO-Engineering) strategy. A selected PSC line is engineered with a designated lentivector delivering all genes of interests (MAIT TCR gene together with / out additional CAR and IL 15 genes) to establish a master engineered PSC line, followed by a streamlined 3-Stage Ex Vivo Culture to generate a designated PSC-derived MAIT cell product. A master PSC line engineered with a Lenti / MAIT vector encoding a human MAIT TCR gene can give rise to regular MAIT cells (denoted asPSCMAIT cells, a master PSC line engineered with a Lenti / MAIT -BCAR vector encoding a human MAIT TCR gene together with a BCAR gene can give rise to BCAR-armed MAIT cells (denoted aspscBCAR-MAIT cells), while a master PSC line engineered with a Lenti / MAIT-BCAR-IL15 vector encoding a human MAIT TCR gene together with a BCAR gene and a IL- 15 gene can give rise to B CAR / IL- 15 -armed MAIT cells (denoted aspscl5BCAR-MAIT cells). BCAR, a BCMA-targeting chimeric antigen receptor. (B) FACS monitoring ofPSCMAIT cell development over time. An Hl ESC line engineered with the Lenti / MAIT vector was used to initiate the Ex Vivo culture. (C) FACS monitoring ofpscl5BCAR-MAIT cell development overtime. An Hl ESC line engineered with the Lenti / MAIT-BCAR-IL15 vector was used to initiate the Ex Vivo culture. (D) Estimated yield of the Hl ESC derivedPSCMAIT,pscBCAR-MAIT, andpscl5BCAR-MAIT cell products based on fold expansion (n = 5; n indicates biological replicates). Data are presented as the mean ± SEM. Note the robust and high yield of all three cell products.Figure 7. Characterization of PSC-derived MAIT cells and their CAR / IL- 15-armed derivatives (AO-Engineering strategy).PSCMAIT andpscl5BCAR-MAITcell products derived from Hl ESC via an AO-Engineering strategy were studied for their in vitro anti-tumor efficacy and mechanism of action (MOA). Killing of 0VCAR3-FG (BCMA-), a human ovarian cancer cell line, and MM.1 S-FG (BCMA+), a human multiple myeloma cell line, were studied. (A and B) In vitro killing of 0VCAR3-FG and MM.1 S-FG tumor cells byPSCMAIT cells. (A) Experimental design. (B) Tumor cell killing data collected at 24 hrs post co-culture with / out the addition of 5-OP-RU. Healthy donor PBMC derived T (denoted as T) cells were included as a control (n = 3).(C and D) In vitro killing of MM.1 S-FG tumor cells byPSCMAIT andpscl5BCAR- MAIT cells. (C) Experimental design. (D) Tumor cell killing data collected at 24 hrs post co-culture. Healthy donor PBMC derived non-engineered T and BCAR- engineered T cells (denoted as T and BCAR-T cells, respectively) were included as controls (n = 3). (E and F) In vitro killing of 0VCAR3-FG and MM.1 S-FG byPSCMAIT cells with / out the addition of NKG2D / DNAM-1 blocking antibody (10 pg / ml). (E) Experimental design. (F) OVCAR3-FG (E:T ratio = 0.5: 1; n = 3) and MM. 1 S-FG (E:T ratio = 5 : 1 ; n = 3) killing data collected at 24 hrs post co-culture. Data are presented as the mean ± SEM. ns, not significant, *P < 0.05, ***p < 0.001, ****p < 0.0001, by one-way ANOVA (F).Figure 8. Generation of diverse PSC-derived CAR / IL-15-armed MAIT and conventional T cell products (AL-Engineering strategy). (A) Schematics showing the experimental design to generate diverse PSC-derived T cell products using various PSC sources and an “Assembly-Line” engineering (AL-Engineering) strategy, wherein the gene engineering can occur at both the PSC and CD34+ HSPC stages. Examples shown include the generation of Hl ESC derivedpscl5BCAR-MAIT cells, iPS21 derivedpscl5BCAR-MAIT cells, MAIT-iPSC derivedpscl5BCAR-MAIT cells, and T- iPSC derivedpscl5BCAR-Tc cells. Note in these examples, a single lentivector was used to deliver all transgenes at the CD34+ HSPC stage. PSC-derived CD34+ HSPCs were premade and cryopreserved, then thawed for lentivector transduction followed by continued culture. (B) FACS plots showing the characteristics of the indicatedPSCT cellproducts. (C) Estimated yield of the indicatedPSCT cell products based on fold expansion (n = 5; n indicates biological replicates). Data are presented as the mean ± SEM.Figure 9. Pharmacology study-pscl5BCAR-MAIT cells (AL-Engineering strategy).PSC15BCAR-MAIT cells derived from the Hl ESC using an AL-Engineering strategy (as shown in Figure 8A) were studied. Healthy donor PBMC-derived conventional T cells and MAIT cells, as well as conventional T cells engineered to express the same BCMA-targeting CAR (denoted as PBMC-T, BCAR-T, and PBMC-MAIT cells, respectively) were included as controls. Representative FACS plots are presented. Note thatPSC15BCAR-MAIT cells display a phenotype (e.g., expression of CD28 costimulatory molecule, mixed T / NK markers, and high levels of effector molecules like cytokines and cytotoxic molecules) similar to that of the PBMC-MAIT cells, with heightened memory features (e.g., enhanced expression of memory markers like CD45RA and CD62L).Figure 10. In vitro antitumor efficacy and mechanism of action (MOA) study-pscl5BCAR-MAIT cells (AL-Engineering strategy).pscl5BCAR-MAIT cells derived from Hl ESC using an AL-Engineering strategy (as shown in Figure 8A) were studied. Healthy donor PBMC-derived conventional T cells with or without engineering to express the same BCMA-targeting CAR (denoted as T and BCAR-T cells, respectively) were included as controls. Tumor cell lines used in this study include: MM. 1S-FG (BCMA+), a human multiple myeloma (MM) cell line engineered to express the firefly luciferase and green fluorescent protein (FG) dual-reporters;Bt IKOMM.1 S-FG (BCMA-), MM.1S-FG tumor cell line knocked-out of BCMA gene via CRISPR; and K562-FG, a human chronic myeloid leukemia (CML) cell line engineered to express the FG dual reporters. (A and B) Studying the in vitro antitumor efficacy ofpscl5BCAR-MAIT cells against the MM.1S-FG tumor cells, alongside other PSC- derived T cell products generated using the AL-Engineering strategy including iPS21 derivedpscl5BCAR-MAIT, T-iPSC derivedpscl5BCAR-Tc, and MAIT-iPSC derivedPSC15BCAR-MAIT cells (as shown in Figure 19A). (A) Experimental design. (B) Tumor cell killing data at 24 h (E:T ratio = 0.5: 1; n = 4). Note that all PSC-derived BCAR- armed T cell products, despite their diverse PSC sources, exhibit potent antitumor efficacy comparable to that of the conventional BCAR-T cells. (C and D) Studying the in vitro antitumor efficacy ofpscl5BCAR-MAIT cells again the MM.1S-FG tumor cells under repeating tumor challenge. (C) Experimental design.pscl5BCAR-MAIT cells were mixed with MM.1S-FG (E:T ratio = 1 : 1) and re-challenged every 2 days. Tumor cell killing data were collected on the day of re-challenge. (D) Tumor cell killing data (n = 4). Note that thepscl5BCAR-MAIT cells exhibit potent and durable antitumor efficacy comparable to that of the conventional BCAR-T cells. (E and F) Studying the tumor targeting mechanism ofpscl5BCAR-MAIT cells mediated by TCR and CAR. (E) Experimental design. 5-OP-RU is an agonist antigen recognized by the MAIT TCR. (F) Tumor cell killing data at 24 h (n = 3). (G and H) Studying the tumor targeting mechanism ofpscl5BCAR-NKT cells mediated by NKRs (i.e., NKG2D and DNAM-1). (G) Experimental design. (H) Tumor cell killing data at 24 h (E:T ratio = 5: 1 forBCMA'KOMM.1 S-FGand 2: 1 for K562-FG; n = 4). (I) Schematic showing the CAR / TCR / NKR triple-targeting mechanisms utilized by thepscl5BCAR-MAIT cells to attack the BCMA+ tumor cells. Data are presented as the mean ± SEM. ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, by one-way ANOVA (A, H).Figure 11. In vivo antitumor efficacy study-pscl5BCAR-MAIT cells (AL- Engineering strategy).pscl5BCAR-MAIT cells derived from El HSC using an AL- Engineering strategy (as shown in Figure 8A) were studied. Healthy donor PBMC- derived conventional T cells engineered to express the same BCMA-targeting CAR (denoted as BCAR-T cells) were included as a benchmark control. (A) Experimental design. An MM.1S-FG human MM xenograft NSG mouse model was used. BLI: live animal bioluminescence imaging. (B) BLI images showing the presence of tumor cells in experimental mice over time. (C and D) Quantification of B shown as the average tumor load in all experimental mice of each treatment group over time (C) or shown as the tumor load in individual experimental mice of each treatment group (D). N = 5.TBL, total body luminescence. Data are presented as the mean ± SEM. ****p < 0.0001, by one-way ANOVA (C). Note thatpscl5BCAR-MAIT cells exhibit potent in vivo antitumor efficacy, comparable to that of the conventional BCAR-T cells.Figure 12. Safety & immunogenicity study-pscl5BCAR-MAIT cells (AL- Engineering strategy).pscl5BCAR-MAIT cells derived from El HSC using an AL- Engineering strategy (as shown in Figure 19A) were studied. Healthy donor PBMC- derived conventional T cells engineered to express the same BCMA-targeting CAR (denoted as BCAR-T cells) were included as a benchmark control. (A and B) Studying the graft-versus-host (GvH) response ofpscl5BCAR-MAIT cells using an In Vitro Mixed Lymphocyte Reaction (MLR) Assay. Irradiated PBMCs from random mismatched healthy donors were used as stimulator cells. Data from 3 representative donors are presented. (A) Experimental design. (B) ELISA analyses of IFN-y production on day 4 (n = 3). N, no addition of stimulator PBMCs. (C-E) Studying the host-versus-graft (HvG) response ofpscl5BCAR-MAIT cells using an In Vitro Mixed Lymphocyte Reaction (MLR) Assay. PBMCs from random mismatched healthy donors were used as responder cells. Data from one of three representative donors are presented. (C) Experimental design. (D) ELISA analyses of IFN-y production on day 4 (n = 3). None, no addition of stimulator therapeutic cells. (E) FACS measurements of HLA-I and HLA-II expression on the indicated therapeutic cells. MFI, mean fluorescence intensity. Data are presented as the mean ± SEM. ns, not significant, *P < 0.05, **p < 0.01, ***P < 0.001, ****p < 0.0001, by one-way ANOVA (B, D). Note thatPSC15BCAR-MAIT cells induced no GvH response, in sharp contrast to the control conventional BCAR-T cells, likely attributed to the monoclonal MAIT TCR expressed on thesepscl5BCAR-MAIT cells that recognizes non-polymorphic MHC-I like molecular called MR1. Also note thatpscl5BCAR-MAIT cells triggered significantly reduced HvG response, compared to that triggered by the conventional BCAR-T cells, likely attributed to their significant surface expression of HLA-I / II molecules. These “low-GvHD risk” and “low immunogenicity” features may renderpscl5BCAR-MAIT cells safety as well as resistance to allorej ection by the host T cells, and therefore canbe attractive for an allogeneic “off-the-shelf’ application of thesepscl5BCAR-MAIT cells.Figure 13. Tumor microenvironment (TME) targeting study-pscl5BCAR- MAIT cells (AL-Engineering strategy).pscl5BCAR-MAIT cells derived from El HSC using an AL-Engineering strategy (as shown in Figure 8A) were studied. Primary bone marrow (BM) samples collected from multiple myeloma (MM) patients were used as tumor samples in the study. (A) Experimental design to study TME targeting by therapeutic cells. Primary BM samples collected from MM patients were co-cultured with the therapeutic cells (i.e.,pscl5BCAR-MAIT cells) for 24 hours. (B) FACS measurements of surface MR1 expression on the indicated TME component cells in the MM patient BM samples. TAM, tumor associated macrophages; MDSC, myeloid- derived suppressor cell; T, T cell; B, B cell; NK, natural killer cell. MFI, mean fluorescence intensity. (C) Killing of the indicated TME component cells byPSC15BCAR-MAIT cells at 24 hours (n = 4; n indicates different MM patient BM samples).Data are presented as the mean ± SEM. ns, not significant, ****p < 0.0001, by Student’s t test (C). Note thatpscl5BCAR-MAIT cells effectively and selectively depleted the immunosuppressive TAMs and MDSCs but spared other immune cells in the TME of primary MM patient BM samples, likely attributed to the high surface expression of MR1 on these TAMs and MDSCs that is recognized by the MAIT TCR. The immunosuppressive TME, largely mediated by TAMs and MDSCs, is considered a critical hurdle limiting cancer immunotherapy. The capacity of PSC-engineered CAR- MAIT cells to target and alter the immunosuppressive TME is attractive, offering these cells a unique opportunity for cancer therapy applications especially against solid tumors.Figure 14. Generation and characterization of CD4 single-positive (CD4 SP) and CD8 single-positive (CD8 SP)pscl5BCAR-MAIT cells (AL-Engineering strategy). (A) Schematics showing the experimental design to generate Hl ESC derivedpscl5BCAR-MAIT cells that are either CD4 single-positive (CD4 SP) or CD8single-positive (CD8-SP), using an AL-Engineering strategy. Note that a “CD4 Induction” step is added between Stage 2 (Ex Vivo T Cell Differentiation) and Stage 3 (Ex Vivo T Cell Expansion) cultures to dictate the CD4 SP vs. CD8 SPpscl5BCAR- MAIT cell product. (B) FACS monitoring of the transition of week 4pscl5BCAR- MAIT cells from CD4 / CD8 double positive (DP) to CD4 SP following inducer treatment, or their retention as CD4 / CD8 DP in the absence of inducer treatment by week 5 of T cell differentiation. The week 4 cell culture was treated with CD4 inducer for 16 hours, followed by changed back to fresh media devoid of CD4 inducer and further cultured for an additional 6 days. Note, the CD4 SP lineage commitment transcription factor ThPOK was detected in CD4 SPpscl5BCAR-MAIT cells. (C) FACS plots showing the characteristics of CD4 SP and CD8 SPpscl5BCAR-MAIT final cell products. (D) Estimated yield of CD4 SP and CD8 SPpscl5BCAR-MAIT cell products based on fold expansion (n = 3; n indicates biological replicates).Figure 15. Generation and characterization of ThO / Thl-like and Th2-like CD4 SPpscl5BCAR-MAIT cells (AL-Engineering strategy). (A) Schematics showing the experimental design to generate Hl ESC derived CD4 SPpscl5BCAR- MAIT cells that are ThO / Thl-like or Th2-like, using an AL-Engineering strategy. Note that a “CD4 Induction” step is added between Stage 2 (Ex Vivo T Cell Differentiation) and Stage 3 (Ex Vivo T Cell Expansion) to dictate the CD4 SPpscl5BCAR-MAIT lineage commitment, and an optional “Th2 Polarization” step is added in Stage 3 to dictate the ThO / Thl-like or Th2-like function of the final CD4 SPpscl5BCAR-MAIT cell product. Stage 3 CD4 SPpscl5BCAR-MAIT cells were stimulated with 1 pg / ml anti-CD3 / CD28 antibodies for 7 days in the presence of human IL-2 and irradiated healthy donor PBMCs as antigen presenting cells, resulting in a ThO / Thl-like CD4 SPPSC15BCAR-MAIT final cell product. For Th2 polarization, Stage 3 CD4 SPPSC15BCAR-MAIT cells were stimulated under the same condition but with the addition of ImmunoCult Human Th2 Differentiation Supplement (StemCell Technologies) containing human IL-4 and anti-human IFN-y, resulting in a Th2-like CD4 SPPSC15BCAR-MAIT final cell product. (B) FACS analyses of intracellular production ofcytokines and effector molecules by the indicated cells. Various Hl ESC-derivedPSC15BCAR-MAIT cell products were analyzed, including those induced to be CD8 SP, ThO / Thl-like CD4 SP, and Th2-like CD4 SP. Healthy donor PBMC-derived CD8 SP and CD4 SP conventional BCAR-T cells were included as controls. Note Th2 -polarized CD4 SPPSC15BCAR-MAIT cells exhibit a typical Th2-like function, evidenced by their reduced production of Thl cytokines like IFN-y while enhanced production of Th2 cytokines line IL-4, in sharp contrast to the non-Th2-polarized CD4 SPpscl5BCAR- MAIT cells that exhibit a typical ThO / Thl-like function (high production of IFN-y while low production of IL-4). On the other hand, the CD8 SPpscl5BCAR-MAIT cells exhibit a typical cytotoxic function similar to that of the CD8 SP BCAR-T cells (high production of IFN-y as well as cytotoxic molecules like Perforin and Granzyme B).Figure 16. CMC study- generation ofpscBCAR-iNKT cells from iPSCs. (A) Experimental design to generate the PSC-engineered BCAR-armed invariant natural killer T (pscBCAR-iNKT) cell product. BCMA, B-cell maturation antigen; CAR, chimeric antigen receptor; BCAR, BCMA-targeting CAR. (B) Schematics of Lenti / iNKT-BCAR-(GFP) lentivector encoding a human iNKT TCR gene, a BCAR gene, and an optional GFP reporter gene. (C) FACS plots showing the retention of pluripotency markers (i.e., TRA-1-60R and SSEA-5) on gene-engineered DMD iPSC master cell line. Note the co-expression of the GFP reporter. (D-E) Differentiation of gene-engineered iPSC master cell line into HSCs at the HSC Differentiation Stage. (D) Microscope images showing the cell cultures over time. (E) FACS plots showing the detection of HSC markers (i.e., CD34, CD43, and CD31) on cells from day 12 culture. (F-G) Differentiation of iPSC-derived HSCs intopscBCAR-iNKT cells at the iNKT Differentiation Stage. (F) FACS plots showing the generation of human iNKT cells (identified as CD3+iNKT TCR+) overtime. (G) FACS plots showing the co-expression of iNKT TCR, BCAR, and the GFP reporter on maturepscBCAR-iNKT cells collected at day 28 of the iNKT Differentiation Stage culture.Figure 17. CMC study- generation ofpscBCAR-iNKT cells from ESCs. (A) DNA gel image showing the detection of transgene in Lenti / iNKT-BCAR-GFP vector-transduced PSC master cell lines (Hl ESC line and DMD iPSC line). Genomic DNA was extracted from the indicated PSC master cell line and subjected to polymerase chain reaction (PCR) to amplify a ~2,700bp partial transgene fragment. (B) FACS plots showing the retention of pluripotency markers (i.e., TRA-1-60R and SSEA-5) on gene- engineered Hl ESC master cell line. Note the co-expression of the GFP reporter. (C- D) Differentiation of gene-engineered ESC master cell line into HSCs at the HSC Differentiation Stage. (D) Microscope images showing the cell cultures over time. (E) FACS plots showing the detection of HSC markers (i.e., CD34, CD43, and CD31) on cells from day 12 culture. (E-F) Differentiation of ESC-derived HSCs intopscBCAR- iNKT cells at the iNKT Differentiation Stage. (E) FACS plots showing the generation of human iNKT cells (identified as CD3+iNKT TCR+) over time. (F) FACS plots showing the co-expression of iNKT TCR, BCAR, and the GFP reporter on maturepscBCAR-iNKT cells collected at day 28 of the iNKT Differentiation Stage culture.Figures 18. Pharmacology study -pscBCAR-iNKT cells. Representative FACS plots showing the analysis of phenotype (surface markers) and functionality (intracellular production of effector molecules) ofpscBCAR-iNKT cells. Data of DMD iPSC-derivedpscBCAR-iNKT cells are presented. Healthy donor PBMC-derived conventional aP T cells engineered to express the same BCAR (denoted as BCAR-T cells) were included as a benchmark control.Figure 19. In vitro efficacy study -psciNKT cells. In vitro direct killing of human tumor cells bypsciNKT cells were studied. Healthy donor PBMC-derived conventional aP T (PBMC-T) cells were included as a control. Data of DMD iPSC- derivedpsciNKT cells are presented. Four human tumor cell lines were studied: A375 (melanoma), MDA (breast cancer), 0VCAR8 (ovarian cancer), and PC3 (prostate cancer). All four tumor cell lines were engineered to express firefly luciferase and green fluorescence protein (FG) dual-reporters. N = 4. (A) Experimental design. (B-E) Tumor cell killing data at 24-hours. Also presented are FACS plots showing the upregulation of surface activation markers (i.e., CD69) and intracellular cytotoxic molecules (i.e., Perforin and Granzyme B) inpsciNKT cells post co-culturing withtumor cells, as well as the ELISA analyses of cell culture supernatants showing increased IFN-y secretion bypsciNKT cells post co-culturing with tumor cells. Data are presented as the mean ± SEM. ns, not significant, *P < 0.05, **P < 0.01, ***p < 0.001, ****p < 0.0001, by Student’s t test.Figure 20. MOA study -psciNKT Cells. Direct killing of human tumor cells bypsciNKT cells via NK pathway was studied. Data of DMD iPSC-derivedpscBCAR- iNKT cells are presented. Healthy donor PBMC-derived conventional aP T (PBMC-T) cells were included as a control. Four human tumor cell lines were studied: A375 (melanoma), MDA (breast cancer), OVCAR8 (ovarian cancer), and PC3 (prostate cancer). All four tumor cell lines were engineered to express firefly luciferase and green fluorescence protein (FG) dual-reporters. (A) Experimental design. The NK activating receptor DNAM-1 mediated pathway was studied. (B) Tumor cell killing data at 24- hours (turn or: effector cell ratio 1 :2; n = 3). Data are presented as the mean ± SEM. ns, not significant, *P < 0.05, **P < 0.01, ***p < 0.001, ****p < 0.0001, by 1-way ANOVA. (C) FACS plots showing the detection of DNAM-1 ligands stress molecules (i.e., Nectin-2 / CDl 12 and PVR / CD155) on the indicated human tumor cell lines.Figure 21. In vitro efficacy and MOA study-pscBCAR-iNKT cells. Data of DMD iPSC-derivedpscBCAR-iNKT cells are present. (A-B) In vitro direct killing of MM.1S-FG tumor cells. (A) Experimental design. (B) Tumor killing data at 24-hours (n = 3). (C-D) / / ? vitro direct killing of MM.1 S -CD Id-FG tumor cells. (C) Experimental design. (D) Tumor killing data at 24-hours (n = 3). MM. IS, human multiple myeloma cell line; MM.1S-FG, MM. IS cell line engineered to express firefly luciferase and green fluorescence protein dual-reporters; MM.lS-CDld-FG, MM. I S cell line engineered to express human CD Id as well as the firefly luciferase and green fluorescence protein dual-reporters. Four effector cells were studied: healthy donor PBMC-derived conventional aP T (PBMC-T) cells, BCMA-targeting CAR-engineered PBMC-T (BCAR-T) cells, PSC-derived iNKT (psciNKT) cells, and PSC-derived BCMA-targeting CAR-armed iNKT (pscBCAR-iNKT) cells. Data are presented as themean ± SEM. ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****p < 0.0001, by 1-way ANOVA.Figure 22. Safety & immunogenicity study-pscBCAR-iNKT cells. Data of DMD iPSC-derivedpscBCAR-iNKT cells are present. (A-B) In Vitro Mixed Lymphocyte Reaction (MLR) Assay studying graft-versus-host (GvH) response.pscBCAR-iNKT cells were studied as responder cells; healthy donor PBMC-derived conventional BCAR-T cells were included as a responder control; irradiated donor- mismatched PBMCs from 3 random healthy donors were used as stimulator cells. (A) Experimental design. (B) ELISA analyses of IFN-y production at day 4 (n = 3). N, no stimulator cells. (C-D) In Vitro Mixed Lymphocyte Reaction (MLR) Assay studying t-versus-host (GvH) response. IrradiatedpscBCAR-iNKT were studied as stimulator cells; irradiated healthy donor PBMC-derived conventional BCAR-T cells were included as a stimulator control; donor-mismatched PBMCs from 3 random healthy donors were used as responder cells. (C) Experimental design. (D) ELISA analyses of IFN-y production at day 4 (n = 3). (E) FACS plots showing the detection of surface HLA-I(B2M) and HLA-II molecules onpscBCAR-iNKT cells. Healthy donor PBMC- derived conventional BCAR-T cells were included as a control. (F) Quantification of E (n = 3). Data are presented as the mean ±SEM. ns, not significant, *P < 0.05, **P < 0.01, ****p < 0.0001, by 1-way ANOVA (B and D) or Student’s t test (F).Figure 23. CMC study- generation ofpscy6T cells. (A) Experimental design to generate thepscy6T cell product. (B) Schematics of Lenti / y5T lentivector encoding a pair of human y9 and 52 TCR genes. (C) DNA gel image showing the detection of transgene in Lenti / y5T vector-transduced Hl ESC master cell line. Genomic DNA was extracted from the y5 TCR gene-engineered Hl ESC master cell line and subjected to polymerase chain reaction (PCR) to amplify a partial transgene fragment. Nonengineered Hl ESC line was included as a control. Detection of HSC markers (i.e., CD34, CD43, and CD31) on cells from day 12 culture. (D) Differentiation of y5 TCR gene-engineered ESC master cell line into HSCs at the HSC Differentiation Stage. FACS plots are presented showing the detection of HSC markers (i.e., CD34, CD43,and CD31) on cells from day 12 culture. (E-F) Differentiation of ESC-derived HSCs into PSCyST cells at the y6T Differentiation Stage. (E) FACS plots showing the generation of human y8T cells (identified as CD3+V52 TCR+) over time. (F) FACS plots showing the detection of CD 16 expression on maturepscy5T cells collected at day 28 of the y5T Differentiation Stage culture.Figure 24. Pharmacology study -pscy6T cells. Data of Hl ESC-derivedpscy5T cells are presented. Representative FACS plots are presented, showing the analysis of phenotype (surface markers) and functionality (intracellular production of effector molecules) ofpscy6T cells. Healthy donor PBMC-derived conventional yST (PBMC-T) and yST (PBMC-yST) cells were included as staining controls.Figure 25. In vitro efficacy and MOA study-pscy5T cells. Data of Hl ESC- derivedpscy6T cells are presented. (A-B) In vitro direct killing of A375-FG tumor cells. (A) Experimental design. (B) Tumor killing data at 24-hours (n = 3). (C-D) In vitro direct killing of MM.1S-FG tumor cells. (C) Experimental design. (D) Tumor killing data at 24-hours (n = 3). ZOL: zoledronate, a Vy9Vy52 TCR stimulator. A375-FG, a human melanoma cell line A375 engineered to express the firefly luciferase and green fluorescence protein dual -reporters; MM.1S-FG, a human multiple myeloma cell line MM. 1 S engineered to express the firefly luciferase and green fluorescence protein dualreporters. PSC-derived human y6T (pscy6T) cells were studied; healthy donor PBMC- derived conventional yST (PBMC-T) cells were included as a control. Data are presented as the mean ± SEM. ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, by Student’s t test (B) or by 1-way ANOVA (D).Figure 26. Safety & immunogenicity study-pscy6T cells. Data of Hl ESC- derivedpscy6T cells are presented. (A-B) In Vitro Mixed Lymphocyte Reaction (MLR) Assay studying graft-versus-host (GvH) response.pscy6T cells were studied as responder cells; healthy donor PBMC-derived conventional yST (PBMC-T) and yST (PBMC-yST) cells were included as responder controls; irradiated donor-mismatched PBMCs from 3 random healthy donors were used as stimulator cells. (A) Experimentaldesign. (B) ELISA analyses of IFN-y production at day 4 (n = 3). N, no stimulator cells. (C) FACS measurements of surface HLA-I / II molecules on the indicated cells (n = 3). (D-E) In Vitro Mixed Lymphocyte Reaction (MLR) Assay studying host-versus-graft (HvG) response. Irradiatedpscy6T were studied as stimulator cells; irradiated healthy donor PBMC-T and PBMC-y5T cells were included as stimulator controls; donor- mismatched PBMCs from a random healthy donor were used as responder cells. (D) Experimental design. (E) ELISA analyses of IFN-y production at day 4 (n = 3). Data are presented as the mean ± SEM. ns, not significant, *P < 0.05, **P < 0.01, ****p < 0.0001, by 1-way ANOVA (B, C, E).DETAILED DESCRIPTION

[0015] Unless otherwise defined, all terms of art, notations, and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings may be defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional methodology by those skilled in the art. As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out in accordance with manufacturer defined protocols and / or parameters unless otherwise noted.

[0016] As discussed below, we have discovered that pluripotent stem cells (PSCs) can be genetically engineered and differentiated into different types of immune cells, thereby providing an unlimited resource for developing off-the-shelf cell therapies. As is known in the art, pluripotent stem cells include, for example, embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). For review of current stages of PSC-based cell therapies, see, e.g., Zhou Y, Li M, Zhou K, Brown J, Tsao T, Cen X,Husman T, Baipai A, Dunn ZS, and Yang L. Engineering Induced Pluripotent Stem Cells for Cancer Immunotherapy. Cancers. 2022, 14:2266.

[0017] T cells play a central role in mediating and orchestrating immune responses against cancer; therefore they are attractive therapeutic targets for treating cancer and other diseases (see, e.g., Couzin-Frankel, J. 2013. Breakthrough of the year 2013. Cancer immunotherapy. Science 342: 1432-1433. Lim, W.A., and C.H. June. 2017. The Principles of Engineering Immune Cells to Treat Cancer. Cell 168:724-740 ; Rosenberg, S.A., and N.P. Restifo. 2015. Adoptive cell transfer as personalized immunotherapy for human cancer. Science 348:62-68; Vivier, E., Ugolini, S., Blaise, D., Chabannon, C. & Brossay, L. Targeting natural killer cells and natural killer T cells in cancer. Nat Rev Immunol 12, 239-52 (2012); Meraviglia S., Lo Presti E., Dieli F., Stassi G. 2015. T cell-based anticancer immunotherapy: progress and possibilities. Immunotherapy 7:949-951; and Godfrey D.I., Le Nours J., Andrew D.M., Uldrich A.P., and Rossjohn J. 2018. Unconventional T cell targets for cancer immunotherapy. Immunity 48, March 20, 2018). T cells recognize antigens through their surface T cell receptor (TCR) molecules (e.g. an a.p TCR; a y5 TCR; an invariant NK TCR, a noninvariant NKT TCR, and / or a mucosal associated invariant TCR). Typically, TCR polypeptide molecules displayed by a T cell are encoded by a single TCR gene (comprising two genes encoding two subunits of a TCR molecules; referred to as a TCR gene in this material). The TCR gene of a T cell can be generated through a random genomic V / D / J recombination process during T cell development, and therefore is unique for each T cell. Based on the genomic components of their TCR genes, T cells can be divided into two large categories, alpha-beta T (a|3 T) cells and gamma-delta T (y5 T) cells. Alpha-beta T cells can be further divided into subtypes: 1) conventional a|3 T cells that include CD4+helper T cells (CD4 T cells; or TH cells) and CD8+cytotoxic T cells (CD8 T cells; or CTL) cells; and 2) unconventional a|3 T cells that include Type 1 invariant natural killer T (iNKT) cells, Type 2 natural killer T (Type 2 NKT) cells, and mucosal associated invariant T (MAIT) cells, and the like.

[0018] Illustrative TCRs are useful in embodiments of the invention are discussed below.

[0019] Conventional aB CD8 T (CD8 T) cells

[0020] CD8 T cells recognize protein peptide antigens presented by polymorphic major histocompatibility complex (MHC) Class I molecules. CD8 T cells are potent cytotoxic cells for killing target pathogenic cells. CD8 T cells are also named cytotoxic T lymphocytes (CTLs).

[0021] Conventional aB CD4 T (CD4 T) cells

[0022] CD4 T cells recognize protein peptide antigens presented by polymorphic MHC Class II molecules. CD4 T cells are helper T (TH) cells orchestrating the immune responses. Based on their specialized functions, CD4 T cells can be classified into further subtypes: THI, TH2, TH17, TFH, TH9, TREG, and more.

[0023] Type 1 invariant natural killer T (iNKT) cells

[0024] iNKT cells recognize glycolipid antigens presented by a non-polymorphic non-classical MHC Class I-like molecule CD Id. Consequently, iNKT cells do not cause graft-versus-host disease (GvHD) when adoptively transferred into allogeneic recipients. iNKT TCR comprises an invariant alpha chain (Val4-Jal8 in mouse; Va24-J l8 in human), and a limited selection of beta chains (predominantly VP8 / VP7 / VP2 in mouse; predominantly Vpi l in human). Both mouse and human iNKT cells respond to a synthetic agonist glycolipid ligand, alpha-Galactosylceramide (aGC, or a-GC, or a-GalCer).

[0025] Type 2 natural killer T (NKT) cells

[0026] Type 2 NKT cells are also restricted to CD Id. Type 2 NKT cells have a more diverse TCR repertoire and their antigens are less well defined.

[0027] MAIT cellsHuman mucosal-associated invariant T (MAIT) cells are characterized by their expression of an invariant TCRa chain Va7.2- Ja33 / Ja20 / Jal2 paired with a restricted TCR0 chain. MAIT cells recognize microbial peptides presented by the highly conserved MHC class I-like molecule MR1 and bridge the innate and acquired immunesystems to mediate augmented immune responses. Upon activation, MAIT cells rapidly proliferate, produce a variety of cytokines and cytotoxic molecules, and trigger efficient antitumor immunity. Administration of a representative MAIT cell ligand 5-OP-RU effectively activates MAIT cells and enhances their antitumor capacity.

[0028] T cellsThe human y5 T subsets that have been most comprehensively studied are V51+ and V52+ T cells. y5 T cells that express the Vy9 chains paired with the V52 chains (Vy9V52 T cells) are the major y5 T cell population in human peripheral blood. Vy9V52 T cells react to cells with accumulated intracellular phosphoantigens (pAgs), intermediate metabolites produced by infected or transformed cells. These metabolites include isopentenyl pyrophosphate (IPP) formed by the mevalonate (MV A) pathway of tumor cells, and (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP) produced by microbial isoprenoid biosynthesis. Overproduction of IPP in cancer cells as a result of dysregulated MVA pathway leads to activation of Vy9V52 T cells. Stimulation of Vy9V52 T cells can be achieved with bisphosphonates, a class of drugs that prevent or slow down bone loss, such as zoledronate (ZOL). V51+ subset has been found to recognize CD1 proteins, although recognition of lipid antigens on CD1 molecules is typically associated with NKT cells.See, e.g., Lee D, Rosenthal CJ, Penn NE, Dunn ZS, Zhou Y, and Yang L. Human yd T

[0029] Other T cells

[0030] See, e.g. Godfrey D.I., Le Nours J., Andrew D.M., Uldrich A.P., and Rossjohn J. Unconventional T cell targets for cancer immunotherapy. Immunity. 2018, 48(3):453.

[0031] CD4 helper T cell subsetsIn human peripheral blood, CD4 T cells represent approximately two-thirds of the total T cell population, while CD8 T cells constitute the remaining third. All helper T (TH) cells express CD4 co-receptors. Upon encountering antigens, naive TH cells become activated, undergo differentiation, and release cytokines to facilitate and modulate immune reactions. Depending on the cytokine milieu, naive CD4 TH cells have the capacity to polarize into various subsets, including THI, TH2, THI7, TH9, T follicular helper cells (TFH), and regulatory T (TREG) cells. See, e.g., Zhu X, Zhu J. CD4 T Helper Cell Subsets and Related Human Immunological Disorders. Int J Mol Sci. 2020.21(2I):8011.THI cellsTHI cells are involved in cellular immunity. They are characterized by the transcription factor T-bet and signal transducer and activator of transcription (STAT) 4, and the production of IL-2, IFN-y, and TNF-a.TH2 cellsTH2 cells are mediators of humoral immunity. They develop into IL-4-, IL-5-, and IL- 13-producing cells, and are characterized by expressing transcription factor GATA-3 and STAT6.THI? cellsTHI? cells produce IL-17 and regulate tissue inflammation in host defense and chronic diseases. They are characterized by expressing transcription factor RORy.TFH cellsTFH cells are essential for germinal center formation, affinity maturation, and development of most high-affinity antibodies and memory B cells. They secrete IL-6, IL-10, IL-12, IL-21, and are characterized by expressing transcriptional factor Bcl6. TH9 cellsTH9 cells produce IL-9 and play a role in defense against helminth infections, in allergic responses, in autoimmunity, and tumor suppression. They are characterized by expressing transcriptional factor PU.1.TREG cellsTREG cells are known as suppressor T cells, are a subpopulation of T cells that modulate the immune system, maintain tolerance to self-antigens, and prevent autoimmune disease. They secrete anti-inflammatory cytokines IL-10, and TGF-P, and are characterized by expressing transcriptional factor Foxp3.

[0032] The invention disclosed herein has a number of embodiments. Embodiments of the invention include methods of making monoclonal TCR-Armed Gene-Engineered T (TARGET) cells comprising (a) introducing into TARGET cells a selected monoclonal T cell receptor (TCR) gene as an endogenous TCR gene when a T cell- reprogrammed induced PSC (T-iPSC) line is used to make the TARGET cells, or (b) introducing into TARGET cells a selected monoclonal TCR gene as an exogeneous TCR transgene when a non-T-iPSC PSC line is used to make the TARGET cells. A TCR transgene can comprise nucleic acid molecule encoding a TCR selected from but not limited to: an a|3 TCR (a conventional CD4 a|3 TCR, a conventional CD8 a|3 TCR, or an unconventional a|3 TCR); a y5 TCR (a Vy9V52 TCR, a 51 TCR, or other y5 TCRs); an invariant NKT TCR (iNKT TCR); a non-invariant NKT TCR; and / or a mucosal associated invariant TCR (MAIT TCR). Embodiments of the invention include monoclonal TCR-Armed Gene-Engineered T (TARGET) cells made by the methods disclosed herein.

[0033] In certain embodiments of the invention, the TARGET cell comprises a gene expression profile characterized as being at least one of: monoclonal TCR-positive CD3-postive; HLA-I-low / negative; HLA-II-low / negative; expression of immune modulatory and / or suicide / marker transgene(s); and / or disrupted expression of endogenous immune modulatory gene(s). In certain embodiments of the invention, the transgene(s) delivered into the TARGET cells can encode any of the following: immune targeting molecules (e.g., chimeric antigen receptors, CARs; T-cell receptors, TCRs; native or synthetic receptor / ligands, and others), immune regulatory molecules (e.g., IL-2, IL-4, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, IFN-y, TNF-a, TL1A, CD27, CD28, 4-1BB, 0X40, ICOS, DAP10, Bell lb, Batf3, ThPOK, FOXP3, Runx3, and others), immune allorejection resistance molecules (e.g., HLA-C, HLA-E, HLA-G,CD47, and others), and / or suicide control and imaging marker molecules (e.g., sr39TK, iCasp9, CD20, and others). In certain embodiments of the invention, the endogenous gene(s) disrupted in the TARGET cells can encode any of the following: immune checkpoint molecules (e.g, PD-1, PD-L1, CTLA-4, LAG-3, TIM-3, TIGIT, B7-H3 / B7- H4, BTLA, VISTA, NKG2A, A2aR, PVRIG, IDO, CD73, CD39, CD96, CD161, and others), immune regulatory molecules (e.g, TET2, PI3K5 / y, DGK, DNMT3a, Suv39hl, and others), or / and immune allorejection molecules (e.g, HLA-I / II, B2M, CIITA, and others).

[0034] In the methods of the invention, a single transgene or multiple transgenes can be incorporated into a TARGET cell product via any of a wide variety of gene delivery vectors (e.g, lentivector, retrovector, adenovector, AAV, and others) and / or vector-free systems (e.g, CRISPR, TALEN, Zinc-Finger, and others), while a single endogenous gene or multiple endogenous genes of a TARGET cell product can be disrupted from expression via any of a wide variety of gene editing tools (e.g, CRISPR, TALEN, Zinc- Finger, and others). In some embodiments of the invention, an All-in-One engineering (AO-Engineering) strategy can be employed when all the desired gene modifications intended for a designated TARGET cell product are integrated in a master PSC line. In some embodiments of the invention, an Assembly-Line engineering (AL- Engineering) strategy can be employed when all the desired gene modifications intended for a designated TARGET cell product occur stepwise on a master PSC line as well as its progeny hematopoietic stem and progenitor cells (HSPCs or HSCs; both terms are alternatively used in this invention document).

[0035] PSCs refer to human pluripotent stem cells, that can be embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). iPSCs can be reprogrammed from T cells (resulting in T-iPSCs) or from non-T cells such as CD34+ HSPCs (resulting in HSPC-iPSCs) and others (e.g, fibroblasts, NK cells, macrophages). Genetically engineered PSCs (as well as their derived HSPCs) can be used to establish master cell banks, as an unlimited supply to generate the intended “off-the-shelf’ immune cell products via Ex Vivo culture.

[0036] In the methods of the invention, PSCs are cultured ex vivo to produce TARGET cells. In certain embodiments of the invention, the ex vivo culture can be divided into three stages: Stage 0 (PSC master cell bank generation and maintenance), Stage 1 (ex vivo PSC-HSPC differentiation), Stage 2 (ex vivo HSPC-TARGET cell differentiation), and Stage 3 (ex vivo TARGET cell expansion). In certain embodiments of the invention, an additional “CD4-Induction Step” can be added between the Stage 2 and Stage 3 cultures to enable the generation of CD4+ TARGET cells, and another additional “Tn-Polarization Step” can be further added in Stage 3 culture to enable the generation of Tn-polarized CD4+ TARGET cells. In certain embodiments of the invention, all three culture Stages can be feeder-free and / or serum-free, while in other embodiments of the invention, the Stage 3 culture can contain feeder cells (e.g., artificial antigen presenting cells; APCs). In certain embodiments of the invention, the cell culture media can comprise a base medium supplemented with one or more factors selected to facilitate the PSC-derived TARGET cell differentiation, expansion, and sublineage commitment (see the Detailed Description Section of this invention document). In certain embodiments of the invention, all three stages (Stages 1, 2, and 3) of ex vivo culture can achieve high purity, eliminating the need for in-process purification steps. In certain embodiments of the invention, the PSC-derived HSPC and / or HSPC-derived TARGET intermediate cell product(s) can be cultured freshly or cryopreserved and then thawed for continued culture. In certain embodiments of the invention, an All-in-One engineered (AO-Engineered) master PSC line is cultured ex vivo to produce a designated TARGET cell product, without the need for additional gene engineering steps; in other embodiments, an Assembly-Line engineered (AL- Engineered) master PSC line is cultured ex vivo to make a designated TARGET cell product, requiring additional gene engineering step(s) on the PSC-derived HSPCs and / or other TARGET cell progenitors.

[0037] In the methods of the invention, the Stage 0 culture can support PSC master cell bank generation and maintenance. In some embodiments, a selected PSC line can be cultured in a suitable cell culture vessel (e.g., in Matrigel, or on laminin-coated plate)with a serum-free PSC Culture Media for 12-72 hours, followed by a gene engineering step interacting with a single or multiple transgene delivery vectors (e.g., lentivector, retrovector, adenovector, AAV, and others) and / or vector-free systems (e.g., CRISPR, TALEN, Zinc-Finger, and others) and / or gene editing tools (e.g., CRISPR, TALEN, Zinc-Finger, and others), and culturing for an additional 1-7 days. The PSC Culture Media can comprise a base medium (e.g., mTeSR™ Plus Medium, CTS™ Essential 8™ Medium, StemFit®AK03N Medium) and supplements such as bFGF, TGF0, FLT3L, Noggin, activin, Bio, LIF, and others. After verification (and sorting and single cloning if needed), a verified gene-engineered PSC line can then be used to establish master cell bank, that can be cryopreserved for storage or / and maintained in cell culture via passaging. In some embodiments, all gene modifications of a PSC mast line can occur at a single gene-engineering step; while in other embodiments, the gene modifications can occur stepwise, resulting in intermediate PSC master lines that can be used for convenient “plug-in” engineering designs.

[0038] In the methods of the invention, the Stage 1 culture can support PSCs to differentiate into CD34+ HSPCs. In some embodiments, a gene-engineered PSC master cell line generated and maintained from the Stage 0 culture can be gently dissociated to single cells (e.g., via cell dissociation reagent such as Accutase, Versene, or TrypLE) and then transferred to a suitable cell culture vessel (e.g., Ultra-Low Attachment plate or AggreWell) and culture with a serum-free HSPC Differentiation Media A for 12- 48 hours to form embryoid bodies (EBs) or monolayers, fresh media can then be added to the cell culture till day 3-4. The HSPC Differentiation Media A can comprise a base medium (e.g., APEL Medium, StemPro-34 SFM, Ham's F-12 Nutrient Mix, Iscove's Modified Dulbecco's Medium, and others) and supplements such as GlutaMAX, Non- essential amino acids, Vitamin C (e.g., L- Ascorbic acid, L- Ascorbic acid 2-phosphate sesquimagnesium salt hydrate, and its other forms), Monothioglycerol, Transferrin, Activin A, BMP -4, bFGF, VEGF, GSK3 Inhibitors / WNT Activators (e.g., Wnt3A, CHLR99021, AR-A014418, TWS119, LY2090314, 9-ING-41, and others), and ROCK inhibitors (e.g., Y-27632, Thiazovivin, H-1152, and others). On day 3-4, half media isremoved from the cell culture and replaced with a serum-free HSPC Differentiation Media B followed by culturing for additional 6-10 days. A half media change is performed every other day using fresh HSPC Differentiation Media B. The HSPC Differentiation Media B can comprise a base medium (e.g., APEL Medium, StemPro- 34 SFM, Ham's F-12 Nutrient Mix, Iscove's Modified Dulbecco's Medium, and others) and supplements such as GlutaMAX, Non-essential amino acids, Vitamin C (e.g., L- Ascorbic acid, L-Ascorbic acid 2-phosphate sesquimagnesium salt hydrate, and its other forms), Monothioglycerol, human transferrin, heparin, serum-free supplement (e.g., recombinant human albumin, BIT 9500 serum substitute, B-27 supplement, and others), TGF-P inhibitors (e.g., SB431542, SB505124, SB525334, A 83-01, and others), aryl hydrocarbon receptor inhibitors (e.g., PD98059, BAY-218, StemRegenin 1, CH-223191, and others), BMP-4, bFGF, VEGF, SCF, TPO, Flt3 ligand, IL-3, IL-6, IL-11, IGF-1, IGF-2, EPO, SDF-la, and others. At the end of the Stage 1 culture, the resulting CD34+HSPCs can be harvested and proceed to the Stage 2 culture freshly, or be cryopreserved for future usage.

[0039] In the methods of the invention, the Stage 2 culture can support PSC-derived HSPCs generated from the Stage 1 culture to differentiate into mature TARGET cells over a period of 4-10 weeks in the absence of feeders. In some embodiments, freshly harvested PSC-derived HSPCs can directly proceed to the Stage 2 culture; in other embodiments, cryopreserved PSC-derived HSPCs can be recovered and then proceed to the Stage 2 culture. In some embodiments, fresh or cryo-recovered PSC-derived HSPCs can be cultured in a non-tissue culture-treated plate coated with a TARGET Culture Coating (TARGETc) Material (e.g., DLL-1 / 4, VCAM-1 / 5, Retronectin, and others) and with a TARGET Expansion (TARGETe) Media for 12-14 days. The TARGETe Media can comprise a base medium (e.g., Iscove's Modified Dulbecco's Medium, RPMI 1640, SFEMII, aMEM, and others) and supplements such as serum- free supplement (e.g., recombinant human albumin, BIT 9500 serum substitute, B-27 supplement, and others), Insulin-transferrin-selenium, 2-mercaptoethanol, Vitamin C (e.g., L-Ascorbic acid, L-Ascorbic acid 2-phosphate sesquimagnesium salt hydrate, andits other forms), human low density lipoproteins, IL-7, SCF, TPO, IL-3, IL-6, Flt3 ligand, SDF-la, HSC self-renewal agonists (e.g., UM171, UM729, and others), aryl hydrocarbon receptor inhibitors (e.g., PD98059, BAY-218, StemRegenin 1, CH- 223191, and others), p38 MAPK inhibitors (e.g., Adezmapimod, Doramapimod, Losmapimod, SB202190, and others), and others additives. TARGETe Medium can be refreshed every 3-4 days. Cells can then be collected and resuspended in a TARGET Maturation (TARGETm) Media and cultured for another 14-28 days. The TARGETm media can comprise a base medium (e.g., Iscove's Modified Dulbecco's Medium, RPMI 1640, SFEMII, aMEM, and others) and supplements such as serum- free supplement (e.g., recombinant human albumin, BIT 9500 serum substitute, B-27 supplement, and others), Insulin-transferrin-selenium, 2-mercaptoethanol, Vitamin C (e.g., L-Ascorbic acid, L-Ascorbic acid 2-phosphate sesquimagnesium salt hydrate, and its other forms), human low density lipoproteins, IL-7, SCF, Flt3 ligand, IL-2, IL-15, IL-21, TGF-P, SDF-la, HSC self-renewal agonists (e.g., UM171, UM729, and others), aryl hydrocarbon receptor inhibitors (e.g., PD98059, BAY-218, StemRegenin 1, CH- 223191, and others), p38 MAPK inhibitors (e.g., Adezmapimod, Doramapimod, Losmapimod, SB202190, and others), and other additives. TARGETm Medium can be refreshed every 3-4 days. In some embodiments, an All-in-One Engineering (AO- Engineering) strategy can be applied when no additional gene engineering steps are performed in the Stage 2 culture; while in other embodiments, an Assembly-Line Engineering (AL-Engineering) strategy can be applied when additional gene engineering step(s) are performed in the Stage 2 culture. In some embodiments, the gene engineering step(s) can occur at the beginning of the Stage 2 culture when the cells are cultured in the TARGETe Media. In some embodiments, the gene engineering steps can involve the interaction of the cultured cells with a single or multiple transgene delivery vectors (e.g., lentivector, retrovector, adenovector, AAV, and others) and / or vector-free systems (e.g., CRISPR, TALEN, Zinc-Finger, and others) and / or gene editing tools (e.g., CRISPR, TALEN, Zinc-Finger, and others). The resulting mature TARGET cells generated at the end of the Stage 2 culture can be harvested and proceedto the Stage 3 culture freshly, or be cryopreserved for future usage; the intermediate TARGET progenitor cells generated in the middle of the Stage 2 culture, can also proceed along the Stage 2 culture freshly, or be cryopreserved for future usage.

[0040] In the methods of the invention, the Stage 3 culture can support the expansion of mature TARGET cells generated from the Stage 2 culture over a period of about 1-4 weeks, resulting in the final TARGET cell product. In some embodiments, fresh or cryo-recovered mature TARGET cells generated from the Stage 2 culture can be stimulated with TCR cognate antigens (e.g., proteins, peptides, lipids, phosphoantigens, small molecules, and others) or non-specific TCR stimulatory reagents (e.g., anti-CD3 / anti-CD28 antibodies or antibody-coated beads, Concanavalin A, PMA / Ionomycin, and others), with or without the presence of antigen-presenting cells (e.g., irradiated healthy donor PBMCs, artificial APCs, and others), and expanded for up to one month in a T Cell Culture Media. The T Cell Culture Media can comprise a base medium (e.g., CTS OpTmizer, TexMACS, RPMI, DMEM, X-Vivol5, and others) with supplements such as T cell supporting cytokines (e.g., IL-2, IL-4, IL-7, IL-12, IL- 15, IL-18, IL-21, TNFa, TL-1A, SDF-la, TGF-P, and others), as well as small molecules and additives like Wnt activators or glycogen synthase kinase-3 (GSK-3) inhibitors (e.g., Wnt3A and others), CHIR99021, AR-A014418, TWS119, LY2090314, 9-ING-41, lithium chloride (LiCl), BIO (6-bromoindirubin-3 -oxime, 6- Bromoindirubin-3 '-oxime, tyrosine kinase inhibitors (e.g., dasatinib, ibrutinib, acalabrutinib, and zanubrutinib, and others), and caspase inhibitors (e.g., Emricasan, Z- VAD-FMK, Z-VKD-FMK, and others). At the end of the Stage 3 culture, expanded TARGET cells can then be harvested and formulated into the final TARGET cell product, that can be used freshly or cryopreserved for future usage as an “off-the-shelf’ supply.

[0041] In certain embodiments of the invention, an additional “CD4-Induction Step” can be added between the Stage 2 and Stage 3 cultures to enable the generation of CD4+ TARGET cells, and another additional “Tn-Polarization Step” can be further added in Stage 3 culture to enable the generation of Tn-polarized CD4+ TARGET cells.In some embodiments, at the end of the Stage 2 culture, when the developing TARGET cells reach the CD4+CD8+ double-positive (DP) stage, they can be induced to become CD4 SP TARGET cells by switching to a TARGETc-coated plate and culturing with a CD4 Induction (CD4i) Media for a period of about 10-48 hours. The CD4i Media can comprise the TARGETm Media supplemented with T cell activation molecules (e.g., anti-CD3 / CD28 / CD2 antibodies, anti-CD3 / CD28 / CD2 beads, TCR stimulating antigens, phorbol 12-myristate 13-acetate [PMA] and ionomycin, phytohaemagglutinin [PHA], and others). Following this transit CD4 Induction Step, developing TARGET cells can then be returned to fresh TARGETm Media and proceed with the Stage 2 culture. In some embodiments, induced CD4 SP TARGET cells can be further polarized to a designated TH subtype via a “TH-Polarization Step” during the Stage 3 culture. In some embodiments, specific Tn-polarization reagents can be added into the Stage 3 T Cell Culture Media to produce CD4 SP TARGET cells of designated Tn-like subtypes, including but not limited to TnO / THl-like TARGET cells (by adding no Tn-polarization reagents), Tn l -like TARGET cells (by adding Tn l -polarization reagents such as IL-12, IL- 18, and anti -IL-4 antibody), Tn2-like TARGET cells (by adding TH2-polarization reagents such as IL-4 and anti-IFN-y antibody), Tnl7-like TARGET cells (by adding TH17-polarization reagents such as TGF-P, IL-ip, IL-6, IL-21, IL-23), TFH-like TARGET cells (by adding TFH-polarization reagents such as IL-12 with TGF-P or activin A), and TREG-like TARGET cells (by adding TREG-polarization reagents such as IL-2 and TGF-P). At the end of the Stage 3 culture, the resulting non-polarized or TH- polarized CD4 SP TARGET cells can then be harvested and formulated into the final TARGET cell product, that can be used freshly or cryopreserved for future usage as an “off-the-shelf’ supply.

[0042] In some embodiments, a TARGET cell product produced by the methods described herein can be cryopreserved. In some embodiments, the cryo-recovered cell product can be stable at room temperature for at least one hour. In some embodiments, the cryo-recovered cell product is stable at room temperature for at least 1, 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, 24, 30, or 48 hours (or any derivable range therein). In certainembodiments, a cell product contains a solution comprising dextrose, one or more electrolytes, albumin, dextran, and / or DMSO. In further embodiments, a cell product is in a solution that is sterile, nonpyogenic, and isotonic.

[0043] Embodiments of the invention include methods of making monoclonal TCR- Armed Gene-Engineered T (TARGET) cells comprising (a) selecting T cell- reprogrammed induced PSC (T-iPSC) cells comprising an endogenous T cell receptor (TCR); or (b) transducing pluripotent stem cells (e.g. from a PSC cell line) with at least one exogenous nucleic acid molecule encoding a TCR such that the cells transduced by the at least one exogenous nucleic acid molecule express a functional TCR encoded by the exogenous nucleic acid molecule; and differentiating the cells of (a) or (b) so as to generate the monoclonal TCR-Armed Gene-Engineered T (TARGET) cells.Embodiments of the invention further include monoclonal TCR-Armed Gene- Engineered T (TARGET) cells made by the methods disclosed herein.

[0044] As noted above, in certain embodiment of the invention, pluripotent stem cells are gene-engineered via an All-in-One engineering (AO-Engineering) strategy where all gene modifications are integrated in a master PSC line. In alternative embodiments, pluripotent stem cells are gene-engineered via an Assembly-Line engineering (AL-Engineering) strategy when the desired gene modifications occur stepwise on the master PSC line as well as its progeny hematopoietic stem and progenitor cells. In some embodiments of the invention, the PSC comprises a T cell- reprogrammed induced PSC (T-iPSC) and the TCR comprises an endogenous TCR. In some embodiments of the invention, the genetic modifications are made prior to disposing PSC cells in a differentiation media. In certain embodiments of the invention, the genetic modifications are made after disposing PSC cells in a differentiation media.

[0045] Assembly line embodiments of the invention include methods of making monoclonal TCR-Armed Gene-Engineered T (TARGET) cells by: (a) disposing the pluripotent stem cells in a serum-free, feeder free PSC culture media comprising at least one of bFGF, TGFP, FLT3L, Noggin, activin and Bio for at least 3, 6 or 12 hours; (b) combining the pluripotent stem cells from (a) with the at least one exogenous nucleicacid molecule disposed in an expression vector, culturing the PSC cells for at least 3, 6 or 12 hours, and identifying pluripotent stem cells transduced with the expression vector; (c) disposing pluripotent stem cells transduced with the expression vector from (b) that are dissociated to single cells into a serum-free, feeder free HSC differentiation culture media A comprising at least one of: glutamax, ascorbic acid, monothioglycerol, Insulin-Transferrin-Selenium, Activin A, BMP-4, bFGF, VEGF, SB431542, CHIR99021, and a ROCK inhibitor for at least 3, 6 or 12 hours such that the cells form embryonic bodies; (d) disposing the embryonic bodies of (c) in a serum-free, feeder free cell PSC differentiation culture media B comprising at least one of BMP-4, FGF, SCF, TPO, FLT3L, IL-6, IL-11, IGF-1, SB203580, and EPO for at least 2, 4 or 6 days so as to form CD34+ hematopoietic stem cells; (e) collecting and / or enriching CD34+hematopoietic stem cells obtained from (d); (f) disposing the CD34+hematopoietic stem cells obtained from (e) in a serum-free, feeder free TARGET expansion cell culture media comprising at least one of serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, SCF, TPO, IL-3, IL-6, Flt3 ligand, human LDL, and UM171 for at least 1, 2 or 3 days; and (g) disposing the CD34+hematopoietic stem cells obtained from (f) in a serum-free, feeder free cell TARGET maturation culture media comprising at least one of serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, SCF, TPO, IL-3, IL-6, IL-7, IL-15, Flt3 ligand, and ascorbic acid; such that the monoclonal TCR-Armed Gene-Engineered T (TARGET) cells are made. Some embodiments of the invention further comprise disposing the monoclonal TCR-Armed Gene-Engineered T (TARGET) cells into a target cell expansion media comprising at least one of a TCR cognate antigen or a non-specific TCR stimulatory reagent. Optionally, the target cell expansion media comprises feeder cells (e.g. a media comprising at least one of IL-2, IL-7 and IL-15). In some embodiments of this methodology, the methods further comprise cryopreserving cells used in the methodology, for example the CD34+ hematopoietic stem cells made in step (d).

[0046] In certain embodiments of the invention, the method generates TARGET cells expressing at least 2,000 exogenous TCR polypeptides on the surface of the cell. In typical embodiments of the invention, the exogenous nucleic acid molecule encodes at least one T cell receptor selected from: an alpha beta TCR; a gamma delta TCR; an invariant NKT (TARGET) TCR, a non-invariant NKT TCR, and / or a mucosal associated invariant TCR. In some embodiments of the invention, the exogenous nucleic acid molecule encoding a T cell receptor comprises a promoter selected for its ability to resist silencing in the TARGET cells, for example a human ubiquitin promoter. In some embodiments of the invention, the exogenous nucleic acid molecule is contained in a lentiviral expression vector; and / or the exogenous nucleic acid molecule further encodes a polypeptide that stimulates T cells, a polypeptide that disrupts T cell inhibitory factors, and / or a polypeptide comprising a further receptor. Optionally the polypeptide encoded by the exogenous nucleic acid comprises at least one of a chimeric antigen receptor (CAR), IL-2, IL-7, IL-15, IFN-y, TNF-a, CD28, 4- 1BB, 0X40, ICOS, and FOXP3.

[0047] One illustrative embodiment of the invention comprises a method of making monoclonal TCR-Armed Gene-Engineered T (TARGET) cells comprising: transducing pluripotent stem cells with at least one exogenous nucleic acid molecule encoding a T cell receptor (TCR) such that the cells transduced by the at least one exogenous nucleic acid molecule express a functional TCR encoded by the exogenous nucleic acid molecule, wherein the exogenous nucleic acid molecule encodes at least one T cell receptor selected from a mucosal associated invariant TCR alpha chain Va7.2- Ja33 / Ja20 / Jal2 and / or a restricted TCR beta chain; and differentiating the transduced cells so as to generate the monoclonal TCR-Armed Gene-Engineered T (TARGET) cells. See, e.g., Li et al., Mol Ther. 2023 Mar 1;31(3):631-64. In certain of these embodiments, the transduced cells are cultured in a medium comprising one or more of: vitamin B2 or a vitamin B2 precursor (e.g., 5-(2-oxopropylideneamino)-6-D- ribitylaminouracil (5-OP-RU) and 5-(2-oxoethylideneamino)-6-D-ribitylaminouracil(5-OE-RU) ); 5-amino-6-ribitylamino-2,4-(lH,3H)-pyrimidinedione (5-A-RU); a TLR9 agonist, IL-12, and / or IL-18, vitamin B2 or a vitamin B2 precursor.

[0048] Methods of treating patients with a TARGET cell product are also provided. In certain embodiments, the patient has a cancer. In other embodiments, the patient has a viral, bacterial, fungal or parasitic infection. In some embodiments, the patient has a disease or condition involving inflammation, which, in some embodiments, excludes cancer. In specific embodiments, the patient has an autoimmune disease or condition. In some embodiments, the TARGET cell product is allogeneic with respect to the patient. In additional embodiments, the patient does not exhibit signs of rejection or depletion of the TARGET cells. Some therapeutic methods further include administering to the patient a stimulatory reagent that activates TARGET cells, or a reagent that triggers the suicide gene kill-switch.

[0049] Figures 1-26 collectively present data demonstrating the feasibility and therapeutic potential of the disclosed PSC-engineered TARGET cell technology. Various PSC resources (ESC and various iPSCs including T-iPSCs), gene engineering strategies (All-in-One and Assembly-Line Engineering strategies), gene modifications (TCR, CAR, IL- 15, reporters), and TARGET cell types (MAIT, iNKT, y5T, Tc) and subtypes (CD8 SP, CD4 SP, and Tn-subtypes) are validated. The culture method is largely feeder-free and serum-free and therefore is readily scalable. The CMC procedure is robust and the yield is impressive: 1 x 106input PSCs can potentially give rise to ~2 x 106CD34+ HSPCs and eventually ~1013TARGET cells or their derivatives. The resulting PSC-derived TARGET cell products display typical memory T cell and NK features, demonstrate potent in vitro and in vivo antitumor efficacy, and exhibit unique attributes such as low GvHD risk, resistance to allorej ection, and capacity to alter the immunosuppressive TME. These unique attributes can be attractive for potential “off-the-shelf’ cell therapy applications against a large variety of diseases including various cancers especially solid tumors, as well as infections and autoimmune diseases.

[0050] Figure 1 shows an overview of the PSC-T invention.

[0051] Figure 2 shows the successful generation of MAIT cell-reprogrammed iPSC (MAIT-iPSC) lines with high efficiency, as well as the validation and characterization of the resulting MAIT-iPSC lines.

[0052] Figure 3 shows successful differentiation of various sources of PSCs into CD34+ HSPCs in a scalable ex vivo feeder-free / serum-free culture. The tested PSCs include ESC, fibroblast-reprogrammed iPSC, CD34+ HSPC-reprogrammed iPSC, conventional T cell -reprogrammed iPSC, and MAIT cell -reprogrammed iPSC. The resulting CD34+ HSPCs are of high yield (~2 x 106CD34+ HSPCs from 1 x 106input PSCs) and high purity (>60% CD34+CD31+CD45+CD44+CD43+CD144 CD235a ), eliminating the needs for additional purification. These CD34+ HSPCs are also suitable for cryopreservation, that can be valuable for CMC development.

[0053] Figure 4 shows the successful differentiation of PSC-derived CD34+ HSPCs into T cells in a scalable ex vivo feeder-free / serum-free culture. Notably, when a non- T-iPSC line is used as the starting PSC, this PSC-HSPC differentiation culture can support the successful rearrangement of endogenous TCR genes and the generation of both alpha-beta and gamma-delta T cells, highlighting the capacity and potential of this culture method.

[0054] Figure 5 shows the characterization of various PSC-derived T cells (differentiated from ESC, T-iPSC, and MAIT-iPSC), demonstrating their functionality as evidenced by significant expansion in response to TCR antigen stimulation.

[0055] Figures 6 and 7 demonstrate the successful generation and characterization of PSC-derived MAIT cells and their CAR / IL- 15 -armed derivatives produced using an “All-in-One” Engineering (AO-Engineering) strategy. The various product designs tested all worked robustly and of high yield. The AO-engineeredpscl5BCAR-MAIT cell product demonstrated robust in vitro tumor cell killing efficacy and the utilization of TCR / CAR / NKR triple-targeting mechanisms.

[0056] Figures 8-13 demonstrate the successful generation and characterization of PSC-derived MAIT cells and their CAR / IL- 15 -armed derivatives produced using an “Assembly-Line” Engineering (AL-Engineering) strategy. The various product designstested all worked robustly and of high yield. The AL-engineeredpscl5BCAR-MAIT cell product demonstrated robust antitumor efficacy and TCR / CAR / NKR triple-targeting mechanisms. Importantly, the AL-engineeredpscl5BCAR-MAIT cell product demonstrated an in vivo antitumor efficacy comparable to or better than that of the conventional BCAR-T cells, and attractive attributes including free-of-GvHD risk, resistant to allorej ection, and a unique MAIT TCR-mediated TME-targeting capacity that are superior to conventional BCAR-T cells.

[0057] Figures 14 and 15 demonstrate the successful generation and characterization of PSC-derived CD4 single-positive (CD4 SP) MAIT cells as well as their TH2- polarized subtype.

[0058] Figures 16-22 demonstrate the successful generation and promising “off-the- shelf’ cancer therapy potential of PSC-derived iNKT cells and their CAR-armed derivatives.

[0059] Figures 23-26 demonstrate the successful generation and promising “off-the- shelf’ cancer therapy potential of PSC-derived y5T cells.

[0060] The term “exogenous TCR” refers to a TCR gene or TCR gene derivative that is transferred (i.e. by way of gene transfer / transduction / transfection techniques) into the cell or is the progeny of a cell that has received a transfer of a TCR gene or gene derivative. The exogenous TCR genes are inserted into the genome of the recipient cell. In some embodiments, the insertion is random insertion. Random insertion of the TCR gene is readily achieved by methods known in the art. In some embodiments, the TCR genes are inserted into an endogenous loci (such as an endogenous TCR gene loci). In some embodiments, the cells comprise one or more TCR genes that are inserted at a loci that is not the endogenous loci. In some embodiments, the cells further comprise heterologous sequences such as a marker or resistance gene.

[0061] The term “chimeric antigen receptor” or “CAR” refers to engineered receptors, which graft an arbitrary specificity onto an immune effector cell. These receptors are used to graft the specificity of a monoclonal antibody onto a T cell; withtransfer of their coding sequence facilitated by retroviral or lentiviral vectors. The receptors are called chimeric because they are composed of parts from different sources. The most common form of these molecules are fusions of single-chain variable fragments (scFv) derived from monoclonal antibodies, fused to CD3-zeta transmembrane and endodomain; CD28 or 4 IBB intracellular domains, or combinations thereof. Such molecules result in the transmission of a signal in response to recognition by the scFv of its target. An example of such a construct is 14g2a-Zeta, which is a fusion of a scFv derived from hybridoma 14g2a (which recognizes disialoganglioside GD2). When T cells express this molecule (as an example achieved by oncoretroviral vector transduction), they recognize and kill target cells that express GD2 (e.g. neuroblastoma cells). To target malignant B cells, investigators have redirected the specificity of T cells using a chimeric immunoreceptor specific for the B-lineage molecule, CD 19. The variable portions of an immunoglobulin heavy and light chain are fused by a flexible linker to form a scFv. This scFv is preceded by a signal peptide to direct the nascent protein to the endoplasmic reticulum and subsequent surface expression (this is cleaved). A flexible spacer allows the scFv to orient in different directions to enable antigen binding. The transmembrane domain is a typical hydrophobic alpha helix usually derived from the original molecule of the signaling endodomain which protrudes into the cell and transmits the desired signal.

[0062] The term “antigen” refers to any substance that causes an immune system to produce antibodies against it, or to which a T cell responds. In some embodiments, an antigen is a peptide that is 5-50 amino acids in length or is at least, at most, or exactly 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 250, or 300 amino acids, or any derivable range therein.

[0063] The term “allogeneic to the recipient” is intended to refer to cells that are not isolated from the recipient. In some embodiments, the cells are not isolated from the patient. In some embodiments, the cells are not isolated from a genetically matched individual (such as a relative with compatible genotypes).

[0064] The term “inert” refers to one that does not result in unwanted clinical toxicity. This could be either on-target or off-target toxicity. “Inertness” can be based on known or predicted clinical safety data.

[0065] The term “xeno-free (XF)” or “animal component-free (ACF)” or “animal free,” when used in relation to a medium, an extracellular matrix, or a culture condition, refers to a medium, an extracellular matrix, or a culture condition which is essentially free from heterogeneous animal-derived components. For culturing human cells, any proteins of a non-human animal, such as mouse, would be xeno components. In certain aspects, the xeno-free matrix may be essentially free of any non-human animal -derived components, therefore excluding mouse feeder cells or Matrigel™. Matrigel™ is a solubilized basement membrane preparation extracted from the Engelbreth-Holm- Swarm (EHS) mouse sarcoma, a tumor rich in extracellular matrix proteins to include laminin (a major component), collagen IV, heparin sulfate proteoglycans, and entactin / nidogen.

[0066] The term “defined,” when used in relation to a medium, an extracellular matrix, or a culture condition, refers to a medium, an extracellular matrix, or a culture condition in which the nature and amounts of approximately all the components are known.

[0067] A “chemically defined medium” refers to a medium in which the chemical nature of approximately all the ingredients and their amounts are known. These media are also called synthetic media. Examples of chemically defined media include TeSR™.

[0068] Cells are “substantially free” of certain reagents or elements, such as serum, signaling inhibitors, animal components or feeder cells, exogenous genetic elements or vector elements, as used herein, when they have less than 10% of the element(s), and are “essentially free” of certain reagents or elements when they have less than 1% of the element(s). However, even more desirable are cell populations wherein less than 0.5% or less than 0.1% of the total cell population comprise exogenous genetic elements or vector elements.

[0069] A culture, matrix or medium are “essentially free” of certain reagents or elements, such as serum, signaling inhibitors, animal components or feeder cells, when the culture, matrix or medium respectively have a level of these reagents lower than a detectable level using conventional detection methods known to a person of ordinary skill in the art or these agents have not been extrinsically added to the culture, matrix or medium. The serum-free medium may be essentially free of serum.

[0070] Embodiments of the invention use pluripotent stem “cell lines”. As is known in the art, a cell line is a cell culture that is derived from one cell or set of cells of the same type and in which under certain conditions the cells proliferate indefinitely in the laboratory. In this way, cell lines differ from primary cells isolated from individuals in that they are immortalized. Cell lines further differ from primary cells isolated from individuals in that they are clonal (e.g. monoclonal or polyclonal). Because the physiology of cell lines is different from the physiology of primary cells, those of skill in the art cannot predict how cell lines will act solely from studies of primary cells. In some embodiments of the invention, the pluripotent stem cell line is Hl (see, e.g., Getachew et al., Stem Cell Res. 2021 Jul;54: 102401. In other embodiments of the invention, the pluripotent stem cell line is the UCLA DMD1001R; UCLA iPS-21 stem cell line. Other illustrative stem cell lines are disclosed, for example, in Sullivan et al., Regen Med. 2018 Oct;13(7):859-866. doi: 10.2217 / rme-2018-0095; Capowski et al., Development. 2019 Jan 9; 146(1); Ortman et al., Curr Opin Genet Dev. 2017 Oct;46: 179-185; Kattman et al., Cell Stem Cell. 2011 Feb 4;8(2):228-40; Yu et al., Genes Dev. 2008 Aug 1;22(15): 1987-97; Chhabra Stem Cell Rev Rep. 2017 Dec;13(6):757-773; Stacey et al., Nucleic Acids Res. 2016 Jan 4;44; and US Patent Application Publications: 20210310020, 20190153386, 20170226482, 20140154800, 20120083032, 20080311625, 20080267874, 20060160215, 20050095703 and 20030003088.

[0071] A "vector" or "construct" (sometimes referred to as gene delivery or gene transfer "vehicle") refers to a macromolecule, complex of molecules, or viral particle,comprising a polynucleotide to be delivered to a host cell, either in vitro or in vivo. The polynucleotide can be a linear or a circular molecule.

[0072] A “plasmid”, a common type of a vector, is an extra-chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently of the chromosomal DNA. In certain cases, it is circular and doublestranded.

[0073] By "expression construct" or "expression cassette" is meant a nucleic acid molecule that is capable of directing transcription. An expression construct includes, at the least, a promoter or a structure functionally equivalent to a promoter. Additional elements, such as an enhancer, and / or a transcription termination signal, may also be included.

[0074] The term "exogenous," when used in relation to a protein, gene, nucleic acid, or polynucleotide in a cell or organism refers to a protein, gene, nucleic acid, or polynucleotide which has been introduced into the cell or organism by artificial means, or in relation a cell refers to a cell which was isolated and subsequently introduced to other cells or to an organism by artificial means. An exogenous nucleic acid may be from a different organism or cell, or it may be one or more additional copies of a nucleic acid which occurs naturally within the organism or cell. An exogenous cell may be from a different organism, or it may be from the same organism. By way of a non-limiting example, an exogenous nucleic acid is in a chromosomal location different from that of natural cells, or is otherwise flanked by a different nucleic acid sequence than that found in nature.

[0075] The term "corresponds to" is used herein to mean that a polynucleotide sequence is homologous (z.e., is identical, not strictly evolutionarily related) to all or a portion of a reference polynucleotide sequence, or that a polypeptide sequence is identical to a reference polypeptide sequence. In contradistinction, the term "complementary to" is used herein to mean that the complementary sequence is homologous to all or a portion of a reference polynucleotide sequence. For illustration,the nucleotide sequence "TATAC" corresponds to a reference sequence "TATAC" and is complementary to a reference sequence "GT ATA".

[0076] A "gene," "polynucleotide," "coding region," "sequence," "segment," "fragment," or "transgene" which "encodes" a particular protein, is a nucleic acid molecule which is transcribed and optionally also translated into a gene product, e.g., a polypeptide, in vitro or in vivo when placed under the control of appropriate regulatory sequences. The coding region may be present in either a cDNA, genomic DNA, or RNA form. When present in a DNA form, the nucleic acid molecule may be single-stranded (z.e., the sense strand) or double-stranded. The boundaries of a coding region are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A gene can include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and synthetic DNA sequences. A transcription termination sequence will usually be located 3' to the gene sequence.

[0077] The term "cell" is herein used in its broadest sense in the art and refers to a living body which is a structural unit of tissue of a multicellular organism, is surrounded by a membrane structure which isolates it from the outside, has the capability of selfreplicating, and has genetic information and a mechanism for expressing it. Cells used herein may be naturally-occurring cells or artificially modified cells (e.g., fusion cells, genetically modified cells, etc.).

[0078] As used herein, the term "stem cell" refers to a cell capable of self-replication and pluripotency or multipotency. Typically, stem cells can regenerate an injured tissue. Stem cells herein may be, but are not limited to, embryonic stem (ES) cells, induced pluripotent stem cells or tissue stem cells (also called tissue-specific stem cell, or somatic stem cell).

[0079] “Embryonic stem (ES) cells” are pluripotent stem cells derived from early embryos. An ES cell was first established in 1981, which has also been applied to production of knockout mice since 1989. In 1998, a human ES cell was established, which is currently becoming available for regenerative medicine.

[0080] “ Induced pluripotent stem cells,” commonly abbreviated as iPS cells or iPSCs, refer to a type of pluripotent stem cell artificially prepared from a non- pluripotent cell, typically an adult somatic cell, or terminally differentiated cell, such as fibroblast, a hematopoietic cell, a myocyte, a neuron, an epidermal cell, or the like, by introducing certain factors, referred to as reprogramming factors.

[0081] As used herein, “isolated” for example, with respect to cells and / or nucleic acids means altered or removed from the natural state through human intervention.

[0082] “Pluripotency” refers to a stem cell that has the potential to differentiate into all cells constituting one or more tissues or organs, or particularly, any of the three germ layers: endoderm (interior stomach lining, gastrointestinal tract, the lungs), mesoderm (muscle, bone, blood, urogenital), or ectoderm (epidermal tissues and nervous system). “Pluripotent stem cells” used herein refer to cells that can differentiate into cells derived from any of the three germ layers, for example, direct descendants of totipotent cells or induced pluripotent cells.

[0083] By "operably linked" with reference to nucleic acid molecules is meant that two or more nucleic acid molecules (e.g., a nucleic acid molecule to be transcribed, a promoter, and an enhancer element) are connected in such a way as to permit transcription of the nucleic acid molecule. "Operably linked" with reference to peptide and / or polypeptide molecules is meant that two or more peptide and / or polypeptide molecules are connected in such a way as to yield a single polypeptide chain, z.e., a fusion polypeptide, having at least one property of each peptide and / or polypeptide component of the fusion. The fusion polypeptide is particularly chimeric, z.e., composed of heterologous molecules.

[0084] Embodiments of the disclosure concern cells from a pluripotent stem cell line engineered to function as TARGET cells with a T cell receptor (TCR) and that also have imaging and suicide targeting capabilities and are resistant to host immune cell- targeted depletion. Such cells are generated in a scalable PSC-T ex vivo culture system that supports the production of a TARGET cell product from a pluripotent stem cell line at high-efficiency, high yield, and high purity.

[0085] In some embodiments, the engineered cell is a functional TARGET cell. In some embodiments, the engineered cell is capable of producing one or more cytokines and / or chemokines such as IFN-gamma, TNF-alpha, TGF-beta, GM-CSF, IL-2, IL-4, IL-5, IL-6, IL-10, IL-13, IL-17, IL-21, RANTES, Eotaxin, MIP-l-alpha, MIP-l-beta, and the like.

[0086] In some embodiments, the TARGET cell from which the TCR-alpha chain is obtained and the TARGET cell from which the TCR-beta chain is obtained are from the same donor. In some embodiments, the donor of the TARGET cell from which the TCR-alpha chain is obtained is different from the donor of the TARGET cell from which the TCR-beta chain is obtained. In some embodiments, the sequence encoding the TCR-alpha chain and / or the sequence encoding the TCR-beta chain of a TCR clone is modified. In some embodiments, the modified sequence may encode the same polypeptide sequence as the unmodified TCR clone, e.g., the sequence is codon optimized for expression. In some embodiments, the modified sequence may encode a polypeptide that has a sequence that is different from the unmodified TCR clone, e.g., the modified sequence encodes a polypeptide sequence having one or more amino acid substitutions, deletions, and / or truncations.

[0087] In particular embodiments, TARGET cells produced from pluripotent stem cell lines cells are further modified to have one or more characteristics, including to render the cells suitable for allogeneic use or more suitable for allogeneic use than if the cells were not further modified to have one or more characteristics. The present disclosure encompasses TARGET cells that are suitable for allogeneic use, if desired. In some embodiments, the TARGET cells are non-alloreactive and express an exogenous iNTK TCR. These cells are useful for “off the shelf’ cell therapies and do not require the use of the patient’s own TARGET or other cells. Therefore, the current methods provide for a more cost-effective, less labor-intensive cell immunotherapy.

[0088] In specific embodiments, TARGET cells are engineered to be HLA-negative to achieve safe and successful allogeneic engraftment without causing graft-versus-host disease (GvHD) and being rejected by host immune cells (HvG rejection). In specificembodiments, allogeneic TARGET cells do not express endogenous TCRs and do not cause GvHD, because the expression of the transgenic TARGET TCR gene blocks the recombination of endogenous TCRs through allelic exclusion. In particular embodiments, allogeneic TARGET cells do not express HLA-I and / or HLA-II molecules on cell surface and resist host CD8+and CD4+T cell-mediated allograft depletion and sr39TK immunogen-targeting depletion.

[0089] Thus, in certain embodiments the engineered TARGET cells do not express surface HLA-I or -II molecules, achieved through disruption of genes encoding proteins relevant to HLA-I / II expression, including but not limited to beta-2-microglobulin (B2M), major histocompatibility complex II transactivator (CIITA), or HLA-I / II molecules. In some cases, the HLA-I or HLA-II are not expressed on the surface of TARGET cells because the cells were manipulated by gene editing, which may or may not involve CRISPR-Cas9.

[0090] In cases wherein the TARGET cells have been modified to exhibit one or more characteristics of any kind, the TARGET cells may comprise nucleic acid sequences from a recombinant vector that was introduced into the cells. The vector may be a non-viral vector, such as a plasmid, or a viral vector, such as a lentivirus, a retrovirus, an adeno-associated virus (AAV), a herpesvirus, or adenovirus.

[0091] The TARGET cells of the disclosure may or may not have been exposed to one or more certain conditions before, during, or after their production. In specific cases, the cells are not or were not exposed to media that comprises animal serum. The cells may be frozen. The cells may be present in a solution comprising dextrose, one or more electrolytes, albumin, dextran, and / or DMSO. Any solution in which the cells are present may be a solution that is sterile, nonpyogenic, and isotonic. The cells may have been activated and expanded by any suitable manner, such as activated with alphagalactosylceramide (a-GC), for example.

[0092] Aspects of the disclosure relate to a human cell comprising: i) an exogenous expression or activity inhibitor of; or ii) a genomic mutation of: one or more of P2 microglobin (B2M), CIITA, TRAC, TRBC1, or TRBC2. In some embodiments, thecell comprises a genomic mutation. In some embodiments, the genomic mutation comprises a mutation of one or more endogenous genes in the cell’s genome, wherein the one or more endogenous genes comprise the B2M, CIITA, TRAC, TRBC1, or TRBC2 gene. In some embodiments, the mutation comprises a loss of function mutation. In some embodiments, the inhibitor is an expression inhibitor. In some embodiments, the inhibitor comprises an inhibitory nucleic acid. In some embodiments, the inhibitory nucleic acid comprises one or more of a siRNA, shRNA, miRNA, or an antisense molecule. In some embodiments, the cells comprise an activity inhibitor. In some embodiments, following modification the cell is deficient in any detectable expression of one or more of B2M, CIITA, TRAC, TRBC1, or TRBC2 proteins. In some embodiments, the cell comprises an inhibitor or genomic mutation of B2M. In some embodiments, the cell comprises an inhibitor or genomic mutation of CIITA. In some embodiments, the cell comprises an inhibitor or genomic mutation of TRAC. In some embodiments, the cell comprises an inhibitor or genomic mutation of TRBC1. In some embodiments, the cell comprises an inhibitor or genomic mutation of TRBC2. In some embodiments, at least 90% of the genomic DNA encoding B2M, CIITA, TRAC, TRBC1, and / or TRBC2 is deleted. In some embodiments, at least or at most 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99, or 100% (or any range derivable therein) of the genomic DNA encoding B2M, CIITA, TRAC, TRBC1, and / or TRBC2 is deleted. In other embodiments, a deletion, insertion, and / or substitution is made in the genomic DNA. In some embodiments, the cell is a progeny of the human stem or progenitor cell.

[0093] The TARGET cells that are modified to be HLA-negative may be genetically modified by any suitable manner. The genetic mutations of the disclosure, such as those in the CIITA and / or B2M genes can be introduced by methods known in the art. In certain embodiments, engineered nucleases may be used to introduce exogenous nucleic acid sequences for genetic modification of any cells referred to herein. Genome editing, or genome editing with engineered nucleases (GEEN) is a type of genetic engineering in which DNA is inserted, replaced, or removed from a genome usingartificially engineered nucleases, or "molecular scissors." The nucleases create specific double-stranded break (DSBs) at desired locations in the genome, and harness the cell’s endogenous mechanisms to repair the induced break by natural processes of homologous recombination (HR) and nonhomologous end-joining (NHEJ). Nonlimiting engineered nucleases include: Zinc finger nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), the CRISPR / Cas9 system, and engineered meganuclease re-engineered homing endonucleases. Any of the engineered nucleases known in the art can be used in certain aspects of the methods and compositions.

[0094] The engineered TARGET cells may be modified using methods that employ RNA interference. It is commonly practiced in genetic analysis that in order to understand the function of a gene or a protein function one interferes with it in a sequence-specific way and monitors its effects on the organism. However, in some organisms it is difficult or impossible to perform site-specific mutagenesis, and therefore more indirect methods have to be used, such as silencing the gene of interest by short RNA interference (siRNA). However, gene disruption by siRNA can be variable and incomplete. Genome editing with nucleases such as ZFN is different from siRNA in that the engineered nuclease is able to modify DNA-binding specificity and therefore can in principle cut any targeted position in the genome, and introduce modification of the endogenous sequences for genes that are impossible to specifically target by conventional RNAi. Furthermore, the specificity of ZFNs and TALENs are enhanced as two ZFNs are required in the recognition of their portion of the target and subsequently direct to the neighboring sequences.

[0095] Meganucleases may be employed to modify engineered TARGET cells. Meganucleases, found commonly in microbial species, have the unique property of having very long recognition sequences (>14bp) thus making them naturally very specific. This can be exploited to make site-specific DSB in genome editing; however, the challenge is that not enough meganucleases are known, or may ever be known, to cover all possible target sequences. To overcome this challenge, mutagenesis and highthroughput screening methods have been used to create meganuclease variants that recognize unique sequences. Others have been able to fuse various meganucleases and create hybrid enzymes that recognize a new sequence. Yet others have attempted to alter the DNA interacting amino acids of the meganuclease to design sequence specific meganucelases in a method named rationally designed meganuclease (U.S. Patent 8,021,867, incorporated herein by reference). Meganuclease have the benefit of causing less toxicity in cells compared to methods such as ZFNs likely because of more stringent DNA sequence recognition; however, the construction of sequence specific enzymes for all possible sequences is costly and time consuming as one is not benefiting from combinatorial possibilities that methods such as ZFNs and TALENs utilize. So there are both advantages and disadvantages.

[0096] As opposed to meganucleases, the concept behind ZFNs and TALENs is more based on a non-specific DNA cutting enzyme which would then be linked to specific DNA sequence recognizing peptides such as zinc fingers and transcription activator-like effectors (TALEs). One way was to find an endonuclease whose DNA recognition site and cleaving site were separate from each other, a situation that is not common among restriction enzymes. Once this enzyme was found, its cleaving portion could be separated which would be very non-specific as it would have no recognition ability. This portion could then be linked to sequence recognizing peptides that could lead to very high specificity. An example of a restriction enzyme with such properties is Fokl. Additionally FokI has the advantage of requiring dimerization to have nuclease activity and this means the specificity increases dramatically as each nuclease partner would recognize a unique DNA sequence. To enhance this effect, Fokl nucleases have been engineered that can only function as heterodimers and have increased catalytic activity. The heterodimer functioning nucleases would avoid the possibility of unwanted homodimer activity and thus increase specificity of the DSB.

[0097] Although the nuclease portion of both ZFNs and TALENs have similar properties, the difference between these engineered nucleases is in their DNA recognition peptide. ZFNs rely on Cys2-His2 zinc fingers and TALENs on TALEs.Both of these DNA recognizing peptide domains have the characteristic that they are naturally found in combinations in their proteins. Cys2-His2 Zinc fingers typically happen in repeats that are 3 bp apart and are found in diverse combinations in a variety of nucleic acid interacting proteins such as transcription factors. TALEs on the other hand are found in repeats with a one-to-one recognition ratio between the amino acids and the recognized nucleotide pairs. Because both zinc fingers and TALEs happen in repeated patterns, different combinations can be tried to create a wide variety of sequence specificities. Zinc fingers have been more established in these terms and approaches such as modular assembly (where Zinc fingers correlated with a triplet sequence are attached in a row to cover the required sequence), OPEN (low-stringency selection of peptide domains vs. triplet nucleotides followed by high-stringency selections of peptide combination vs. the final target in bacterial systems), and bacterial one-hybrid screening of zinc finger libraries among other methods have been used to make site specific nucleases.

[0098] Thus, embodiments of the disclosure may or may not include the targeting of endogenous sequences to reduce or knock out expression of one or more certain endogenous sequences. In specific embodiments, disruption of one or more of the following genes may block the rearrangement of endogenous TCRs.

[0099] Inhibitory nucleic acids or any ways of inhibiting gene expression of CIITA and / or B2M known in the art are contemplated in certain embodiments. Examples of an inhibitory nucleic acid include but are not limited to siRNA (small interfering RNA), short hairpin RNA (shRNA), double-stranded RNA, an antisense oligonucleotide, a ribozyme and a nucleic acid encoding thereof. An inhibitory nucleic acid may inhibit the transcription of a gene or prevent the translation of a gene transcript in a cell. An inhibitory nucleic acid may be from 16 to 1000 nucleotides long, and in certain embodiments from 18 to 100 nucleotides long. The nucleic acid may have nucleotides of at least or at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 50, 60, 70, 80, 90 or any range derivable therefrom. An siRNA naturally present in a living animal is not“isolated,” but a synthetic siRNA, or an siRNA partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated siRNA can exist in substantially purified form, or can exist in a non-native environment such as, for example, a cell into which the siRNA has been delivered.

[0100] Inhibitory nucleic acids are well known in the art. For example, siRNA and double-stranded RNA have been described in U.S. Patents 6,506,559 and 6,573,099, as well as in U.S. Patent Publications 2003 / 0051263, 2003 / 0055020, 2004 / 0265839, 2002 / 0168707, 2003 / 0159161, and 2004 / 0064842, all of which are herein incorporated by reference in their entirety.

[0101] Particularly, an inhibitory nucleic acid may be capable of decreasing the expression of the protein or mRNA by at least 10%, 20%, 30%, or 40%, more particularly by at least 50%, 60%, or 70%, and most particularly by at least 75%, 80%, 90%, 95% or more or any range or value in between the foregoing.

[0102] In further embodiments, there are synthetic nucleic acids that are protein inhibitors. An inhibitor may be between 17 to 25 nucleotides in length and comprises a 5’ to 3’ sequence that is at least 90% complementary to the 5’ to 3’ sequence of a mature mRNA. In certain embodiments, an inhibitor molecule is 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, or any range derivable therein. Moreover, an inhibitor molecule has a sequence (from 5’ to 3’) that is or is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 or 100% complementary, or any range derivable therein, to the 5’ to 3’ sequence of a mature mRNA, particularly a mature, naturally occurring mRNA, such as a mRNA to B2M, CIITA, TRAC, TRBC1, or TRBC2. One of skill in the art could use a portion of the probe sequence that is complementary to the sequence of a mature mRNA as the sequence for an mRNA inhibitor. Moreover, that portion of the probe sequence can be altered so that it is still 90% complementary to the sequence of a mature mRNA.

[0103] In cases wherein the engineered TARGET cells comprise one or more suicide genes for subsequent depletion upon need, the suicide gene may be of any suitable kind. The TARGET cells of the disclosure may express a suicide gene productthat may be enzyme-based, for example. Examples of suicide gene products include herpes simplex virus thymidine kinase (HSV-TK), purine nucleoside phosphorylase (PNP), cytosine deaminase (CD), carboxypetidase G2, cytochrome P450, linamarase, beta-lactamase, nitroreductase (NTR), carb oxy peptidase A, or inducible caspase 9. Thus, in specific cases, the suicide gene may encode thymidine kinase (TK). In specific cases, the TK gene is a viral TK gene, such as a herpes simplex virus TK gene. In particular embodiments, the suicide gene product is activated by a substrate, such as ganciclovir, penciclovir, or a derivative thereof.

[0104] In some embodiments, the engineered TARGET cells are able to be imaged or otherwise detected. In particular cases, the cells comprise an exogenous nucleic acid encoding a polypeptide that has a substrate that may be labeled for imaging, and the imaging may be fluorescent, radioactive, colorimetric, and so forth. In specific cases, the cells are detected by positron emission tomography. The cells in at least some cases express sr39TK gene that is a positron emission tomography (PET) reporter / thymidine kinase gene that allows for tracking of these genetically modified cells with PET imaging and elimination of these cells through the sr39TK suicide gene function.

[0105] Encompassed by the disclosure are populations of engineered TARGET cells. In particular aspects, TARGET clonal cells comprise an exogenous nucleic acid encoding an TARGET T-cell receptor (T-cell receptor) and lack surface expression of one or more HLA-I or HLA-II molecules. The TARGET cells may comprise an exogenous nucleic acid encoding a suicide gene, including an enzyme-based suicide gene such as thymidine kinase (TK). The TK gene may be a viral TK gene, such as a herpes simplex virus TK gene. In the cells of the population the suicide gene may be activated by a substrate, such as ganciclovir, penciclovir, or a derivative thereof, for example. The cells may comprise an exogenous nucleic acid encoding a polypeptide that has a substrate that may be labeled for imaging, and in some cases a suicide gene product is the polypeptide that has a substrate that may be labeled for imaging. In specific aspects, the suicide gene is sr39TK.

[0106] In certain embodiments of the TARGET cell population, the TARGET cells do not express surface HLA-I or -II molecules because of disrupted expression of genes encoding beta-2-microglobulin (B2M), major histocompatibility complex class II transactivator (CIITA), and / or HLA-I or HLA-II molecules, for example. The HLA-I or HLA-II molecules are not expressed on the cell surface of TARGET cells because the cells were manipulated by gene editing, in specific cases. The gene editing may or may not involve CRISPR-Cas9.

[0107] In particular cases for the TARGET cell population, the TARGET cells comprise nucleic acid sequences from a recombinant vector that was introduced into the cells, such as a viral vector (including at least a lentivirus, a retrovirus, an adeno- associated virus (AAV), a herpesvirus, or adenovirus).

[0108] In certain embodiments, the cells of the TARGET cell population may or may not have been exposed to, or are exposed to, one or more certain conditions. In certain cases, for example, the cells of the population not exposed or were not exposed to media that comprises animal serum. The cells of the population may or may not be frozen. In some cases the cells of the population are in a solution comprising dextrose, one or more electrolytes, albumin, dextran, and / or DMSO. The solution may comprise dextrose, one or more electrolytes, albumin, dextran, and DMSO. The cells may be in a solution that is sterile, nonpyogenic, and isotonic. In specific cases the TARGET cells have been activated, such as activated with alpha-galactosylceramide (a-GC). In specific aspects, the cell population comprises at least about 102- 106clonal cells. The cell population may comprise at least about 106-1013total cells, in some cases.

[0109] In particular embodiments there is a TARGET cell population comprising: clonal TARGET cells comprising one or more exogenous nucleic acids encoding an TARGET T-cell receptor (T-cell receptor) and a thymidine kinase suicide, wherein the clonal TARGET cells have been engineered not to express functional beta-2- microglobulin (B2M), major histocompatibility complex class II transactivator (CIITA), and / or HLA-I and HLA-II molecules and wherein the cell population is atleast about 106-1013total cells and comprises at least about 102-106clonal cells. In some cases the cells are frozen in a solution.II. Formulations and Culture of the Cells

[0110] In particular embodiments, the TARGET cells and / or precursors thereto may be specifically formulated and / or they may be cultured in a particular medium at any stage of a process of generating the TARGET cells. The cells may be formulated in such a manner as to be suitable for delivery to a recipient without deleterious effects.

[0111] The medium in certain aspects can be prepared using a medium used for culturing animal cells as their basal medium, such as any of AIM V, X-VIVO-15, NeuroBasal, EGM2, TeSR, BME, BGJb, CMRL 1066, Glasgow MEM, Improved MEM Zinc Option, IMDM, Medium 199, Eagle MEM, aMEM, DMEM, Ham, RPMI- 1640, and Fischer's media, as well as any combinations thereof, but the medium may not be particularly limited thereto as far as it can be used for culturing animal cells. Particularly, the medium may be xeno-free or chemically defined.

[0112] The medium can be a serum-containing or serum-free medium, or xeno-free medium. From the aspect of preventing contamination with heterogeneous animal- derived components, serum can be derived from the same animal as that of the stem cell(s). The serum-free medium refers to medium with no unprocessed or unpurified serum and accordingly, can include medium with purified blood-derived components or animal tissue-derived components (such as growth factors).

[0113] The medium may contain or may not contain any alternatives to serum. The alternatives to serum can include materials which appropriately contain albumin (such as lipid-rich albumin, bovine albumin, albumin substitutes such as recombinant albumin or a humanized albumin, plant starch, dextrans and protein hydrolysates), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolgiycerol, or equivalents thereto. The alternatives to serum can be prepared by the method disclosed in International Publication No. 98 / 30679, for example (incorporated herein in its entirety). Alternatively, any commercially available materials can be used for more convenience. The commerciallyavailable materials include knockout Serum Replacement (KSR), Chemically-defined Lipid concentrated (Gibco), and Glutamax (Gibco).

[0114] In further embodiments, the medium may be a serum-free medium that is suitable for cell development. For example, the medium may comprise B-27® supplement, xeno-free B-27® supplement (available at world wide web at thermofisher.com / us / en / home / technical-resources / media-formulation.250.html),NS21 supplement (Chen et al., J Neurosci Methods, 2008 Jun 30; 171(2): 239-247, incorporated herein in its entirety), GS21™ supplement (available at world wide web at amsbio.com / B-27.aspx), or a combination thereof at a concentration effective for producing T cells from the 3D cell aggregate.

[0115] In certain embodiments, the medium may comprise one, two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more of the following: Vitamins such as biotin; DL Alpha Tocopherol Acetate; DL Alpha- Tocopherol; Vitamin A (acetate); proteins such as BSA (bovine serum albumin) or human albumin, fatty acid free Fraction V; Catalase; Human Recombinant Insulin; Human Transferrin; Superoxide Dismutase; Other Components such as Corticosterone; D-Galactose; Ethanolamine HC1; Glutathione (reduced); L-Carnitine HC1; Linoleic Acid; Linolenic Acid; Progesterone; Putrescine 2HC1; Sodium Selenite; and / or T3 (tri odo-I-thy ronine) .

[0116] In some embodiments, the medium further comprises vitamins. In some embodiments, the medium comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 of the following (and any range derivable therein): biotin, DL alpha tocopherol acetate, DL alpha-tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, folic acid nicotinamide, pyridoxine, riboflavin, thiamine, inositol, vitamin B12, or the medium includes combinations thereof or salts thereof. In some embodiments, the medium comprises or consists essentially of biotin, DL alpha tocopherol acetate, DL alpha-tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, folic acid nicotinamide, pyridoxine, riboflavin, thiamine, inositol, and vitamin B12. In some embodiments, the vitamins include or consist essentially of biotin, DL alphatocopherol acetate, DL alpha-tocopherol, vitamin A, or combinations or salts thereof. In some embodiments, the medium further comprises proteins. In some embodiments, the proteins comprise albumin or bovine serum albumin, a fraction of BSA, catalase, insulin, transferrin, superoxide dismutase, or combinations thereof. In some embodiments, the medium further comprises one or more of the following: corticosterone, D-Galactose, ethanolamine, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, or triodo-I-thyronine, or combinations thereof. In some embodiments, the medium comprises one or more of the following: a B-27® supplement, xeno-free B-27® supplement, GS21™ supplement, or combinations thereof. In some embodiments, the medium comprises or further comprises amino acids, monosaccharides, inorganic ions. In some embodiments, the amino acids comprise arginine, cystine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine, or combinations thereof. In some embodiments, the inorganic ions comprise sodium, potassium, calcium, magnesium, nitrogen, or phosphorus, or combinations or salts thereof. In some embodiments, the medium further comprises one or more of the following: molybdenum, vanadium, iron, zinc, selenium, copper, or manganese, or combinations thereof. In certain embodiments, the medium comprises or consists essentially of one or more vitamins discussed herein and / or one or more proteins discussed herein, and / or one or more of the following: corticosterone, D-Galactose, ethanolamine, glutathione, L-camitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, or triodo-I-thyronine, a B-27® supplement, xeno-free B- 27® supplement, GS21™ supplement, an amino acid (such as arginine, cystine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine), monosaccharide, inorganic ion (such as sodium, potassium, calcium, magnesium, nitrogen, and / or phosphorus) or salts thereof, and / or molybdenum, vanadium, iron, zinc, selenium, copper, or manganese.

[0117] In further embodiments, the medium may comprise externally added ascorbic acid. The medium can also contain one or more externally added fatty acids orlipids, amino acids (such as non-essential amino acids), vitamin(s), growth factors, cytokines, antioxidant substances, 2-mercaptoethanol, pyruvic acid, buffering agents, and / or inorganic salts.

[0118] One or more of the medium components may be added at a concentration of at least, at most, or about 0.1, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 180, 200, 250 ng / L, ng / ml, pg / ml, mg / ml, or any range derivable therein.

[0119] The medium used may be supplemented with at least one externally added cytokine at a concentration from about 0.1 ng / mL to about 500 ng / mL, more particularly 1 ng / mL to 100 ng / mL, or at least, at most, or about 0.1, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 180, 200, 250 ng / L, ng / ml, pg / ml, mg / ml, or any range derivable therein. Suitable cytokines, include but are not limited to, FLT3 ligand (FLT3L), interleukin 7 (IL-7), stem cell factor (SCF), thrombopoietin (TPO), IL-2, IL-4, IL-6, IL-15, IL-21, TNF-alpha, TGF- beta, interferon-gamma, interferon-lambda, TSLP, thymopentin, pleotrophin, and / or midkine. Particularly, the culture medium may include at least one of FLT3L and IL-7. More particularly, the culture may include both FLT3L and IL-7.

[0120] Other culturing conditions can be appropriately defined. For example, the culturing temperature can be about 20 to 40°C, such as at least, at most, or about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40°C (or any range derivable therein), though the temperature may be above or below these values. The CO2 concentration can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% (or any range derivable therein), such as about 2% to 10%, for example, about 2 to 5%, or any range derivable therein. The oxygen tension can be at least or about 1, 5, 8, 10, 20%, or any range derivable therein.

[0121] In specific embodiments, the allogeneic HSC-engineered HLA-negative TARGET cells are specifically formulated. They may or may not be formulated as a cell suspension. In specific cases they are formulated in a single dose form. They may be formulated for systemic or local administration. In some cases, the cells areformulated for storage prior to use, and the cell formulation may comprise one or more cryopreservation agents, such as DMSO (for example, in 5% DMSO). The cell formulation may comprise albumin, including human albumin, with a specific formulation comprising 2.5% human albumin. The cells may be formulated specifically for intravenous administration; for example, they are formulated for intravenous administration over less than one hour. In particular embodiments the cells are in a formulated cell suspension that is stable at room temperature for 1, 2, 3, or 4 hours or more from time of thawing.

[0122] In some embodiments, the method further comprises priming the TARGET cells. In some embodiments, the TARGET cells are primed with antigen presenting cells. In some embodiments, the antigen presenting cells present tumor antigens.

[0123] In particular embodiments, the exogenous TCR of the TARGET cells may be of any defined antigen specificity. In some embodiments, it can be selected based on absent or reduced alloreactivity to the intended recipient (examples include certain virus-specific TCRs, xeno-specific TCRs, or cancer-testis antigen-specific TCRs). In the example where the exogenous TCR is non-alloreactive, during T cell differentiation the exogenous TCR suppresses rearrangement and / or expression of endogenous TCR loci through a developmental process called allelic exclusion, resulting in T cells that express only the non-alloreactive exogenous TCR and are thus non-alloreactive. In some embodiments, the choice of exogenous TCR may not necessarily be defined based on lack of alloreactivity. In some embodiments, the endogenous TCR genes have been modified by genome editing so that they do not express a protein. Methods of gene editing such as methods using the CRISPR / Cas9 system are known in the art and described herein.

[0124] In some embodiments, the isolated TARGET cell or population thereof comprise a one or more chimeric antigen receptors (CARs). Examples of tumor cell antigens to which a CAR may be directed include at least 5T4, 8H9, avPe integrin, BCMA, B7-H3, B7-H6, CAIX, CA9, CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFRfamily including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, ERBB3, ERBB4, ErbB3 / 4, EPCAM, EphA2, EpCAM, folate receptor-a, FAP, FBP, fetal AchR, FRa, GD2, G250 / CAIX, GD3, Glypican-3 (GPC3), Her2, IL-13Ra2, Lambda, Lewis-Y, Kappa, KDR, MAGE, MCSP, Mesothelin, Mucl, Mucl6, NCAM, NKG2D Ligands, NY- ESO-1, PRAME, PSC1, PSCA, PSMA, R0R1, SP17, Survivin, TAG72, TEMs, carcinoembryonic antigen, HMW-MAA, AFP, CA-125, ETA, Tyrosinase, MAGE, laminin receptor, HPV E6, E7, BING-4, Calcium-activated chloride channel 2, Cyclin- Bl, 9D7, EphA3, Telomerase, SAP-1, BAGE family, CAGE family, GAGE family, MAGE family, SAGE family, XAGE family, NY-ES0-1 / LAGE-1, PAME, SSX-2, Melan-A / MART-1, GP100 / pmell7, TRP-1 / -2, P. polypeptide, MC1R, Prostatespecific antigen, P-catenin, BRCA1 / 2, CML66, Fibronectin, MART-2, TGF-PRII, or VEGF receptors (e.g., VEGFR2), for example. The CAR may be a first, second, third, or more generation CAR. The CAR may be bispecific for any two nonidentical antigens, or it may be specific for more than two nonidentical antigens.III. Additional Modifications and Polypeptide Embodiments

[0125] Additionally, the polypeptides of the disclosure may be chemically modified. Glycosylation of the polypeptides can be altered, for example, by modifying one or more sites of glycosylation within the polypeptide sequence to increase the affinity of the polypeptide for antigen (U.S. Pat. Nos. 5,714,350 and 6,350,861).

[0126] Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein, and may be designed to modulate one or more properties of the polypeptide, with or without the loss of other functions or properties. Substitutions may be conservative, that is, one amino acid is replaced with one of similar shape and charge. Conservative substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine,leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine. Alternatively, substitutions may be non-conservative such that a function or activity of the polypeptide is affected. Non-conservative changes typically involve substituting a residue with one that is chemically dissimilar, such as a polar or charged amino acid for a nonpolar or uncharged amino acid, and vice versa.

[0127] Proteins may be recombinant, or synthesized in vitro. Alternatively, a nonrecombinant or recombinant protein may be isolated from bacteria. It is also contemplated that bacteria containing such a variant may be implemented in compositions and methods. Consequently, a protein need not be isolated.

[0128] The term “functionally equivalent codon” is used herein to refer to codons that encode the same amino acid, such as the six codons for arginine or serine, and also refers to codons that encode biologically equivalent amino acids.

[0129] It also will be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids, or 5' or 3' sequences, respectively, and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where protein expression is concerned. The addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences flanking either of the 5' or 3' portions of the coding region.

[0130] The following is a discussion based upon changing of the amino acids of a protein to create an equivalent, or even an improved, second-generation molecule. For example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity. Structures such as, for example, an enzymatic catalytic domain or interaction components may have amino acid substituted to maintain such function. Since it is the interactive capacity and nature of a protein that defines that protein’s biological functional activity, certain amino acid substitutions can be made in a protein sequence, and in its underlying DNA codingsequence, and nevertheless produce a protein with like properties. It is thus contemplated by the inventors that various changes may be made in the DNA sequences of genes without appreciable loss of their biological utility or activity.

[0131] In other embodiments, alteration of the function of a polypeptide is intended by introducing one or more substitutions. For example, certain amino acids may be substituted for other amino acids in a protein structure with the intent to modify the interactive binding capacity of interaction components. Structures such as, for example, protein interaction domains, nucleic acid interaction domains, and catalytic sites may have amino acids substituted to alter such function. Since it is the interactive capacity and nature of a protein that defines that protein’s biological functional activity, certain amino acid substitutions can be made in a protein sequence, and in its underlying DNA coding sequence, and nevertheless produce a protein with different properties. It is thus contemplated by the inventors that various changes may be made in the DNA sequences of genes with appreciable alteration of their biological utility or activity.

[0132] In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.

[0133] It also is understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Patent 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still produce a biologically equivalent and immunologically equivalent protein.

[0134] As outlined above, amino acid substitutions generally are based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take into consideration the various foregoing characteristics are well known and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

[0135] In specific embodiments, all or part of proteins described herein can also be synthesized in solution or on a solid support in accordance with conventional techniques. Various automatic synthesizers are commercially available and can be used in accordance with known protocols. See, for example, Stewart and Young, (1984); Tam et al., (1983); Merrifield, (1986); and Barany and Merrifield (1979), each incorporated herein by reference. Alternatively, recombinant DNA technology may be employed wherein a nucleotide sequence that encodes a peptide or polypeptide is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultivated under conditions suitable for expression.

[0136] One embodiment includes the use of gene transfer to cells, including microorganisms, for the production and / or presentation of proteins. The gene for the protein of interest may be transferred into appropriate host cells followed by culture of cells under the appropriate conditions. A nucleic acid encoding virtually any polypeptide may be employed. The generation of recombinant expression vectors, and the elements included therein, are discussed herein. Alternatively, the protein to be produced may be an endogenous protein normally synthesized by the cell used for protein production.IV. Methods of Producing the TARGET Cells

[0137] The example provided herein are merely illustrative TARGET cells may be produced by a variety of suitable method(s). The method(s) may utilize one or more successive steps for one or more modifications to cells and / or utilize one or more simultaneous steps for one or more modifications to cells. In specific embodiments, a starting source of cells from a cell line are modified to become functional as TARGETcells followed by one or more steps to add one or more additional characteristics to the cells, such as the ability to be imaged, and / or the ability to be selectively killed, and / or the ability to be able to be used allogeneically. In specific embodiments, at least part of the process for generating TARGET cells occurs in a specific in vitro culture system. An example of a specific in vitro culture system is one that allows differentiation of certain cells at high efficiency and high yield.

[0138] In specific cases, TARGET cells may be generated by the following: 1) genetic modification of cells from a pluripotent stem cell line so that they express TARGET TCRs (for example, via lentiviral vectors) and optionally to eliminate expression of HLA-I / II molecules (for example, via CRISPR / Cas9-based gene editing); 2) in vitro differentiation into TARGET cells via culture, 3) in vitro TARGET cell purification and expansion, and 4) formulation and cryopreservation and / or use.

[0139] Specific aspects of the disclosure relate to a novel three dimensional cell culture system to produce TARGET cells from less differentiated cell lines such as embryonic stem cell lines, pluripotent stem cell lines, hematopoietic stem or progenitor cell lines, induced pluripotent stem (iPS) cell lines, or stem or progenitor cell lines.

[0140] In particular embodiments, the system involves using serum-free medium. In certain aspects, the system uses a serum-free medium that is suitable for cell development for culturing of a three-dimensional cell aggregate. Such a system produces sufficient amounts of TARGET cells. In embodiments of the disclosure, the 3D cell aggregate is cultured in a serum-free medium comprising insulin for a time period sufficient for the in vitro differentiation of stem or progenitor cells to TARGET cells or precursors to TARGET cells.

[0141] Embodiments of a cell culture composition comprise an culture that uses highly-standardized, serum-free components and a stromal cell line to facilitate robust and highly reproducible T cell differentiation from human HSCs. In certain embodiments, cell differentiation mimics endogenous thymopoiesis and, in contrast to monolayer co-cultures, supported efficient positive selection of functional TARGET cells. Certain aspects of the 3D culture compositions use serum-free conditions, avoidthe use of human thymic tissue or proprietary scaffold materials, and facilitate positive selection and robust generation of fully functional, mature human TARGET cells from source cells.

[0142] In methods of preparing a population of clonal TARGET cells, selecting TARGET cells lacking surface expression of HLA-I and HLA-II molecules may comprise contacting the TARGET cells with magnetic beads that bind to and positively select for TARGET cells and negatively select for HLA-I / II-negative cells. In specific embodiments, the magnetic beads are coated with monoclonal antibodies recognizing human TARGET TCRs, HLA-I molecules, or HLA-II molecules. In particular embodiments, the monoclonal antibodies are Clone 6B11 (recognizing human TCR Va24-Jal8 thus recognizing human TARGET invariant TCR alpha chain), Clone 2M2 (recognizing human B2M thus recognizing cell surface-displayed human HLA-I molecules), Clone W6 / 32 (recognizing HLA-A,B,C thus recognizing human HLA-I molecules), and Clone Tu39 (recognizing human HLA-DR, DP, DQ thus recognizing human HLA-II molecules).

[0143] Cells produced by the preparation methods may be frozen. The produced cells may be in a solution comprising dextrose, one or more electrolytes, albumin, dextran, and DMSO. The solution may be sterile, nonpyogenic, and isotonic.

[0144] In particular embodiments, the system utilizes feeder cells that may comprise CD34- cells.

[0145] Preparation methods may further comprise activating and expanding the selected TARGET cells; for example, the selected TARGET cells have been activated with alpha-galactosylceramide (a-GC). The feeder cells may have been pulsed with a- GC.

[0146] Preparation methods of the disclosure may produce a population of clonal TARGET cells comprising at least about 102- 106clonal TARGET cells. The method may produce a cell population comprising at least about 106-1012total cells. The produced cell population may be frozen and then thawed. In some cases of the preparation method, the method further comprises introducing one or more additionalnucleic acids into the frozen and thawed cell population, such as the one or more additional nucleic acids encoding one or more therapeutic gene products, for example.

[0147] For example, aggregation is achieved by centrifugation of the mixed cell suspension (“compaction aggregation”) followed by aspiration of the cell-free supernatant. In particular embodiments, the cell pellet may then be aspirated as a slurry in 5-10 ul of a differentiation medium and transferred as a droplet onto 0.4 um nylon transwell culture inserts, which are floated in a well of differentiation medium, allowing the bottom of the insert to be in contact with medium and the top with air.

[0148] Variations in the protocol permit the use of alternative components with varying impact on efficacy, specifically:

[0149] Base medium RPMI may be substituted for several commercially available alternatives (e.g. IMDM)

[0150] The stromal cell line used is MS-5, a previously described murine bone marrow cell line (Itoh et al, 1989), however MS-5 may be substituted for similar murine stromal cell lines (e.g. OP9, SI 7), human stromal cell lines (e.g. HS-5, HS-27a), primary human stromal cells, or human pluripotent stem cell-derived stromal cells.

[0151] The stromal cell line can be transduced with a lentivirus encoding human DLL1 cDNA; however the method of gene delivery, as well as the Notch ligand gene, may be varied. Alternative Notch ligand genes include DLL4, JAGl, JAG2, and others. Notch ligands also include those described in U.S. Patent Nos. 7,795,404 and 8,377,886, which are herein incorporated by reference. Notch ligands further include Delta 1, 3, and 4 and Jagged 1, 2.

[0152] Cytokine conditions can be varied: e.g. levels of FLT3L and IL-7 may be changed to alter T cell differentiation kinetics; other hematopoietic cytokines such as Stem Cell Factor (SCF / KIT ligand), thrombopoietin (TPO), IL-2, IL- 15 may be added.

[0153] Genetic modification may also be introduced to certain components to generate antigen-specific T cells, and to model positive and negative selection. Examples of these modifications include: transduction of HSCs with a lentiviral vector encoding an antigen-specific T cell receptor (TCR) or chimeric antigen receptor (CAR)for the generation of antigen-specific, allelically excluded naive T cells; transduction of HSCs with gene / s to direct lineage commitment to specialized lymphoid cells. For example, transduction of HSCs with an invariant natural killer T cell (TARGET) associated TCR to generate functional TARGET cells; transduction of the cells with human MHC genes (e.g. human CD Id gene) to enhance positive selection and maturation of both TCR engineered or non- engineered T cells; and / or transduction of the cell line with an antigen plus costimulatory molecules or cytokines to enhance the positive selection of CAR T cells.

[0154] In producing the engineered TARGET cells, CD34+ cells may be modified by introducing certain exogenous gene(s) and by knocking out certain endogenous gene(s). The methods may further comprise culturing selected CD34+ cells in media prior to introducing one or more nucleic acids into the cells. The culturing may comprise incubating the selected CD34+ cells with medium comprising one or more growth factors, in some cases, and the one or more growth factors may comprise c-kit ligand, flt-3 ligand, and / or human thrombopoietin (TPO), for example. The growth factors may or may not be at a certain concentration, such as between about 5 ng / ml to about 500 ng / ml / .

[0155] In particular methods the nucleic acid(s) to be introduced into the cells are one or more nucleic acids that comprise a nucleic acid sequence encoding an a-TCR and a P-TCR. The methods may further comprise introducing into the selected CD34+ cells a nucleic acid encoding a suicide gene. In specific aspects, one nucleic acid encodes both the a-TCR and the P-TCR, or one nucleic acid encodes the a-TCR, the P- TCR, and the suicide gene. The suicide gene may be enzyme-based, such as thymidine kinase (TK) including a viral TK gene such as one from herpes simplex virus TK gene. The suicide gene may be activated by a substrate, such as ganciclovir, penciclovir, or a derivative thereof. The cells may be engineered to comprise an exogenous nucleic acid encoding a polypeptide that has a substrate that may be labeled for imaging. In some cases, a suicide gene product is a polypeptide that has a substrate that may be labeled for imaging, such as sr39TK.

[0156] The cells may be engineered to lack surface expression of HLA-I and / or HLA-II molecules, for example by disrupting the functional expression of genes encoding beta-2-microglobulin (B2M), major histocompatibility complex class II transactivator (CIITA), and / or HLA-I and HLA-II molecules. In the production methods, eliminating surface expression of one or more HLA-I / II molecules in the isolated human CD34+ cells may comprise introducing CRISPR and one or more guide RNAs (gRNAs) corresponding to B2M, CIITA, or individual HLA-I or HLA-II molecules into the cells. CRISPR or the one or more gRNAs are transfected into the cell by electroporation or lipid-mediated transfection in some cases. In specific embodiments, the nucleic acid encoding the TCR receptor is introduced into the cell using a recombinant vector such as a viral vector including at least a lentivirus, a retrovirus, an adeno-associated virus (AAV), a herpesvirus, or adenovirus, for example.

[0157] In manufacturing the engineered TARGET cells, the cells may be present in a particular serum-free medium, including one that comprises externally added ascorbic acid. In specific aspects, the serum-free medium further comprises externally added FLT3 ligand (FLT3L), interleukin 7 (IL-7), stem cell factor (SCF), thrombopoietin (TPO), stem cell factor (SCF), thrombopoietin (TPO), IL-2, IL-4, IL-6, IL-15, IL-21, TNF-alpha, TGF-beta, interferon-gamma, interferon-lambda, TSLP, thymopentin, pleotrophin, midkine, or combinations thereof. The serum-free medium may further comprise vitamins, including biotin, DL alpha tocopherol acetate, DL alpha-tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, folic acid nicotinamide, pyridoxine, riboflavin, thiamine, inositol, vitamin B12, or combinations thereof or salts thereof. The serum-free medium may further comprise one or more externally added (or not) proteins, such as albumin or bovine serum albumin, a fraction of BSA, catalase, insulin, transferrin, superoxide dismutase, or combinations thereof. The serum-free medium may further comprise corticosterone, D-Galactose, ethanolamine, glutathione, L-camitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, or triodo-I-thyronine, or combinations thereof. The serum- free medium may comprise a B-27® supplement, xeno-free B-27® supplement, GS21™supplement, or combinations thereof. Amino acids (including arginine, cysteine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine, or combinations thereof), monosaccharides, and / or inorganic ions (including sodium, potassium, calcium, magnesium, nitrogen, or phosphorus, or combinations or salts thereof, for example) may be present in the serum- free medium. The serum-free medium may further comprise molybdenum, vanadium, iron, zinc, selenium, copper, or manganese, or combinations thereof.

[0158] Cell culture conditions may be provided for the culture of 3D cell aggregates described herein and for the production of T cells and / or positive / negative selection thereof. In certain aspects, starting cells of a selected population may comprise at least or about 104, 105, 106, 107, 108, 109, IO10, 1011, 1012, 1013cells or any range derivable therein. The starting cell population may have a seeding density of at least or about 10, 101, 102, 103, 104, 105, 106, 107, 108cells / ml, or any range derivable therein.

[0159] A culture vessel used for culturing the 3D cell aggregates or progeny cells thereof can include, but is particularly not limited to: flask, flask for tissue culture, dish, petri dish, dish for tissue culture, multi dish, micro plate, micro-well plate, multi plate, multi -well plate, micro slide, chamber slide, tube, tray, Cell STACK® Chambers, culture bag, and roller bottle, as long as it is capable of culturing the stem cells therein. The stem cells may be cultured in a volume of at least or about 0.2, 0.5, 1, 2, 5, 10, 20, 30, 40, 50 ml, 100 ml, 150 ml, 200 ml, 250 ml, 300 ml, 350 ml, 400 ml, 450 ml, 500 ml, 550 ml, 600 ml, 800 ml, 1000 ml, 1500 ml, or any range derivable therein, depending on the needs of the culture. In a certain embodiment, the culture vessel may be a bioreactor, which may refer to any device or system that supports a biologically active environment. The bioreactor may have a volume of at least or about 2, 4, 5, 6, 8, 10, 15, 20, 25, 50, 75, 100, 150, 200, 500 liters, 1, 2, 4, 6, 8, 10, 15 cubic meters, or any range derivable therein.

[0160] The culture vessel can be cellular adhesive or non-adhesive and selected depending on the purpose. The cellular adhesive culture vessel can be coated with any of substrates for cell adhesion such as extracellular matrix (ECM) to improve theadhesiveness of the vessel surface to the cells. The substrate for cell adhesion can be any material intended to attach stem cells or feeder cells (if used). The substrate for cell adhesion includes collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, and fibronectin and mixtures thereof for example Matrigel™, and lysed cell membrane preparations.

[0161] Various defined matrix components may be used in the culturing methods or compositions. For example, recombinant collagen IV, fibronectin, laminin, and vitronectin in combination may be used to coat a culturing surface as a means of providing a solid support for pluripotent cell growth, as described in Ludwig et al. (2006a; 2006b), which are incorporated by reference in its entirety.

[0162] A matrix composition may be immobilized on a surface to provide support for cells. The matrix composition may include one or more extracellular matrix (ECM) proteins and an aqueous solvent. The term “extracellular matrix” is recognized in the art. Its components include one or more of the following proteins: fibronectin, laminin, vitronectin, tenascin, entactin, thrombospondin, elastin, gelatin, collagen, fibrillin, merosin, anchorin, chondronectin, link protein, bone sialoprotein, osteocalcin, osteopontin, epinectin, hyaluronectin, undulin, epiligrin, and kalinin. Other extracellular matrix proteins are described in Kleinman et al.. (1993), herein incorporated by reference. It is intended that the term “extracellular matrix” encompass a presently unknown extracellular matrix that may be discovered in the future, since its characterization as an extracellular matrix will be readily determinable by persons skilled in the art.

[0163] In some aspects, the total protein concentration in the matrix composition may be about 1 ng / mL to about 1 mg / mL. In some embodiments, the total protein concentration in the matrix composition is about 1 pg / mL to about 300 pg / mL. In more preferred embodiments, the total protein concentration in the matrix composition is about 5 pg / mL to about 200 pg / mL.

[0164] The extracellular matrix (ECM) proteins may be of natural origin and purified from human or animal tissues. Alternatively, the ECM proteins may begenetically engineered recombinant proteins or synthetic in nature. The ECM proteins may be a whole protein or in the form of peptide fragments, native or engineered. Examples of ECM protein that may be useful in the matrix for cell culture include laminin, collagen I, collagen IV, fibronectin and vitronectin. In some embodiments, the matrix composition includes synthetically generated peptide fragments of fibronectin or recombinant fibronectin.

[0165] In still further embodiments, the matrix composition includes a mixture of at least fibronectin and vitronectin. In some other embodiments, the matrix composition preferably includes laminin.

[0166] The matrix composition preferably includes a single type of extracellular matrix protein. In some embodiments, the matrix composition includes fibronectin, particularly for use with culturing progenitor cells. For example, a suitable matrix composition may be prepared by diluting human fibronectin, such as human fibronectin sold by Becton, Dickinson & Co. of Franklin Lakes, N.J. (BD) (Cat#354008), in Dulbecco's phosphate buffered saline (DPBS) to a protein concentration of 5 pg / mL to about 200 pg / mL. In a particular example, the matrix composition includes a fibronectin fragment, such as RetroNectin®. RetroNectin® is a ~63 kDa protein of (574 amino acids) that contains a central cell-binding domain (type III repeat, 8,9,10), a high affinity heparin-binding domain II (type III repeat, 12,13,14), and CS1 site within the alternatively spliced IIICS region of human fibronectin.

[0167] In some other embodiments, the matrix composition may include laminin. For example, a suitable matrix composition may be prepared by diluting laminin (Sigma-Aldrich (St. Louis, Mo.); Cat#L6274 and L2020) in Dulbecco's phosphate buffered saline (DPBS) to a protein concentration of 5 pg / ml to about 200 pg / ml.

[0168] In some embodiments, the matrix composition is xeno-free, in that the matrix is or its component proteins are only of human origin. This may be desired for certain research applications. For example, in the xeno-free matrix to culture human cells, matrix components of human origin may be used, wherein any non-human animal components may be excluded. In certain aspects, Matrigel™ may be excluded as asubstrate from the culturing composition. Matrigel™ is a gelatinous protein mixture secreted by mouse tumor cells and is commercially available from BD Biosciences (New Jersey, USA). This mixture resembles the complex extracellular environment found in many tissues and is used frequently by cell biologists as a substrate for cell culture, but it may introduce undesired xeno antigens or contaminants.

[0169] In certain embodiments, cells containing an exogenous nucleic acid may be identified in vitro or in vivo by including a marker in the expression vector or the exogenous nucleic acid. Such markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression vector. Generally, a selection marker may be one that confers a property that allows for selection. A positive selection marker may be one in which the presence of the marker allows for its selection, while a negative selection marker is one in which its presence prevents its selection. An example of a positive selection marker is a drug resistance marker.

[0170] Usually the inclusion of a drug selection marker aids in the cloning and identification of transformants, for example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are useful selection markers. In addition to markers conferring a phenotype that allows for the discrimination of transformants based on the implementation of conditions, other types of markers including screenable markers such as GFP, whose basis is colorimetric analysis, are also contemplated. Alternatively, screenable enzymes as negative selection markers such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may be utilized. One of skill in the art would also know how to employ immunologic markers, possibly in conjunction with FACS analysis. The marker used is not believed to be important, so long as it is capable of being expressed simultaneously with the nucleic acid encoding a gene product. Further examples of selection and screenable markers are well known to one of skill in the art.

[0171] Selectable markers may include a type of reporter gene used in laboratory microbiology, molecular biology, and genetic engineering to indicate the success of a transfection or other procedure meant to introduce foreign DNA into a cell. Selectablemarkers are often antibiotic resistance genes; cells that have been subjected to a procedure to introduce foreign DNA are grown on a medium containing an antibiotic, and those cells that can grow have successfully taken up and expressed the introduced genetic material. Examples of selectable markers include: the Abicr gene or Neo gene from Tn5, which confers antibiotic resistance to geneticin.

[0172] A screenable marker may comprise a reporter gene, which allows the researcher to distinguish between wanted and unwanted cells. Certain embodiments of the present invention utilize reporter genes to indicate specific cell lineages. For example, the reporter gene can be located within expression elements and under the control of the ventricular- or atrial -selective regulatory elements normally associated with the coding region of a ventricular- or atrial-selective gene for simultaneous expression. A reporter allows the cells of a specific lineage to be isolated without placing them under drug or other selective pressures or otherwise risking cell viability.

[0173] Examples of such reporters include genes encoding cell surface proteins (e.g., CD4, HA epitope), fluorescent proteins, antigenic determinants and enzymes (e.g., P-galactosidase). The vector containing cells may be isolated, e.g., by FACS using fluorescently-tagged antibodies to the cell surface protein or substrates that can be converted to fluorescent products by a vector encoded enzyme.

[0174] In specific embodiments, the reporter gene is a fluorescent protein. A broad range of fluorescent protein genetic variants have been developed that feature fluorescence emission spectral profiles spanning almost the entire visible light spectrum. Mutagenesis efforts in the original Aequorea victoria jellyfish green fluorescent protein have resulted in new fluorescent probes that range in color from blue to yellow, and are some of the most widely used in vivo reporter molecules in biological research. Longer wavelength fluorescent proteins, emitting in the orange and red spectral regions, have been developed from the marine anemone, Discosoma striata, and reef corals belonging to the class Anthozoa. Still other species have been mined to produce similar proteins having cyan, green, yellow, orange, and deep red fluorescenceemission. Developmental research efforts are ongoing to improve the brightness and stability of fluorescent proteins, thus improving their overall usefulness.

[0175] The cells in certain embodiments can be made to contain one or more genetic alterations by genetic engineering of the cells either before or after differentiation (US 2002 / 0168766). A cell is said to be "genetically altered", “genetically modified” or “transgenic” when an exogenous nucleic acid or polynucleotide has been transferred into the cell by any suitable means of artificial manipulation, or where the cell is a progeny of the originally altered cell that has inherited the polynucleotide. For example, the cells can be processed to increase their replication potential by genetically altering the cells to express telomerase reverse transcriptase, either before or after they progress to restricted developmental lineage cells or terminally differentiated cells (U.S. Patent Application Publication 2003 / 0022367).

[0176] In certain embodiments, cells containing an exogenous nucleic acid construct may be identified in vitro or in vivo by including a marker in the expression vector, such as a selectable or screenable marker. Such markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression vector, or help enrich or identify differentiated cardiac cells by using a tissue-specific promoter. For example, in the aspects of cardiomyocyte differentiation, cardiac-specific promoters may be used, such as promoters of cardiac troponin I (cTnl), cardiac troponin T (cTnT), sarcomeric myosin heavy chain (MHC), GATA-4, Nkx2.5, N-cadherin, al- adrenoceptor, ANF, the MEF-2 family of transcription factors, creatine kinase MB (CK-MB), myoglobin, or atrial natriuretic factor (ANF). In aspects of neuron differentiation, neuron-specific promoters may be used, including but not limited to, TuJ-1, Map-2, Dex or Synapsin. In aspects of hepatocyte differentiation, definitive endoderm- and / or hepatocyte-specific promoters may be used, including but not limited to, ATT, Cyp3a4, ASGPR, FoxA2, HNF4a or AFP.

[0177] Generally, a selectable marker is one that confers a property that allows for selection. A positive selectable marker is one in which the presence of the marker allows for its selection, while a negative selectable marker is one in which its presenceprevents its selection. An example of a positive selectable marker is a drug resistance marker.

[0178] Usually the inclusion of a drug selection marker aids in the cloning and identification of transformants, for example, genes that confer resistance to blasticidin, neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are useful selectable markers. In addition to markers conferring a phenotype that allows for the discrimination of transformants based on the implementation of conditions, other types of markers including screenable markers such as GFP, whose basis is colorimetric analysis, are also contemplated. Alternatively, screenable enzymes such as chloramphenicol acetyltransferase (CAT) may be utilized. One of skill in the art would also know how to employ immunologic markers, possibly in conjunction with FACS analysis. The marker used is not believed to be important, so long as it is capable of being expressed simultaneously with the nucleic acid encoding a gene product. Further examples of selectable and screenable markers are well known to one of skill in the art.

[0179] In embodiments wherein cells are genetically modified, such as to add or reduce one or more features, the genetic modification may occur by any suitable method. For example, any genetic modification compositions or methods may be used to introduce exogenous nucleic acids into cells or to edit the genomic DNA, such as gene editing, homologous recombination or non-homologous recombination, RNA- mediated genetic delivery or any conventional nucleic acid delivery methods. Nonlimiting examples of the genetic modification methods may include gene editing methods such as by CRISPR / CAS9, zinc finger nuclease, or TALEN technology.

[0180] Genetic modification may also include the introduction of a selectable or screenable marker that aid selection or screen or imaging in vitro or in vivo. Particularly, in vivo imaging agents or suicide genes may be expressed exogenously or added to starting cells or progeny cells. In further aspects, the methods may involve image-guided adoptive cell therapy.V. Methods of Using the Cells

[0181] The TARGET cells of the disclosure may or may not be utilized directly after production. In some cases they are stored for later purpose. In any event, they may be utilized in therapeutic or preventative applications for a mammalian subject (human, dog, cat, horse, etc.) such as a patient. The patient may be in need of cell therapy for a medical condition of any kind, including allogeneic cell therapy.

[0182] Methods of treating a patient with a therapeutically effective amount of TARGET cells of the disclosure comprise administering the cells or clonal populations thereof to the patient. The cells or cell populations may be allogeneic with respect to the patient. The patient does not exhibit signs of depletion of the cells or cell population, in particular embodiments. The patient may or may not have cancer and / or a disease or condition involving inflammation. In specific embodiments wherein the patient has cancer, tumor cells of the cancer patient are killed after administering the cells or cell population to the patient. In specific cases wherein the patient has inflammation, the inflammation is reduced following administering the cells or cell population to the patient. In specific embodiments of the methods of treatment, the method further comprises administering to the patient a compound that initiates the suicide gene product.

[0183] For patients with cancer, once infused into patients it is expected that this cell product can employ multiple mechanisms to target and eradicate tumor cells. The infused cells can directly recognize and kill CDld+tumor cells through cytotoxicity. They can secrete cytokines such as IFN-y to activate cells to kill HLA-negative tumor cells, and also activate DCs which then stimulate cytotoxic T cells to kill HLA-positive tumor cells. Accordingly, we plan a series of in vitro and in vivo studies to demonstrate the pharmacological efficacy of this cell product for cancer therapy.

[0184] Because the TARGET cells can target a large range of cancers without tumor antigen- and MHC-restrictions, an off-the-shelf TARGET cellular product is useful as a general cancer immunotherapy for treating any type of cancer and a large population of cancer patients. In specific cases, the present therapy is useful for patients withcancers that have been clinically indicated to be subject to TARGET cell regulation, including multiple types of solid tumors (melanoma, colon, lung, breast, and head and neck cancers) and blood cancers (leukemia, multiple myeloma, and myelodysplastic syndromes), for example.

[0185] In some embodiments of any of the above-disclosed methods, the subject has or is at risk of having an autoimmune disease, graft versus host disease (GVHD), or graft rejection. The subject may be one diagnosed with such disease or one that has been determined to have a pre-disposition to such disease based on genetic or family history analysis. The subject may also be one that is preparing to or has undergone a transplant. In some embodiments, the method is for treating an autoimmune disease, GVHD, or graft rejection.

[0186] Individuals treated with the present cell therapy may or may not have been treated for the particular medical condition prior to receiving the TARGET cell therapy. In cases wherein the individual has cancer, the cancer may be primary, metastatic, resistant to therapy, and so forth, patients who have exhausted conventional treatment options.

[0187] In particular embodiments, the cells are provided to the patient at 107-109cells per dose. In specific embodiments, the dosing regimen is a single-dose of allogeneic TARGET cells following lymphodeleting conditioning. The cells may be administered intravenously following lymphodepleting conditioning with fludarabine and cyclophosphamide, for example.

[0188] In cases wherein antitumor efficacy in vivo is characterized for subsequent in vivo therapeutic cases, in vivo pharmacological responses may be measured by treating tumor-bearing NSG mice with escalating doses (IxlO6, 5xl06, 10xl06) of TARGET cells (n = 8 per group); treatment with PBS may be included as a control. Two tumor models may be utilized, as examples. A375.CDld (IxlO6s.c.) may be used as a solid tumor model and MM. IS. Luc (5xl06i.v.) may be used as a hematological malignancy model. Tumor growth can be monitored by either measuring size (A375.CDld) or bioluminescence imaging (MM. IS. Luc). Antitumor immuneresponses can be measured by PET imaging, periodic bleeding, and end-point tumor harvest followed by flow cytometry and qPCR. Inhibition of tumor growth in response to TARGET treatment can indicate the therapeutic efficacy of TARGET cell therapy. Correlation of tumor inhibition with TARGET doses can confirm the therapeutic role of the TARGET cells and indicate an effective therapeutic window for human therapy. Detection of TARGET cell responses to tumors can demonstrate the pharmacological antitumor activities of these cells in vivo.

[0189] Methods may be employed with respect to individuals who have tested positive for a medical condition, who have one or more symptoms of a medical condition, or who are deemed to be at risk for developing such a condition. In some embodiments, the compositions and methods described herein are used to treat an inflammatory or autoimmune component of a disorder listed herein and / or known in the art.

[0190] Certain aspects of the disclosure relate to the treatment of cancer and / or use of cancer antigens. The cancer to be treated or antigen may be an antigen associated with any cancer known in the art or, for example, epithelial cancer, (e.g., breast, gastrointestinal, lung), prostate cancer, bladder cancer, lung (e.g., small cell lung) cancer, colon cancer, ovarian cancer, brain cancer, gastric cancer, renal cell carcinoma, pancreatic cancer, liver cancer, esophageal cancer, head and neck cancer, or a colorectal cancer. In some embodiments, the cancer to be treated or antigen is from one of the following cancers: adenocortical carcinoma, agnogenic myeloid metaplasia, AIDS- related cancers (e.g., AIDS-related lymphoma), anal cancer, appendix cancer, astrocytoma (e.g., cerebellar and cerebral), basal cell carcinoma, bile duct cancer (e.g., extrahepatic), bladder cancer, bone cancer, (osteosarcoma and malignant fibrous histiocytoma), brain tumor (e.g., glioma, brain stem glioma, cerebellar or cerebral astrocytoma (e.g., pilocytic astrocytoma, diffuse astrocytoma, anaplastic (malignant) astrocytoma), malignant glioma, ependymoma, oligodenglioma, meningioma, meningiosarcoma, craniopharyngioma, haemangioblastomas, medulloblastoma, supratentorial primitive neuroectodermal tumors, visual pathway and hypothalamicglioma, and glioblastoma), breast cancer, bronchial adenomas / carcinoids, carcinoid tumor (e.g., gastrointestinal carcinoid tumor), carcinoma of unknown primary, central nervous system lymphoma, cervical cancer, colon cancer, colorectal cancer, chronic myeloproliferative disorders, endometrial cancer (e.g., uterine cancer), ependymoma, esophageal cancer, Ewing's family of tumors, eye cancer (e.g., intraocular melanoma and retinoblastoma), gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor, (e.g., extracranial, extragonadal, ovarian), gestational trophoblastic tumor, head and neck cancer, hepatocellular (liver) cancer (e.g., hepatic carcinoma and hepatoma), hypopharyngeal cancer, islet cell carcinoma (endocrine pancreas), laryngeal cancer, laryngeal cancer, leukemia, lip and oral cavity cancer, oral cancer, liver cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), lymphoid neoplasm (e.g., lymphoma), medulloblastoma, ovarian cancer, mesothelioma, metastatic squamous neck cancer, mouth cancer, multiple endocrine neoplasia syndrome, myelodysplastic syndromes, myelodysplastic / myeloproliferative diseases, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, neuroendocrine cancer, oropharyngeal cancer, ovarian cancer (e.g., ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor), pancreatic cancer, parathyroid cancer, penile cancer, cancer of the peritoneal, pharyngeal cancer, pheochromocytoma, pineoblastoma and supratentorial primitive neuroectodermal tumors, pituitary tumor, pleuropulmonary blastoma, lymphoma, primary central nervous system lymphoma (microglioma), pulmonary lymphangiomyomatosis, rectal cancer, renal cancer, renal pelvis and ureter cancer (transitional cell cancer), rhabdomyosarcoma, salivary gland cancer, skin cancer (e.g., non-melanoma (e.g., squamous cell carcinoma), melanoma, and Merkel cell carcinoma), small intestine cancer, squamous cell cancer, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, tuberous sclerosis, urethral cancer, vaginal cancer, vulvar cancer, Wilms' tumor, and post-transplantlymphoproliferative disorder (PTLD), abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), or Meigs' syndrome.

[0191] Certain aspects of the disclosure relate to the treatment of an autoimmune condition and / or use of an autoimmune-associated antigen. The autoimmune disease to be treated or antigen may be an antigen associated with any autoimmune condition known in the art or, for example, diabetes, graft rejection, GVHC, arthritis (rheumatoid arthritis such as acute arthritis, chronic rheumatoid arthritis, gout or gouty arthritis, acute gouty arthritis, acute immunological arthritis, chronic inflammatory arthritis, degenerative arthritis, type II collagen-induced arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, Still's disease, vertebral arthritis, and juvenile-onset rheumatoid arthritis, osteoarthritis, arthritis chronica progrediente, arthritis deformans, polyarthritis chronica primaria, reactive arthritis, and ankylosing spondylitis), inflammatory hyperproliferative skin diseases, psoriasis such as plaque psoriasis, gutatte psoriasis, pustular psoriasis, and psoriasis of the nails, atopy including atopic diseases such as hay fever and Job's syndrome, dermatitis including contact dermatitis, chronic contact dermatitis, exfoliative dermatitis, allergic dermatitis, allergic contact dermatitis, dermatitis herpetiformis, nummular dermatitis, seborrheic dermatitis, non-specific dermatitis, primary irritant contact dermatitis, and atopic dermatitis, x-linked hyper IgM syndrome, allergic intraocular inflammatory diseases, urticaria such as chronic allergic urticaria and chronic idiopathic urticaria, including chronic autoimmune urticaria, myositis, polymyositis / dermatomyositis, juvenile dermatomyositis, toxic epidermal necrolysis, scleroderma (including systemic scleroderma), sclerosis such as systemic sclerosis, multiple sclerosis (MS) such as spino-optical MS, primary progressive MS (PPMS), and relapsing remitting MS (RRMS), progressive systemic sclerosis, atherosclerosis, arteriosclerosis, sclerosis disseminata, ataxic sclerosis, neuromyelitis optica (NMO), inflammatory bowel disease (IBD) (for example, Crohn's disease, autoimmune-mediated gastrointestinal diseases, colitis such as ulcerative colitis, colitis ulcerosa, microscopic colitis, collagenous colitis, colitis polyposa, necrotizing enterocolitis, and transmural colitis, andautoimmune inflammatory bowel disease), bowel inflammation, pyoderma gangrenosum, erythema nodosum, primary sclerosing cholangitis, respiratory distress syndrome, including adult or acute respiratory distress syndrome (ARDS), meningitis, inflammation of all or part of the uvea, iritis, choroiditis, an autoimmune hematological disorder, rheumatoid spondylitis, rheumatoid synovitis, hereditary angioedema, cranial nerve damage as in meningitis, herpes gestationis, pemphigoid gestationis, pruritis scroti, autoimmune premature ovarian failure, sudden hearing loss due to an autoimmune condition, IgE-mediated diseases such as anaphylaxis and allergic and atopic rhinitis, encephalitis such as Rasmussen's encephalitis and limbic and / or brainstem encephalitis, uveitis, such as anterior uveitis, acute anterior uveitis, granulomatous uveitis, nongranulomatous uveitis, phacoantigenic uveitis, posterior uveitis, or autoimmune uveitis, glomerulonephritis (GN) with and without nephrotic syndrome such as chronic or acute glomerulonephritis such as primary GN, immune- mediated GN, membranous GN (membranous nephropathy), idiopathic membranous GN or idiopathic membranous nephropathy, membrano- or membranous proliferative GN (MPGN), including Type I and Type II, and rapidly progressive GN, proliferative nephritis, autoimmune polyglandular endocrine failure, balanitis including balanitis circumscripta plasmacellularis, balanoposthitis, erythema annulare centrifugum, erythema dyschromicum perstans, eythema multiform, granuloma annulare, lichen nitidus, lichen sclerosus et atrophicus, lichen simplex chronicus, lichen spinulosus, lichen planus, lamellar ichthyosis, epidermolytic hyperkeratosis, premalignant keratosis, pyoderma gangrenosum, allergic conditions and responses, allergic reaction, eczema including allergic or atopic eczema, asteatotic eczema, dyshidrotic eczema, and vesicular palmoplantar eczema, asthma such as asthma bronchiale, bronchial asthma, and auto-immune asthma, conditions involving infiltration of T cells and chronic inflammatory responses, immune reactions against foreign antigens such as fetal A-B- O blood groups during pregnancy, chronic pulmonary inflammatory disease, autoimmune myocarditis, leukocyte adhesion deficiency, lupus, including lupus nephritis, lupus cerebritis, pediatric lupus, non-renal lupus, extra-renal lupus, discoidlupus and discoid lupus erythematosus, alopecia lupus, systemic lupus erythematosus (SLE) such as cutaneous SLE or subacute cutaneous SLE, neonatal lupus syndrome (NLE), and lupus erythematosus disseminatus, juvenile onset (Type I) diabetes mellitus, including pediatric insulin-dependent diabetes mellitus (IDDM), and adult onset diabetes mellitus (Type II diabetes) and autoimmune diabetes. Also contemplated are immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes, sarcoidosis, granulomatosis including lymphomatoid granulomatosis, Wegener's granulomatosis, agranulocytosis, vasculitides, including vasculitis, large-vessel vasculitis (including polymyalgia rheumatica and gianT cell (Takayasu's) arteritis), medium-vessel vasculitis (including Kawasaki's disease and polyarteritis nodosa / periarteritis nodosa), microscopic polyarteritis, immunovasculitis, CNS vasculitis, cutaneous vasculitis, hypersensitivity vasculitis, necrotizing vasculitis such as systemic necrotizing vasculitis, and ANCA-associated vasculitis, such as Churg-Strauss vasculitis or syndrome (CSS) and ANCA-associated small-vessel vasculitis, temporal arteritis, aplastic anemia, autoimmune aplastic anemia, Coombs positive anemia, Diamond Blackfan anemia, hemolytic anemia or immune hemolytic anemia including autoimmune hemolytic anemia (AIHA), Addison's disease, autoimmune neutropenia, pancytopenia, leukopenia, diseases involving leukocyte diapedesis, CNS inflammatory disorders, Alzheimer's disease, Parkinson's disease, multiple organ injury syndrome such as those secondary to septicemia, trauma or hemorrhage, antigen-antibody complex-mediated diseases, anti-glomerular basement membrane disease, anti-phospholipid antibody syndrome, allergic neuritis, Behcet's disease / syndrome, Castleman's syndrome, Goodpasture's syndrome, Reynaud's syndrome, Sjogren's syndrome, Stevens- Johnson syndrome, pemphigoid such as pemphigoid bullous and skin pemphigoid, pemphigus (including pemphigus vulgaris, pemphigus foliaceus, pemphigus mucus-membrane pemphigoid, and pemphigus erythematosus), autoimmune polyendocrinopathies, Reiter's disease or syndrome, thermal injury, preeclampsia, an immune complex disorder such as immune complex nephritis, antibody-mediated nephritis, polyneuropathies, chronic neuropathy such asIgM polyneuropathies or IgM -mediated neuropathy, autoimmune or immune-mediated thrombocytopenia such as idiopathic thrombocytopenic purpura (ITP) including chronic or acute ITP, scleritis such as idiopathic cerato-scleritis, episcleritis, autoimmune disease of the testis and ovary including autoimmune orchitis and oophoritis, primary hypothyroidism, hypoparathyroidism, autoimmune endocrine diseases including thyroiditis such as autoimmune thyroiditis, Hashimoto's disease, chronic thyroiditis (Hashimoto's thyroiditis), or subacute thyroiditis, autoimmune thyroid disease, idiopathic hypothyroidism, Grave's disease, polyglandular syndromes such as autoimmune polyglandular syndromes (or polyglandular endocrinopathy syndromes), paraneoplastic syndromes, including neurologic paraneoplastic syndromes such as Lambert-Eaton myasthenic syndrome or Eaton-Lambert syndrome, stiff-man or stiff-person syndrome, encephalomyelitis such as allergic encephalomyelitis or encephalomyelitis allergica and experimental allergic encephalomyelitis (EAE), experimental autoimmune encephalomyelitis, myasthenia gravis such as thymoma- associated myasthenia gravis, cerebellar degeneration, neuromyotonia, opsoclonus or opsoclonus myoclonus syndrome (OMS), and sensory neuropathy, multifocal motor neuropathy, Sheehan's syndrome, autoimmune hepatitis, chronic hepatitis, lupoid hepatitis, gianT cell hepatitis, chronic active hepatitis or autoimmune chronic active hepatitis, lymphoid interstitial pneumonitis (LIP), bronchiolitis obliterans (nontransplant) vs NSIP, Guillain-Barre syndrome, Berger's disease (IgA nephropathy), idiopathic IgA nephropathy, linear IgA dermatosis, acute febrile neutrophilic dermatosis, subcorneal pustular dermatosis, transient acantholytic dermatosis, cirrhosis such as primary biliary cirrhosis and pneumonocirrhosis, autoimmune enteropathy syndrome, Celiac or Coeliac disease, celiac sprue (gluten enteropathy), refractory sprue, idiopathic sprue, cryoglobulinemia, amylotrophic lateral sclerosis (ALS; Lou Gehrig's disease), coronary artery disease, autoimmune ear disease such as autoimmune inner ear disease (AIED), autoimmune hearing loss, polychondritis such as refractory or relapsed or relapsing polychondritis, pulmonary alveolar proteinosis, Cogan's syndrome / nonsyphilitic interstitial keratitis, Bell's palsy, Sweet's disease / syndrome,rosacea autoimmune, zoster-associated pain, amyloidosis, a non-cancerous lymphocytosis, a primary lymphocytosis, which includes monoclonal B cell lymphocytosis (e.g., benign monoclonal gammopathy and monoclonal gammopathy of undetermined significance, MGUS), peripheral neuropathy, paraneoplastic syndrome, channelopathies such as epilepsy, migraine, arrhythmia, muscular disorders, deafness, blindness, periodic paralysis, and channelopathies of the CNS, autism, inflammatory myopathy, focal or segmental or focal segmental glomerulosclerosis (FSGS), endocrine opthalmopathy, uveoretinitis, chorioretinitis, autoimmune hepatological disorder, fibromyalgia, multiple endocrine failure, Schmidt's syndrome, adrenalitis, gastric atrophy, presenile dementia, demyelinating diseases such as autoimmune demyelinating diseases and chronic inflammatory demyelinating polyneuropathy, Dressier's syndrome, alopecia greata, alopecia totalis, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyl), and telangiectasia), male and female autoimmune infertility, e.g., due to anti-spermatozoan antibodies, mixed connective tissue disease, Chagas' disease, rheumatic fever, recurrent abortion, farmer's lung, erythema multiforme, post-cardiotomy syndrome, Cushing's syndrome, bird-fancier's lung, allergic granulomatous angiitis, benign lymphocytic angiitis, Alport's syndrome, alveolitis such as allergic alveolitis and fibrosing alveolitis, interstitial lung disease, transfusion reaction, leprosy, malaria, parasitic diseases such as leishmaniasis, kypanosomiasis, schistosomiasis, ascariasis, aspergillosis, Sampler's syndrome, Caplan's syndrome, dengue, endocarditis, endomyocardial fibrosis, diffuse interstitial pulmonary fibrosis, interstitial lung fibrosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endophthalmitis, erythema elevatum et diutinum, erythroblastosis fetalis, eosinophilic faciitis, Shulman's syndrome, Felty's syndrome, flariasis, cyclitis such as chronic cyclitis, heterochronic cyclitis, iridocyclitis (acute or chronic), or Fuch's cyclitis, Henoch-Schonlein purpura, human immunodeficiency virus (HIV) infection, SCID, acquired immune deficiency syndrome (AIDS), echovirus infection, sepsis, endotoxemia, pancreatitis, thyroxicosis, parvovirus infection, rubella virus infection, post-vaccination syndromes, congenital rubella infection, Epstein-Barrvirus infection, mumps, Evan's syndrome, autoimmune gonadal failure, Sydenham's chorea, post-streptococcal nephritis, thromboangitis ubiterans, thyrotoxicosis, tabes dorsalis, chorioiditis, gianT cell polymyalgia, chronic hypersensitivity pneumonitis, keratoconjunctivitis sicca, epidemic keratoconjunctivitis, idiopathic nephritic syndrome, minimal change nephropathy, benign familial and ischemia-reperfusion injury, transplant organ reperfusion, retinal autoimmunity, joint inflammation, bronchitis, chronic obstructive airway / pulmonary disease, silicosis, aphthae, aphthous stomatitis, arteriosclerotic disorders, asperniogenese, autoimmune hemolysis, Boeck's disease, cryoglobulinemia, Dupuytren's contracture, endophthalmia phacoanaphylactica, enteritis allergica, erythema nodosum leprosum, idiopathic facial paralysis, chronic fatigue syndrome, febris rheumatica, Hamman-Rich's disease, sensoneural hearing loss, haemoglobinuria paroxysmatica, hypogonadism, ileitis regionalis, leucopenia, mononucleosis infectiosa, traverse myelitis, primary idiopathic myxedema, nephrosis, ophthalmia symphatica, orchitis granulomatosa, pancreatitis, polyradiculitis acuta, pyoderma gangrenosum, Quervain's thyreoiditis, acquired spenic atrophy, non-malignant thymoma, vitiligo, toxic-shock syndrome, food poisoning, conditions involving infiltration of T cells, leukocyte-adhesion deficiency, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes, diseases involving leukocyte diapedesis, multiple organ injury syndrome, antigen-antibody complex-mediated diseases, antiglomerular basement membrane disease, allergic neuritis, autoimmune polyendocrinopathies, oophoritis, primary myxedema, autoimmune atrophic gastritis, sympathetic ophthalmia, rheumatic diseases, mixed connective tissue disease, nephrotic syndrome, insulitis, polyendocrine failure, autoimmune polyglandular syndrome type I, adult-onset idiopathic hypoparathyroidism (AOIH), cardiomyopathy such as dilated cardiomyopathy, epidermolisis bullosa acquisita (EBA), hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosing cholangitis, purulent or nonpurulent sinusitis, acute or chronic sinusitis, ethmoid, frontal, maxillary, or sphenoid sinusitis, an eosinophil- related disorder such as eosinophilia, pulmonary infiltration eosinophilia, eosinophilia-myalgia syndrome, Loffler's syndrome, chronic eosinophilic pneumonia, tropical pulmonary eosinophilia, bronchopneumonic aspergillosis, aspergilloma, or granulomas containing eosinophils, anaphylaxis, seronegative spondyloarthritides, polyendocrine autoimmune disease, sclerosing cholangitis, sclera, episclera, chronic mucocutaneous candidiasis, Bruton's syndrome, transient hypogammaglobulinemia of infancy, Wiskott-Aldrich syndrome, ataxia telangiectasia syndrome, angiectasis, autoimmune disorders associated with collagen disease, rheumatism, neurological disease, lymphadenitis, reduction in blood pressure response, vascular dysfunction, tissue injury, cardiovascular ischemia, hyperalgesia, renal ischemia, cerebral ischemia, and disease accompanying vascularization, allergic hypersensitivity disorders, glomerulonephritides, reperfusion injury, ischemic re-perfusion disorder, reperfusion injury of myocardial or other tissues, lymphomatous tracheobronchitis, inflammatory dermatoses, dermatoses with acute inflammatory components, multiple organ failure, bullous diseases, renal cortical necrosis, acute purulent meningitis or other central nervous system inflammatory disorders, ocular and orbital inflammatory disorders, granulocyte transfusion-associated syndromes, cytokine-induced toxicity, narcolepsy, acute serious inflammation, chronic intractable inflammation, pyelitis, endarterial hyperplasia, peptic ulcer, valvulitis, graft versus host disease, contact hypersensitivity, asthmatic airway hyperreaction, and endometriosis.

[0192] Further aspects relate to the treatment or prevention microbial infection and / or use of microbial antigens. The microbial infection to be treated or prevented or antigen may be an antigen associated with any microbial infection known in the art or, for example, anthrax, cervical cancer (human papillomavirus), diphtheria, hepatitis A, hepatitis B, haemophilus influenzae type b (Hib), human papillomavirus (HPV), influenza (Flu), Japanese encephalitis (JE), lyme disease, measles, meningococcal, monkeypox, mumps, pertussis, pneumococcal, polio, rabies, rotavirus, rubella, shingles (herpes zoster), smallpox, tetanus, typhoid, tuberculosis (TB), varicella (Chickenpox), and yellow fever.

[0193] In some embodiments, the methods and compositions may be for vaccinating an individual to prevent a medical condition, such as cancer, inflammation, infection, and so forth.

[0194] EXAMPLES

[0195] The following examples are included to demonstrate preferred embodiments of the invention. 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 invention, 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 invention.

[0196] Detailed description of the PSC-engineered TARGET cell culture methodsStage 0: PSC master cell bank generationPSC line is cultured in a suitable cell culture vessel (e.g., in Matrigel, or on laminin-coated plate) with serum-free PSC culture media (base medium supplemented with cytokine cocktails including bFGF, TGF0, FLT3L, Noggin, activin, Bio, LIF and others) for 12-72 hours, followed by addition of the TCR gene-delivery vector, and culturing for an additional 1-7 days. After verification (and sorting and single cloning if needed), TCR gene-engineered PSC line is then used to establish master cell bank, that can be cryopreserved for storage or / and maintained in cell culture via passaging.Stage 1: PSC-to-HSPC differentiationTCR gene-engineered PSC cells are gently dissociated to single cells (e.g., via cell dissociation reagent such as Accutase, Versene, or TrypLE) and then transferred to a suitable cell culture vessel (e.g., Ultra-Low Attachment plate or AggreWell) in HSC differentiation medium A (base medium containing GlutaMAX, L-Ascorbic Acid, monothioglycerol, Insulin-Transferrin-Selenium, Activin A, BMP-4, bFGF, VEGF,SB431542, CHIR99021 and others) supplemented with ROCK inhibitor for 12-48 hours to form embryonic bodies (EBs) or monolayers. Then the media is changed to fresh HSC differentiation medium A for the following 24-72 hours. At day 3 to day 5, EBs or monolayers are collected and reseeded into a suitable cell culture vessel (e.g., Ultra-Low Attachment plate or non-treated tissue culture plate) in HSC differentiation medium B (base medium containing GlutaMAX, L-Ascorbic Acid, monothioglycerol, Insulin-Transferrin-Selenium, heparin, BMP -4, bFGF, VEGF, SCF, TPO, Flt3 ligand, IL-3, IL-6, IL-11, IGF-1, IGF-2, SB431542, EPO and others) for 6-12 days. HSC differentiation medium B is refreshed every other day. At the end of culture, the suspending CD34+HSCs are collected and cryopreserved.Stage 2: HSC-to-TARGET cell differentiationTCR gene-engineered PSC-derived HSCs are then differentiated into TARGET cells in a differentiation medium over a period of 4-10 weeks without feeders. Nontissue culture-treated plates are coated with a TARGET Culture Coating (TARGET c) Material (DLL-1 / 4, VCAM-1 / 5, retronectin, and others). CD34+HSCs are suspended in a TARGET Expansion (TARGETe) Medium (base medium containing serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, L- Ascorbic Acid, IL-7, SCF, TPO, IL-3, IL-6, Flt3 ligand, SDF-la, human LDL, UM171, SB203580, and additives), seeded into the coated wells of a plate, and cultured for 12- 14 days. TARGETe Medium is refreshed every 3-4 days. Cells are then collected and suspended in a TARGET Maturation (TARGETm) Medium (base medium containing serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, L- Ascorbic Acid, IL-7, SCF, IL-6, IL-15, Flt3 ligand, SDF-la, human LDL, UM171, SB203580, and additives), seeded into the coated wells of a plate, and cultured for another 14-28 days. TARGETm Medium is refreshed every 3-4 days.Stage 3: TARGET cell expansionDifferentiated TARGET cells are stimulated with TCR cognate antigens (e.g., proteins, peptides, lipids, phospho-antigens, small molecules, and others) or nonspecific TCR stimulatory reagents (e.g., anti-CD3 / anti-CD28 antibodies or antibody- coated beads, Concanavalin A, PMA / Ionomycin, and others), with or without the presence of antigen-presenting cells (e.g., irradiated healthy donor PBMCs, artificial APCs, and others), and expanded for up to 1 month in T cell culture media. The cell culture base media can be, including but not limited to, CTS OpTmizer, TexMACS, RPMI, DMEM, X-Vivol5. The cell culture approach can be serum -free and feeder- free. The culture can be supplemented with T cell supporting cytokines (e.g., IL-2, IL- 4, IL-7, IL-12, IL-15, IL-18, IL-21, TNFa, SDF-la, TGF-P, and others), small molecules and additives (e.g., Wnt activators or glycogen synthase kinase-3 (GSK-3) inhibitors such as Wnt3A, CHIR99021, AR-A014418, TWS119, LY2090314, 9-ING- 41, lithium chloride (LiCl), BIO (6-bromoindirubin-3 -oxime, 6-Bromoindirubin-3'- oxime, or tyrosine kinase inhibitors such as dasatinib, ibrutinib, acalabrutinib, or zanubrutinib).TARGET cell derivativesIn some embodiments, PSC master cell lines / banks and their derived HSCs as well as TARGET cells can be further engineered to express additional transgenes. In one embodiment, such transgenes encode disease targeting molecules such as chimeric antigen receptors (CARs), T-cell receptors (TCRs), and other native or synthetic receptor / ligands. In another embodiment, such transgenes can encode T cell regulatory proteins such as IL-2, IL-4, IL-7, IL-10, IL-12, IL-15, IL-18, IFN-y, TNF-a, TL1A, CD27, CD28, 4- IBB, 0X40, ICOS, DAP 10, and others. In another embodiment, such transgenes can encode transcription factors such as Bell lb, Batf3, ThPOK, FOXP3, Runx3, and others. Transgenes can be introduced into post-expansion TARGET cells or their progenitor cells (HSCs, newly differentiated TARGET cells, in-expansion TARGET cells) at various culture stages.In some embodiments, PSC master cell lines / banks and their derived HSCs as well as TARGET cells can be further engineered to disrupt selected genes using gene editing tools (CRISPR, TALEN, Zinc-Finger, and others). In one embodiment, disrupted genes encode T cell immune checkpoint inhibitors (PD-1, CTLA-4, TIM-3, LAG-3, CD161, and others). In another embodiment, disrupted genes encode T cell regulatory proteins (e.g., TET2, PI3K5 / y, DGK, DNMT3a, Suv39hl, and others). Deficiency of these negative regulatory genes may enhance the disease fighting capacity of TARGET cells, making them resistance to disease-induced anergy and tolerance.In some embodiments, PSC master cell lines / banks and their derived HSCs as well as TARGET cells or enhanced TARGET cells can be further engineered to make them suitable for allogeneic adoptive transfer, thereby suitable for serving as off-the- shelf cellular products. In one embodiment, genes encoding MHC molecules or MHC expression / display regulatory molecules [MHC molecules, B2M, CIITA (Class II transcription activator control induction of MHC class II mRNA expression), and others]. Lack of MHC molecule expression on TARGET cells makes them resistant to allogeneic host T cell-mediated depletion. In another embodiment, MHC class-I deficient TARGET cells will be further engineered to overexpress an HLA-C, HLA-E, HLA-G, and CD47 gene that will endow them resistant to host NK cell-mediated depletion.The gene-engineered PSC master cell lines / banks can be cryopreserved for storage, or maintained in culture via passaging; the PSC-derived HSCs can be used freshly or cryopreserved for future usage; the TARGET cell products and derivatives can also be used freshly or cryopreserved for further usage. Moreover, various intermediate cellular products generated during PSC-to-TARGET cell culture can be paused for cryopreservation, stored and recovered for continued production.Compared with our previous invention on in vitro generation of TARGET cells via culturing gene-engineered primary human CD34+HSCs, this invention offers an in vitro differentiation method that can generate TARGET cells from gene-engineeredPSCs as an unlimited resource. This new method greatly improves the process for the scale-up production and GMP-compatible manufacturing of therapeutic cells for human applications.The cell products, TARGET cells, display phenotypes / functionalities distinct from that of their native counterpart T cells as well as their counterpart T cells generated using other ex vivo culture methods (e.g. ATO culture method) or derived from other starting cells (e.g., primary human CD34+HSCs), making these PSC-derived TARGET cells unique cellular products.Unique features of the PSC-derived TARGET cell differentiation culture include:1) It is Ex Vivo and Feeder-Free;2) No need to purify and sort CD34+ HSC after PSC to HSC differentiation because of high CD34+ purity;3) Without transgenic TCR engineering or using T-iPSCs, robust endogenous TCR recombination (ySTCR and conventional aPTCR) occurs during T cell differentiation. This demonstrates our culture platform resembles the natural T cell development;4) It can generate human CD8 single positive and CD4 single positive T cells. The CD4 single positive T cells can be further differentiated into T-helper 2 (Th2) T cells;5) It has no risk of GvHD, because TARGET cells carry monoclonal innate TCR;6) It supports the synchronized differentiation of transgenic TARGET cells, thereby eliminating the presence of un-differentiated progenitor cells and other lineages of bystander immune cells.7) As a result, the TARGET cell product comprises a homogenous and pure population of monoclonal TCR-armed T cells. No escaped random T cells, no other lineages of immune cells, and no un-differentiated progenitor cells. Therefore, no need for a purification step;8) Unlimited supply of the source PSC and product TARGET cells; and9) Unique phenotype of TARGET cells- monoclonal TCR+ random aP TCR-CD3+.As disclosed herein, proof-of-principle studies have been performed, showing the successful generation of PSC-derived iNKT TCR-engineered TARGET cells (denoted aspsciNKT cells) and gamma-delta TCR-engineered TARGET cells (denoted aspscy6T cells). Further engineering ofpsciNKT cells to additionally express a BCMA CAR (denoted aspscBCAR-iNKT cell product) were also proved successful. Pilot CMC, pharmacology, efficacy, and safety studies were performed analyzing these cell products.“Generation and characterization of off-the-shelf PSC-derived BCMA-targeting CAR-armed invariant natural killer T (pscBCAR-iNKT) cells”Invariant natural killer T (iNKT) cells are a small subpopulation of aP T lymphocytes with the ability to bridge innate and adaptive immunity. Unlike the conventional a|3 T cells, the T cell receptor (TCR) of iNKT cells recognizes lipid antigens presented by CD Id, a major histocompatibility complex (MHC)-like molecule, instead of MHC itself. Because of this unique property, iNKT cells do not cause graft-versus-host disease (GvHD) when transplanted allogeneically. Additionally, iNKT cells have several other unique features that make them ideal cellular carriers for developing off-the-shelf cellular therapy for cancer: 1) they have roles in cancer immunosurveillance; 2) they have the remarkable capacity to target tumors independent of tumor antigen- and major histocompatibility complex (MHC)- restrictions; 3) they can employ multiple mechanisms to attack tumor cells through direct killing and adjuvant effects. However, the development of an allogeneic off-the- shelf iNKT cellular product is greatly hindered by their availability - these cells are of extremely low number and high variability in humans (-0.001-1% in human blood), making it very difficult to produce therapeutic numbers of iNKT cells from blood cells of allogeneic human donors.Two prior methods have been used to generate enough iNKT cells for therapeutic uses. One method is to screen large numbers of donors and find “super donors” who naturally have high percentage of iNKT cells in peripheral blood. iNKTcells are enriched by the magnetic bead-based purification procedure and then expanded by either anti-CD3 / CD28 bead stimulation or co-culture with antigen-presenting cells loaded with alpha-galactosylceramide (aGC). Although expansion can be achieved by this method, the expansion fold is limited, and the expansion is unreliable. Another method is based on the genetic modification of hematopoietic stem cells (HSCs) with iNKT TCRs followed by an artificial thymic organoid (ATO) culture system that supports the in vitro differentiation of human HSCs into iNKT cells. Although this method can generate iNKT cells with high yield, the production requires the use of feeder cells of mouse origin, which poses significant challenges to develop a reliable process for GMP-compatible manufacturing.A novel method that can reliably generate a homogenous monoclonal population of iNKT cells at large quantities with a feeder-free differentiation system is thus pivotal to developing an off-the-shelf iNKT cell therapy.CMC Study-pscBCAR-iNKT Cells (Figures 16&17)PSCs were transduced with a Lenti / iNKT-BCAR-(GFP) vector that encoded a human iNKT TCR gene, a BCMA-targeting CAR gene, and an optional GFP reporter gene, to establish gene-engineered PSC master cell lines / banks, which were then put into the feeder-free / serum-free Ex Vivo PSC-Derived CAR-iNKT Cell Culture to generate PSC-derived BCMA CAR-armed iNKT (pscBCAR-iNKT) cells. Both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) can be used as starting PSC source cells; data generated through using an iPSC cell line (DMD; Figure 16) or ES cell line (Hl; Figure 17) are presented. Note through a 3-stage culture (HSC differentiation, iNKT differentiation, iNKT expansion) over ~5-6 weeks, pure and clonalpscBCAR-iNKT cells were successfully generated.Pharmacology Study-pscBCAR-iNKT Cells (Figure 18)The phenotype and functionality ofpscBCAR-iNKT cells were studied using flow cytometry (Figure 18). iPSC-derived and ESC-derivedpscBCAR-iNKT cellsdisplayed similar phenotype and functionality; data of iPSC-derivedpscBCAR-iNKT cells are presented. Healthy donor PBMC-derived conventional aPT cells engineered to express the same BCAR were included as a benchmark control (denoted as BCAR- T cells). pscBCAR-iNKT cells displayed a typical human iNKT cell phenotype and functionality: they expressed the CD4 and CD8 co-receptors with a mixed pattern (CD4 / CD8 double-negative and CD8 single-positive); they expressed T cell activation markers (CD69hland CD62L10); and they produced exceedingly high levels of effector molecules (e.g., Perforin and Granzyme B). Notably, similar Ex Vivo generated primary human CD34+HSC-derived human iNKT cells,pscBCAR-iNKT cells expressed significantly lower levels of surface HLA-I molecules and nearly undetectable surface HLA-II molecules, suggesting that these cells may resist allo-rej ection mediated by host T cells when adoptively transferred into allogeneic hosts and thus are suitable for “off- the-shelf’ cell therapy.In Vitro Efficacy and MOA Study-psciNKT Cells (Figures 19&20)Even without being engineered to express additional tumor-targeting molecules like Chimeric Antigen Receptors (CARs),psciNKT cells can already target tumor cells through NK receptor-mediated pathways. We established an in vitro tumor cell killing assay to study such tumor killing capacities (Figure 19A). Healthy donor PBMC- derived conventional a|3T cells that were not engineered to express CAR were included as a control (denoted as PBMC-T cells). Various human tumor cell lines were engineered to overexpress the firefly luciferase and enhanced green fluorescence protein dual -reporters (denoted as FG), that facilitate the detection of tumor cell killing using sensitive luciferase activity assay and flow cytometry assay. Four FG reporters- marked human tumor cell lines were used in this study, including a human melanoma cell line A375-FG, a human breast cancer cell line MDA-FG, a human ovarian cancer cell line 0VCAR8-FG, and a prostate cancer cell line PC3-FG.psciNKT cells effectively killed all 4 types of human tumor cells, different from the control PBMC-Tcells (Figures 19B-19E). Blocking of NK activating receptors (e.g., blocking of DNAM-1) reduced the tumor cell killing efficacy ofpsciNKT cells, confirming their NK activating receptor-mediated tumor targeting function (Figures 20A&20B). This mechanism of action (MOA) is supported by the detection of corresponding NK activating receptor ligands (e.g., DNAM-1 ligands like Nectin-2 and PVR) on all 4 types of human tumor cell lines studied, suggesting the capacity ofpsciNKT cells to target a broad range of human tumors via this intrinsic NK function ofpsciNKT cells (Figure 20C).In Vitro Efficacy and MOA Study-pscBCAR-iNKT Cells (Figure 21)We established an in vitro tumor cell killing assay for this study (Figures 21A&21C). BCMA-targeting CAR-armed PSC-derived human iNKT (pscBCAR- iNKT) cells were studied as the therapeutic candidate cells. PSC-derived human iNKT cells not armed with CAR (psciNKT), as well as healthy donor PBMC-derived conventional a^T (PBMC-T) cells and those engineered to express the same BCMA- targeting CAR (BCAR-T cells) were included as control effector cells.Two human tumor cell lines were used in this study: 1) a human MM cell line, MM. IS, that were BCMA+CDld' and served as a target of CAR-mediated killing (Figure 21A); and 2) an MM. IS cell line engineered to overexpress human CDld, MM. lS-CDld, that were BCMA+CDld+and served as a target of both CAR / TCR- mediated killing (Figure 21C). Both human tumor cell lines were engineered to express the firefly luciferase (Flue) and enhanced green fluorescence protein (EGFP) dualreporters. Expression of human CDld enabled the tumor cells to present iNKT TCR cognate glycolipid antigens, such as endogenous tumor lipid antigens or synthetic lipid antigens like aGC, making the CDld+ tumor cells susceptible to iNKT TCR / CDld / gly coantigen-mediated tumor killing pathway. Expression of Flue and EGFP facilitate the detection of tumor cell killing using sensitive luciferase activity assay and flow cytometry assay. The resulting MM.1S-FG and MM.lS-CDld-FG human multiple myeloma cell lines were then utilized in the study.pscBCAR-iNKT cells killed MM.1S-FG tumor cells effectively, at an efficacy comparable to that of BCAR-T cells (Figure 21B). Notably, the none-CAR-armedpsciNKT cells showed certainly efficacy killing the MM.1S-FG tumor cells, unlike the non-CAR-armed PBMC-T cells, presumably through an NK killing path (Figure 21B). pscBCAR-iNKT cells killed MM. IS-hCDld-FG tumor cells effectively, at an efficacy comparable to that of conventional BCAR-T cells (Figure 21D). Importantly, in the presence of a cognate lipid antigen (aGC),pscBCAR-iNKT cells, but not BCAR- T cells, demonstrated enhanced tumor-killing efficacy, likely because of the activation of an additional TCR / CDld / aGC tumor killing path (Figure 21D).Taken together, these results indicate thatpscCAR-iNKT cells can target tumors using three mechanisms: 1) CAR-dependent path, 2) iNKT TCR-dependent path, and 3) NK path. This unique triple-targeting capacity ofpscCAR-iNKT cells is attractive, because it can potentially circumvent antigen escape, a phenomenon that has been reported in autologous CAR-T therapy clinical trials wherein tumor cells down- regulated their expression of CAR-targeting antigen to escape attack from CAR-T cells.Safety & Immunogenicity Study-pscBCAR-iNKT Cells (Figure 22)For allogeneic cell therapies, there are two safety & immunogenicity concerns: a) GvHD responses, and b) host-versus-graft (HvG) responses. We have considered the possible GvHD and HvG risks for the intendedpscBCAR-iNKT cell product (Figure 22). The none-CAR-armedpsciNKT cells were also studied and showed similar results (data not shown).GvHD is the major safety concern. However, because iNKT cells do not react to mismatched HLA molecules and protein autoantigens, they are not expected to induce GvHD1. This notion is evidenced by the lack of GvHD in human clinical experiences in allogeneic HSC transfer and autologous iNKT transfer2,3, and is supported by our In Vitro Mixed Lymphocyte Reaction (MLR) Assay designed to study the GvH response (Figure 22A). Note thatpscBCAR-iNKT cells did not respond torandom allogenic healthy donor PBMCs, in sharp contrast to that of the conventional PBMC-T cells (Figure 22B).On the other hand, HvG risk is largely an efficacy concern, mediated through elimination of allogeneic therapeutic cells by host immune cells, mainly by conventional CD8 and CD4 T cells which recognize mismatched HLA-I and HLA-II molecules. In an In Vitro Mixed Lymphocyte Reaction (MLR) Assay designed to study HvG response, compared to conventional BCAR-T cells,pscBCAR-iNKT cells induced an overall reduced HvG response when cultured with random allogeneic healthy donor PBMCs (Figures 22C&22D), likely because of their expression of significantly lower levels of surface HLA-I molecules and nearly un-detectable HLA- II molecules compared to that of conventional BCAR-T cells (Figures 22E&22F).Taken together, these results strongly supportpscCAR-iNKT cells as an ideal candidate for off-the-shelf cellular therapy that are GvHD-free and HvG-resistant.“Generation and characterization of off-the-shelf PSC-derived gamma-delta T (pscy5T) cells”Gamma delta T (y5 T) cells are a small subpopulation of T lymphocytes with the ability to bridge innate and adaptive immunity. The majority of y5 T cells in adult human blood exhibit Vy9V52 TCR and respond to small phosphorylated nonpeptide antigens, called phosphoantigens (pAgs), which are commonly produced by malignant cells5. Unlike conventional a.p T cells, y5 T cells do not recognize the polymorphic classical major histocompatibility complex (MHC) molecules and are therefore free of GvHD risk when adoptively transferred into allogeneic host. Additionally, y5 T cells have several other unique features that make them ideal cellular carriers for developing off-the-shelf cellular therapy for cancer: 1) they have roles in cancer immune surveillance; 2) they have the remarkable capacity to target tumors independent of tumor antigen and major histocompatibility complex (MHC)-restrictions; 3) they can employ multiple mechanisms to attack tumor cells through direct killing and adjuvant effects; 4) they express a surface receptor, FcyRIII (CD 16), that is involved in antibody -dependent cellular cytotoxicity (ADCC) and can be potentially combined with monoclonal antibody for cancer therapy. However, the development of an allogeneic off-the-shelf y5 T cellular product is greatly hindered by their availability - these cells are of extremely low number and high variability in humans (-1-5% T cells in human blood), making it very difficult to produce therapeutic numbers of y5 T cells from blood cells of allogeneic human donors.The current method of generating y5 T cells, in particular the Vy9V52 subset, for adoptive therapy involves either in vitro or in vivo expansion of peripheral blood mononuclear cell (PBMC)-derived y5 T cells using aminobisphosphonates, such as Zoledronate (ZOL). However, this method generates highly variable yields of y5 T cells depending on PBMC donors; and most importantly, such a y5 T cell product will likely contain bystander ot,p T cells and thereby incurring GvHD risk. A novel method that can reliably generate a homogenous monoclonal population of y5 T cells at large quantities with a feeder-free differentiation system is thus pivotal to developing an off- the-shelf y5 T cell therapy.CMC Study-pscy5T Cells (Figure 23)PSCs were transduced with a Lenti / y5T vector that encoded a pair of human y6T Vy9 and V52 TCR genes, to establish gene-engineered PSC master cell lines / banks, which were then put into the feeder-free / serum-free Ex Vivo PSC-Derived y5T Cell Culture to generate PSC-derived y5T (pscy6T) cells. Both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) can be used as starting PSC source cells; data generated through using an Hl ES cell line (Figure 23) are presented. Note through a 3-stage culture (HSC differentiation, y6T differentiation, y6T expansion) over -5-6 weeks, pure and clonalpscy6T cells were successfully generated.Pharmacology Study-pscy5T Cells (Figure 24)The phenotype and functionality ofpscy6T cells were studied using flow cytometry (Figure 24). Data of Hl ESC-derivedpscy6T cells are presented. Healthydonor PBMC-derived conventional aPT and y5T cells were included as staining controls (denoted as PBMC-T and PBMC-yST cells, respectively). pscy5T cells displayed a typical human y5T cell phenotype and functionality: they expressed T cell activation markers (e.g., CD69hlCD62L10) and inflammatory homing markers (e.g., CXCR3hlCCR5hl), and they produced exceedingly high levels of effector molecules (e.g., cytotoxic molecules as Perforin and Granzyme B). Interestingly,pscy5T cells expressed a broad range of NK activating receptors (e.g., NKG2D, DNAM-1, NKp30, NKp44), at levels much higher than that on native conventional PBMC-T cells and even higher than native PBMC-yST cells, suggesting a strong NK function ofpscy5T cells that may favor their cancer therapy application.In Vitro Efficacy and MOA Study-pscy8T Cells (Figure 25)We established an in vitro tumor cell killing assay for this study (Figures 25A&25C). PSC-derived human y5T (pscy5T) cells were studied as the therapeutic candidate cells. Healthy donor PBMC-derived conventional a|3T (PBMC-T) cells were included as a control.Two human tumor cell lines were used in this study: 1) a human melanoma cell line A375 (Figure 25A); and 2) a human multiple myeloma cell line MM. IS (Figure 25C). Both human tumor cell lines were engineered to express the firefly luciferase and enhanced green fluorescence protein (FG) dual-reporters. Expression of Flue and EGFP facilitate the detection of tumor cell killing using sensitive luciferase activity assay and flow cytometry assay. The resulting A375-FG and MM.1S-FG human tumor cell lines were then utilized in the study. pscy5T cells killed both A375-FG and MM.1S-FG tumor cells effectively, in contrast to the PBMC-T cells that did not show tumor cell killing, indicating an intrinsic capacity ofpscy5T cells to target a broad range of human tumor cells independent of the expression of additional tumor-targeting molecules (e.g., CARs) (Figure 24B&24D) These CAR-independent tumor cell -targeting mechanisms can be mediated through NK and / or y5 TCR paths, as supported by the expression of highlevels of NK activating receptors onpscy6T cells (Figure 24) and by the enhanced tumor cell killing in the presence of a y962 TCR stimulator zoledronate (ZOL) (Figure 25D) The multiple tumor-targeting mechanisms ofpscy6T cells is attractive, because this can potentially circumvent antigen escape, a phenomenon that has been reported in autologous CAR-T therapy clinical trials wherein tumor cells down-regulated their expression of CAR-targeting antigen to escape attack from CAR-T cells.Safety & Immunogenicity Study-pscy6T Cells (Figure 26)For allogeneic cell therapies, there are two immunogenicity concerns: a) Graft- versus-host (GvH)responses, and b) Host-versus-graft (HvG) responses. GvHD is a major safety concern. However, since y5 T cells do not react to mismatched HLA molecules and protein autoantigens, they are not expected to induce GvHD. This notion is evidenced by the lack of GvHD in human clinical experiences in allogeneic HSC transfer and autologous y5 T cell transfer, and is supported by our In Vitro Mixed Lymphocyte Reaction (MLR) Assay (Figure 26A). Note that neither PBMC-y5T cells norpscy5T cells responded to allogenic PBMCs, in sharp contrast to that of the conventional PBMC-T cells (Figure 26B).On the other hand, HvG risk is largely an efficacy concern, mediated through elimination of allogeneic therapeutic cells by host immune cells, mainly by conventional CD8 and CD4 T cells which recognize mismatched HLA-I and HLA-II molecules. Interestingly, compared to PBMC-derived a^T (PBMC-T) cells, PBMC- y5T cells expressed reduced levels of HLA-I and HLA-II molecules, whilepscy6T expressed further reduced levels of HLA-I molecules and nearly undetectable HLA-II molecules (Figure 26C). This HLA-I / II expression pattern suggests that in general native y5T cells exhibit “low-immunogenicity” than a|3T cells, and thatpscy6T exhibit the lowest immunogenicity that may render these cells resistance to allo rejection. Indeed, in an In Vitro MLR assay designated to study HvG response (Figure 26D),pscy6T cells triggered the least allo response, significantly lower than that triggered by the PBMC-T cells as well as PBMC-y5T cells (Figure 26E).Taken together, these results strongly supportpscy5T cells as an ideal candidate for off-the-shelf cellular therapy that are GvHD-free and HvG-resistant.1 Fujii, S. et al. NKT cells as an ideal anti-tumor immunotherapeutic. Front Immunol 4, 409, doi: 10.3389 / fimmu.2013.00409 (2013).2 Haraguchi, K. etal. Recovery of Valpha24+ NKT cells after hematopoietic stem cell transplantation. Bone Marrow Transplant 34, 595-602, doi : 10.1038 / sj .bmt.1704582 (2004).3 de Lalla, C. et al. Invariant NKT cell reconstitution in pediatric leukemia patients given HLA-haploidentical stem cell transplantation defines distinct CD4+ and CD4- subset dynamics and correlates with remission state. J Immunol 186, 4490-4499, doi: 10.4049 / jimmunol.1003748 (2011).

[0197] Although the present disclosure and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the design as defined by the appended claims. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the process, machine, manufacture, composition of matter, means, methods and steps described in the specification. As one of ordinary skill in the art will readily appreciate from the present disclosure, processes, machines, manufacture, compositions of matter, means, methods, or steps, presently existing or later to be developed that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein may be utilized according to the present disclosure. Accordingly, the appended claims are intended to include within their scope such processes, machines, manufacture, compositions of matter, means, methods, or steps.REFERENCES

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Claims

CLAIMSWhat is claimed is:

1. A method of making monoclonal TCR- Armed Gene-Engineered T (TARGET) cells using pluripotent stem cells (PSCs), the method comprising introducing into TARGET cells a selected monoclonal T cell receptor (TCR) gene:(a) as an endogenous TCR gene when a T cell-reprogrammed induced PSC (T-iPSC) line is used to make the TARGET cells; or(b) as an exogeneous TCR transgene when a non-T-iPSC PSC line is used to make the TARGET cells; and differentiating the PSC cells of (a) or (b) so as to generate the TARGET cells.

2. The method of claim 1, wherein the monoclonal TCR gene encodes a TCR selected from: an alpha beta TCR; a gamma delta TCR, an invariant NKT (iNKT) TCR, a non-invariant NKT TCR, and a mucosal associated invariant T (MAIT) TCR.

3. The method of claim 1, wherein a TARGET cell product comprises a gene expression profile characterized as being at least one of: monoclonal TCR-positive CD3-postive,HLA-VII-low / negative, expression of one or more immune modulatory transgene(s), and / or disrupted expression of one or more endogenous immune modulatory gene(s).

4. The method of claim 3, wherein(a) the immune regulatory transgene(s) delivered into a TARGET cell product can encode any one or more of the following: immune targeting molecules, immune regulatory molecules, immune allorej ection resistance molecules, a suicide gene and / or an imaging marker molecules; and(b) the endogenous gene(s) disrupted in the TARGET cells encodes at least one following immune checkpoint agents selected from PD-1, PD-L1, CTLA-4, LAG-3, TIM-3,TIGIT, B7-H3 / B7-H4, BTLA, VISTA, NKG2A, A2aR, PVRIG, IDO, CD73, CD39, CD96, and CD161, and immune regulatory molecule; or / and an immune allorej ection molecule.

5. The method of claim 1, wherein all the desired gene modifications intended for a designated TARGET cell product:(a) are integrated in a master PSC line via an All-in-One engineering (AO-Engineering) strategy,(b) are introduced stepwise to a master PSC line as well as its progeny CD34+ hematopoietic stem and progenitor cells (HSPCs) via an Assembly-Line Engineering (AL- Engineering) strategy,(c) a single transgene or multiple transgenes can be incorporated into a TARGET cell product via a gene delivery vector or va ector-free system; and / or(d) a single endogenous gene or multiple endogenous genes of a TARGET cell product is / are disrupted.

6. The method of claim 1, wherein PSCs are cultured ex vivo to produce TARGET cells through 4 stages and certain optional steps:Stage 0 that supports the PSC master cell bank generation and maintenance, andStage 1 that supports the PSC differentiation into CD34+ HSPCs, andStage 2 that supports the HSPC differentiation into mature TARGET cells, andStage 3 that supports the expansion of TARGET cells, and an optional “CD4-Induction Step” that can be added between the Stage 2 and Stage 3 cultures to enable the generation of CD4 single-positive (CD4 SP) TARGET cells, and another optional “Tu-Polarization Step” that can be further added in Stage 3 culture to enable the generation of Tu-polarized CD4 SP TARGET cells.

7. The method of claim 6, wherein: all 3 culture stages (Stages 1, 2, and 3) can be feeder-free and / or serum-free; or the Stage 3 culture can contain feeder cells; and / or the cell culture media can comprise a base medium supplemented with one or more factors selected to facilitate the PSC-derived TARGET cell differentiation, expansion, and sublineage commitment; and / or all three stages (Stages 1, 2, and 3) of ex vivo culture can achieve high purity, eliminating the need for in-process purification steps; and / orthe PSC-derived HSPC and / or HSPC-derived TARGET intermediate cell product(s) can be cultured freshly or cryopreserved and then thawed for continued culture; and / or an All-in-One engineered (AO-Engineered) master PSC line is cultured ex vivo to produce a designated TARGET cell product, without the need for additional gene engineering steps; and / or an Assembly-Line engineered (AL-Engineered) master PSC line is cultured ex vivo to make a designated TARGET cell product, requiring additional gene engineering step(s) on the PSC-derived HSPCs and / or other TARGET cell progenitors.

8. The method of claim 1, wherein: a TARGET cell product produced by the methods described herein can be cryopreserved; and / or the cryo-recovered cell product can be stable at room temperature for at least one hour; and / or the cryo-recovered cell product is stable at room temperature for at least 1, 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, 24, 30, or 48 hours; and / or a cell product contains a solution comprising dextrose, one or more electrolytes, albumin, dextran, and / or DMSO; and / or a cell product is in a solution that is sterile, nonpyogenic, and isotonic.

9. The method of claim 1, wherein a TARGET cell product can be used for treating patients: the patient has a cancer; and / or the patient has a viral, bacterial, fungal or parasitic infection; and / or the patient has a disease or condition involving inflammation, which, in some embodiments, excludes cancer; and / or the patient has an autoimmune disease or condition; and / or the TARGET cell product is allogeneic with respect to the patient; and / or the patient does not exhibit signs of rejection or depletion of the TARGET cells; and / or some therapeutic methods further include administering to the patient a stimulatory reagent that activates TARGET cells, or a reagent that triggers the suicide gene.

10. A method of using pluripotent stem cells (PSCs) to make monoclonal TCR- Armed Gene-Engineered T (TARGET) cells comprising:- I l l -(a) introducing into TARGET cells a selected monoclonal T cell receptor (TCR) gene as an endogenous TCR gene when a T cell -reprogrammed induced PSC (T-iPSC) line is used to make the TARGET cells; or(b) introducing into TARGET cells a selected monoclonal TCR gene as an exogeneous TCR transgene when a non-T-iPSC PSC line is used to make the TARGET cells.; or and differentiating the PSC cells of (a) or (b) so as to generate the monoclonal TCR-Armed Gene-Engineered T (TARGET) cells.

11. The method of claim 10, wherein genetic modifications are made according to an all- in-one engineering methodology comprising performing all genetic modifications on a master PSC line.

12. The method of claim 10, wherein the method comprises:(a) disposing the pluripotent stem cells in a serum-free, feeder free PSC culture media comprising at least one of bFGF, TGFP, FLT3L, Noggin, activin and Bio for at least 12 hours;(b) combining the pluripotent stem cells from (a) with the at least one exogenous nucleic acid molecule disposed in an expression vector, culturing the PSC cells for at least 12 hours, and identifying pluripotent stem cells transduced with the expression vector;(c) disposing pluripotent stem cells transduced with the expression vector from (b) that are dissociated to single cells into a serum-free, feeder free HSC differentiation culture media A comprising at least one of: glutamax, ascorbic acid, monothioglycerol, Insulin-Transferrin- Selenium, Activin A, BMP-4, bFGF, VEGF, SB431542, CHIR99021, and a ROCK inhibitor for at least 12 hours such that the cells form embryonic bodies;(d) disposing the embryonic bodies of (c) in a serum-free, feeder free cell PSC differentiation culture media B comprising at least one of BMP -4, FGF, SCF, TPO, FLT3L, IL-6, IL-11, IGF-1, SB203580, and EPO for at least 6 days so as to form CD34+ hematopoietic stem cells;(e) collecting and / or enriching CD34+hematopoietic stem cells obtained from (d);(f) disposing the CD34+hematopoietic stem cells obtained from (e) in a serum-free, feeder free TARGET expansion cell culture media comprising at least one of serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, SCF, TPO, IL-3, IL-6, Flt3 ligand, human LDL, and UM171 for at least 3 days; and(g) disposing the CD34+hematopoietic stem cells obtained from (f) in a serum-free, feeder free cell TARGET maturation culture media comprising at least one of serum albumin,recombinant human insulin, human transferrin, 2-mercaptoethanol, SCF, TPO, IL-3, IL-6, IL- 7, IL- 15, Flt3 ligand, and ascorbic acid; such that the monoclonal TCR- Armed Gene-Engineered T (TARGET) cells are made.

13. The method of claim 12, further comprising disposing the monoclonal TCR-Armed Gene-Engineered T (TARGET) cells into a target cell expansion media comprising at least one of a TCR cognate antigen or a non-specific TCR stimulatory reagent.

14. The method of claim 12, wherein the method generates TARGET cells expressing at least 2,000 exogenous TCR polypeptides on the surface of the cell.

15. The method of claim 10, wherein the exogenous nucleic acid molecule encodes at least one T cell receptor selected from: an alpha beta TCR; a gamma delta TCR; an invariant NKT (TARGET) TCR, a non-invariant NKT TCR, and a mucosal associated invariant TCR.

16. The method of claim 10, wherein the exogenous nucleic acid molecule encoding a T cell receptor comprises a promoter selected for its ability to resist silencing in the TARGET cells.

17. The method of claim 13, wherein the promoter is a human ubiquitin promoter.

18. The method of claim 10, wherein: the exogenous nucleic acid molecule is contained in a lentiviral expression vector; and / or the exogenous nucleic acid molecule further encodes a polypeptide that stimulates T cells, a polypeptide that disrupts T cell inhibitory factors, and / or a polypeptide comprising a further receptor.

19. The method of claim 18, wherein the polypeptide comprises at least one of a chimeric antigen receptor (CAR), IL-2, IL-7, IL-15, IFN-y, TNF-a, CD28, 4-1BB, 0X40, ICOS, and FOXP3.

20. The method of claim 11 wherein the genetic modifications are made prior to disposing PSC cells in a differentiation media.

21. The method of claim 11 wherein the genetic modifications are made after disposing PSC cells in a differentiation media.

22. A monoclonal TCR-Armed Gene-Engineered T (TARGET) cell made by the method of claim 11.

23. A monoclonal TCR-Armed Gene-Engineered T (TARGET) cell made by the method of claim 12.

24. The monoclonal target cell of claim 22, wherein the target cell expresses CD4.

25. The monoclonal target cell of claim 23, wherein the target cell expresses CD4.

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