Highly efficient and scalable production of engineered t cells from stem cells

Abstract

This disclosure provides methods for producing T cells with shortened ex vivo manufacturing time. In particular, this disclosure involves the production of T cells from hematopoietic stem cells with the proviso that the process does not involve subsequent in vitro steps of activation and / or expansion of the T cells.

Description

HIGHLY EFFICIENT AND SCALABLE PRODUCTION OF ENGINEERED T CELLSFROM STEM CELLSRELATED APPLICATIONSThis application claims the benefit of U.S. Provisional Application No. 63 / 575,368, filed April 5, 2024, and U.S. Provisional Application No. 63 / 725,783, filed November 27, 2024, each of which is incorporated by reference herein in its entirety.TECHNICAL FIELD

[0001] This disclosure relates to the production of T cells from stem cells.BACKGROUND

[0002] Allogeneic CAR T cell therapy has the potential to transform cancer treatment. The use of pre-made CAR T cells from donors makes cancer treatment immediately available to patients and provides opportunities for redosing or combined use of CAR T cells directed to multiple targets. As such, life-saving treatments are available at earlier stages of disease progression, which improves chances of remission.

[0003] Despite its potential, several key challenges remain for the commercial viability of allogeneic CAR T cells. Presently, manufacture of allogeneic CAR T cells involves lengthy ex- vivo cell culture procedures that are costly and subject to high product variability. Moreover, prolonged ex vivo culture is associated with phenotypic changes that arc poorly characterized and potentially detrimental to therapeutic efficacy. Further, clinical data show engineered T cells with tumor antigen-specific receptors are useful in some patients to cause regression of metastatic cancer. Unfortunately, not all patients benefit from such treatment. One explanation is that during in vitro expansion, some T cells become exhausted or senescent, which limits therapeutic efficacy and persistence in vivo.

[0004] Unless manufacturing processes are developed that reliably and economically produce T cells that are safe and effective, the potential of allogeneic CAR T cell therapy will never translate into clinical application.SUMMARY

[0005] This disclosure provides cost-effective systems and methods for quickly and efficiently producing large numbers of T cells using a shortened ex vivo manufacturing processes. In particular, this disclosure provides methods for producing engineered immune cells (e.g., engineered T cells) from stem cells (e.g., hematopoietic stem cells) using a “deep maturation” or “turbo deep maturation” step, which both include an immune cell “maturation II” step after which an immune cell activation step takes place. Methods for performing the “deep maturation” embodiments of the disclosure include, in order, the steps of: (i) transducing HSPCs; (ii) differentiation of HSPC into T cells; (iii) maturation; (iv) maturation II and (v) activation. As the name implies, the “turbo deep maturation” embodiments of the disclosure involve fewer steps than do the “deep maturation” embodiments. Methods for performing the “turbo deep maturation” embodiments of the disclosure include, in order, the steps of: (i) transducing HSPCs; (ii) differentiation of HSPC into T cells; (iii) maturation II (equivalent to maturation II from the deep turbo method above); and (iv) activation. Thus, the methods of the disclosure for producing engineered immune cells “turbo” embodiments are able to produce engineered immune cells without the two separate maturation steps.

[0006] Advantageously and surprisingly, the present inventors discovered that using the combined maturation Il / activation steps of the deep and turbo deep maturation methods of the disclosure produced engineered immune cells that exhibit, relative to methods that incorporate solely activation or maturation II: (a) improved cell viability; (b) improved cell purity; (c) improved expression of introduced immune cell receptors (e.g., introduced T cell receptors [TCRs] and / or chimeric antigen receptors [CARs]); increased expansion of the engineered immune cells; and (d) improvements in cell profile shown in increases in expression of CCR7, CXCR3, NKp44, and NKG2D. Furthermore, despite using both maturation II and activation steps, the presently disclosed deep maturation and turbo deep maturation methods of the disclosure, the methods are able to produce high numbers of engineered immune cells, with a high purity, within 30-40 days of initially transducing donor cells to produce the resulting engineered immune cells.

[0007] By ensuring the viable manufacture of the desire engineered immune cells within 30-40 days, the methods of the disclosure provide a substantial reduction in the duration of cell culture steps. As such, manufacturing processes of the disclosure produce engineered immune cells usingfewer costly consumables, including those that are generally required for maintenance of immune cells in culture. Moreover, by reducing the duration of ex vivo cell culture, methods of the disclosure minimize opportunities for phenotypic changes to occur during immune cell production. Thus, the presently disclosed systems and methods of the disclosure reduce batch-to-batch variability and improve therapeutic efficacy of the immune cells produced in accordance with the deep and turbo deep maturation methods of the disclosure. Accordingly, methods of the disclosure provide for a rapid and cost-effective approach for producing immune cells, including engineered T cells, which are phenotypically superior and thus more effective in clinical applications compared to those produced using prior methods.

[0008] Moreover, it was also found that the presently disclosed methods could be further improved by the addition of a P13K inhibitor (e.g., Duvelisib) and / or IL-21 to the cells during the manufacturing method, and particularly in the final 1-14 days of the method. Adding the P13K inhibitor and IL-21 was found to improve yields of harvestable cells, help create a pure final product, and improve potency of the harvested cells.

[0009] Methods of the disclosure are useful for the economic production of off-the-shelf CAR-T cell products. Conventional T cell methods of manufacture involve lengthy ex vivo T cell activation steps and culture. In general, T cell activation requires introduction of at least two stimulatory signals: a first signal (e.g., anti-CD3) for activation of the TCR complex, and a second signal (e.g., anti-CD28) for cell proliferation, differentiation, and survival. In prior methods for manufacturing engineered immune cells, following the lengthy ex vivo activation step(s), cells are cultured to proliferate their numbers, which has traditionally required weeks of costly reagents, laboratory space, and technician time for cell maintenance and growth. It is an insight of the disclosure that those prolonged ex vivo activation and expansion steps can be replaced using the deep maturation and turbo deep maturation methods of the disclosure that allow the entire process, from start to end product, to complete within 30-40 days. Advantageously, by omitting the prolonged ex vivo activation and expansion, T cells are subjected to significantly less culture time, which reduces costs associated with manufacture and produces higher-quality cell products, at a faster rate than other methods.

[0010] In addition, these methods are able to produce T cells with long-term cytotoxic efficacy. Prolonged cell culture has been associated with transcriptional and phenotypic changes of certain cell types. Although transcriptional and phenotypic changes of T cells in culture are poorlycharacterized, this disclosure recognizes that unintended changes of cells during prolonged culture may account for observed reductions in therapeutic efficacy and product batch variability. For example, prolonged cell culture of T cells may give rise to elevated levels of exhaustion markers, which reflect loss of effector function. By shortening ex vivo manufacture, methods of the disclosure are useful for consistent production of therapeutically effective T cells.

[0011] Further, the deep maturation and turbo deep maturation methods of the disclosure may be used in systems and methods for quickly and efficiently producing large numbers of T cells with at least two different T cell receptors (TCR) and / or chimeric antigen receptors (CAR). By producing T cells with multiple different TCRs (multi-TCR T cells TCR / CAR cells), methods of the invention are able to produce T cells that recognize multiple different antigens, thereby improving their efficacy as therapeutic treatments. Similarly, the invention provides methods for producing T cells that co-express different TCRs or TCRs and CARs, which provide the cells with optimal phenotypes that confer specific cancer cell targeting properties.

[0012] In particular embodiments, this disclosure provides methods of making multi-TCR or TCR and CAR T cells from stem cells, e.g., hematopoietic stem cells (HSC). By starting with HSCs, systems and methods of the invention leverage the self-regeneration and cellular differentiation capabilities of stem cells in manufacturing the T cells of the invention.

[0013] Advantages of using stem cells (e.g., HSCs) in the methods of the invention, include their abilities for regeneration and expansion. Allogeneic cell therapies often require billions of cells for a single dose of treatment. Due to a potentially limitless ability of stem cells for expansion, methods of the invention are well suited for producing high quality cellular products on a large scale and making those products rapidly available for treatment. Moreover, a hallmark of stem cells is their ability to differentiate into different cell types. In the context of this disclosure, the capacity for differentiation provides a cell manufacturing platform that may produce a broad array of T-cell subtypes, including, for example, natural killer T cells, alpha beta T cells, gamma delta T cells, among others.

[0014] In one aspect, this disclosure provides a method of producing engineered immune cells such as engineered T cells. An exemplary method involves conducting a process involving in vitro differentiation of a hematopoietic stem cells (HSC) into T cells expressing one or more introduced genes; inducing the engineered T cells; and activating the induced T cells. In certain turbo deep maturation embodiments of the disclosure, there is no maturation step prior to maturation II andactivation. Alternatively, in some deep maturation methods of the disclosure, after differentiation, the method further comprises a step of maturating the T cells prior to the inducing step.

[0015] In more preferred aspects of the disclosure, the method for producing engineered immune cells comprises contacting the T cells with a P13K inhibitor. In certain aspects, the T cells are contacted with the P13K inhibitor during the maturing step. In certain aspects, the T cells are contacted with the P13K inhibitor during the inducing step. In certain aspects, the T cells are contacted with the P13K inhibitor during the activating step. In certain aspects, the T cells are contacted with the P13K after the activating step. In preferred aspects, the T cells are contacted with duvelisib only during and / or after the activating step. In more preferred aspects, the T cells are contacted with duvelisib only during the activating step.

[0016] In preferred aspects, the method is performed in less than 40 days. In certain aspects, the method is performed in less than 38 days. In certain aspects, the method is performed in less than 37 days. In certain aspects, the method is performed in less than 36 days. In certain aspects, the T cells are contacted with the P13k inhibitor during the final 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day(s). In preferred aspects, the method includes a maturation process comprising contacting the T cells with duvelisib at around day 25 to around day 40 of the method. In preferred aspects, the method includes a maturation process comprising contacting the T cells with duvelisib at around day 27 to around day 38 of the method. In preferred aspects, the method includes a maturation process comprising contacting the T cells with duvelisib at around day 28 to around day 35 of the method.

[0017] In preferred aspects, the P13K inhibitor is selected from alpelisib (BYL719), Buparlisib (BKM120), Duvelisib, CH5132799 / PA-79, Copanlisib (BAY 80-6946), Idelalisib (GS-1101), Pictilisib (GDC-0941), Pilaralisib (XL-147), Serabelisib (MLN1117), Taselisib (GDC-0032), and Umbralisib. In more preferred aspects, the P13K inhibitor is Duvelisib. Alternatively or additionally to a P13K inhibitor, in some embodiments, the T cells are contacted with an inhibitor of Bruton's tyrosine kinase (BTK), e.g., Ibrutinib (PCI 32765).

[0018] In preferred embodiments, the T cells are contacted with IL-21 concurrent with the P13K inhibitor.

[0019] In preferred embodiments, methods of the disclosure involve an in vitro process as described herein that includes causing HSCs to express at least one TCR and / or one CAR. Expression of the at least one TCR and / or CAR confers certain advantageous therapeuticcapabilities to the cells. The stem cell is preferably an HSC, which may be derived from a progenitor cell. For example, the progenitor cell may be a pluripotent stem cell. In some instances, the HSC is obtained from a body fluid, such as amniotic fluid or umbilical cord fluid.

[0020] Methods of the disclosure may include differentiating stem cells into double negative progenitor T cells. Double negative progenitor T cells involve cells that generally lack expression of co-receptors CD4 and CD8. Methods may further include treating the double negative progenitor cells with a cocktail of cytokines and / or chemokines to produce CD4 positive CD 8 positive T cells.

[0021] Conventional culture practices are often complex and involve many handling steps that are sensitive to human error, compromising the overall reproducibility and effectiveness of T cell therapies. As described herein, the comparatively brief length of the disclosed methods substantially reduces the number of handling steps and reduces overall manufacturing time, which minimizes opportunities for human error. For example, whereas some conventional methods of manufacture require 6 or more weeks, methods of the disclosure produce T cells for clinical use in less than 40 days, less than 39 days, less than 38 days, less than 37 days, less than 36 days, or less than 5 weeks, for example, within 3 or 4 weeks. Accordingly, methods of the disclosure may produce cell products for off-the-shelf therapies in approximately half the amount of time as is currently required. The reduction in time provides substantial cost-savings benefits and, as discussed, may provide for less variability among cells between batches.

[0022] In some instances, administration of adoptive T cell therapies requires billions of T cells. Accordingly, large scale production of T cells by certain methods of the disclosure may involve expanding the T cells. Expanding T cells may involve treating the T cells with one or more cytokines, for example, one or more of IL-2, IL-7, IL- 12, IL- 15, IL-21, or IL- 18, or any combination thereof. For example, the media may contain a combination of IL-7 / 15. In some embodiments, the media contains only IL- 15. In certain aspects, the engineered cells have one or more introduced transgenes. In certain aspects, one or more of the introduced transgenes comprise a cytokine, such as IL- 15. In such methods, the media may not require the addition of IL- 15 due to the expression of the cytokine by an introduced gene.

[0023] Methods of the disclosure are useful for producing allogeneic therapies that are safe and effective. In some instances, methods may involve characterizing cell products at one or more points during manufacture to ensure product quality. In some embodiments, methods of thedisclosure involve analyzing T cells to identify one or more proteins expressed by the T cells. The one or more proteins may include one or more CCR7, CD62L, CD45RA, CXCR3, CD56, NKp44, and NKG2D. The proteins may include markers associated with naive stem cells. Analyzing preferably includes high throughput methods of analyzing cell surface proteins, e.g., methods based on fluorescent signals of individual cells in bulk, such as, FACS.

[0024] On demand availability of treatment is one benefit of allogeneic cell therapies. Since methods may involve manufacture of cells before clinical application, some preferred methods may include cryopreserving T cells. Cryopreserving T cells is useful for safe and effective storage of cells until they are needed by a patient. Cryopreserving is also useful for transportation of cell products to clinical facilities where they can be administered to patients.

[0025] This disclosure provides a platform useful for economical production of therapeutic T cells from stem cells. The platform may be used to produce any type of T cell. Preferably, however, the T cell is an invariant natural killer T (iNKT) cell, which are associated with reduced instances of graft versus host disease (GVHD). The iNKT cell may comprise an alpha / beta iNKT cell, or gamma / delta iNKT cell. In some instances, the cells are programmed to include one or more transgenes in addition to a TCR and / or CAR. The one or more additional transgenes may involve at least one of a cytokine, a checkpoint inhibitor, an inhibitor of transforming growth factor beta signaling, an inhibitor of cytokine release syndrome, or an inhibitor of neurotoxicity. The cytokine can comprise one of IL-2, IL-7, IL- 15, IL- 12, IL- 18, or IL-21.

[0026] In another aspect, this disclosure provides a method for producing a T cell product using a single activation step. The method involves conducting a process comprising in vitro differentiation, maturation and maturation II of an HSC into a T cell with no more than one in vitro T cell activation step and providing the T cell for use in a treatment or research application. Conventional methods of T cell manufacture require at least two separate in vitro activation steps, which can take up to 2-4 weeks of ex vivo cell culture time. Advantageously, producing a T cell product using a single activation step reduces time of cell culture and results in a T cell that presents fewer markers associated with cell exhaustion.

[0027] Accordingly, some embodiments may involve manufacturing a T cell with a single activation step following a maturation II step. The single activation step can be performed by culturing the T cell in activation media, for example, media comprising T cell activation reagentssuch as antibodies. Preferably, the single activation step does not involve changing types of activation reagents during the activation process. The T cell activation step can last 7 days or less.

[0028] In some instances, the single activation step involves a peripheral blood mononuclear cell (PBMC) based T cell activation. Accordingly, the activation step may involve a cell culture of alpha-galactosylceramide (aGC)-loaded PBMCs, soluble anti-CD3 / 28 positive PBMCs, and soluble anti-CD2 / 3 / 28 positive PBMCs. In some instances, the activation step involves an antigen presenting cell (aAPC) based T cell activation step. Accordingly, the activation step can involve aAPCs. The aAPCs are preferably irradiated. The aAPC may be an engineered K562 cell expressing CD80-CD83-CD137L-CAR-antigen. The aAPC may be an aAPC+CDld, and / or aAPC+CDld+ / -aGC. In other instances, the activation step comprises a feeder free-based T cell activation step. The feeder free based T cell activation step can involve introducing soluble activation antibodies, e.g., anti-CD3, anti-CD28, anti-CD2 / 3 / 28, CD3 / 28. In some embodiments, the method involves a culture media comprising one or more of IL-7 / 15, IL-2, IL-2+21, IL-12, or IL-18.

[0029] In more preferred aspects of the disclosure using a single activation step, the method for producing engineered immune cells also comprises contacting the T cells with a P13K inhibitor. In certain aspects, the T cells are contacted with the P13K inhibitor during the maturing step. In certain aspects, the T cells are contacted with the P13K inhibitor during the inducing step. In certain aspects, the T cells are contacted with the P13K inhibitor during the activating step. In certain aspects, the T cells are contacted with the P13K after the activating step. In preferred aspects, the T cells are contacted with duvelisib only during and / or after the activating step. In more preferred aspects, the T cells are contacted with duvelisib only during the activating step.

[0030] In preferred aspects, the method is performed in less than 40 days. In certain aspects, the method is performed in less than 38 days. In certain aspects, the method is performed in less than 37 days. In certain aspects, the method is performed in less than 36 days. In certain aspects, the T cells are contacted with the P13k inhibitor during the final 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day(s). In preferred aspects, the method includes a maturation process comprising contacting the T cells with duvelisib at around day 25 to around day 40 of the method. In preferred aspects, the method includes a maturation process comprising contacting the T cells with duvelisib at around day 27 to around day 38 of the method. In preferred aspects, the method includes amaturation process comprising contacting the T cells with duvelisib at around day 28 to around day 35 of the method.

[0031] In preferred aspects, the P13K inhibitor is selected from alpelisib (BYL719), Buparlisib (BKM120), Duvelisib, CH5132799 / PA-79, Copanlisib (BAY 80-6946), Idelalisib (GS-1101), Pictilisib (GDC-0941), Pilaralisib (XL-147), Serabelisib (MLN1117), Taselisib (GDC-0032), and Umbralisib. In more preferred aspects, the P13K inhibitor is Duvelisib. Alternatively or additionally to a P13K inhibitor, in some embodiments, the T cells are contacted with an inhibitor of Bruton's tyrosine kinase (BTK), e.g., Ibrutinib (PCI 32765).

[0032] In preferred embodiments, the T cells are contacted with IL-21 concurrent with the P13K inhibitor.BRIEF DESCRIPTION OF DRAWINGS

[0033] FIG. 1 diagrams a generalized method for producing T cells using the deep maturation and turbo deep maturation methods of the disclosure.

[0034] FIG. 2 illustrates stages of ex vivo T cell manufacturing without the combination of maturation II and activation of the deep maturation and turbo deep maturation methods of the disclosure.

[0035] FIGS. 3A-3B provided an overview of a scalable process for producing T cells in accordance with the disclosure and producing a finished, cryopreserved process.

[0036] FIGS. 4A-4B show total viable cell counts at thaw and at 2-, 16-, 23-, and 30-days postthaw for engineered T cells made using methods of the disclosure.

[0037] FIG. 5 shows total viable cell (TVC) count, viability, and diameter of cells at week 5 using methods of the disclosure.

[0038] FIG. 6 shows harvest TVC, harvest wash recovery TVC, and number of vials that could be produced from harvest using methods of the disclosure.

[0039] FIG. 7 shows the relative percentages of TCR+, CAR+, and TCR+ / CAR+ cells in a sample made using methods of the disclosure.

[0040] FIG. 8 shows the vector copy number for cells made using methods of the disclosure.

[0041] FIGS. 9A-9B show potency, short term and long-term cytotoxicity data for cells made using the methods of the disclosure.

[0042] FIG. 10 provides certain quality information of final cellular products made using methods of the disclosure.DETAILED DESCRIPTION

[0043] Clinical studies of chimeric antigen receptor (CAR) T cells show remarkable results in treatment of certain pathologies, such as, B-cell malignancies. Presently, however, commercial methods involving CAR T cell therapy involve autologous CAR T cells whose widespread use is limited by logistics and high costs associated with ad hoc generation. Allogeneic CAR T cell therapy address limitations of autologous cells by providing for pre-made cell stocks that are immediately available for patient treatment. Yet, despite its potential, methods for consistent production of therapeutically effective allogeneic CAR T cells have not been established. Most protocols, for example, require long periods of ex vivo culture, with at least two activation steps, which potentially leads to over differentiation, T cell exhaustion, and / or cellular senescence, undermining in vivo efficacy. See, Jafarzadeh, 2020, Prolonged Persistence of Chimeric Antigen Receptor (CAR) T Cell in Adoptive Cancer Immunotherapy: Challenges and Ways Forward, Frontiers in Immunology, 11(702): 1- 17, incorporated by reference.

[0044] This disclosure provides reliable methods for manufacturing T cells with improved phenotype and cellular function. The present disclosure includes methods for manufacturing T cells using a shortened ex vivo culture time, relative to existing methods. Thus, this disclosure provides cost-effective systems and methods for quickly and efficiently producing large numbers of T cells. In particular, this disclosure provides methods for producing engineered immune cells (e.g., engineered T cells) from stem cells (e.g., hematopoietic stem cells) using a “deep maturation” or “turbo deep maturation” step, which both include an immune cell maturation II step after which an immune cell activation step takes place.

[0045] Advantageously and surprisingly, the present inventors discovered that using the combined maturation II / activation steps of in the deep and turbo deep methods of the disclosure produced engineered immune cells that exhibit, relative to methods that incorporate solely activation or maturation: (a) improved cell viability; (b) improved cell purity; (c) improved expression of introduced immune cell receptors (e.g., introduced T cell receptors [TCRs] and / or chimeric antigen receptors [CARs]); increased expansion of the engineered immune cells; and (d) improvements in cell profile shown in increases in expression of CCR7, CXCR3, NKp44, and NKG2D.Furthermore, despite using both maturation TI and activation steps, the presently disclosed deep maturation and turbo deep maturation methods of the disclosure, the methods arc able to produce high numbers of engineered immune cells, with a high purity, within 30-40 days of initially transducing donor cells to produce the resulting engineered immune cells.

[0046] Certain methods of the disclosure reduce ex vivo culture by replacing prolonged ex vivo activation steps of prior methods, with a maturation II step followed by an activation step, which reduces the overall ex vivo manufacturing processes by up to 2-3 weeks. Methods for performing the “deep maturation” embodiments of the disclosure include, in order, the steps of: (i) transducing HSPCs; (ii) differentiation of HSPC into T cells; (iii) maturation; (iv) maturation II; and (v) activation. As the name implies, the “turbo deep maturation” embodiments of the disclosure involve fewer steps than do the “deep maturation” embodiments. Methods for performing the “turbo deep maturation” embodiments of the disclosure include, in order, the steps of: (i) transducing HSPCs; (ii) differentiation of HSPC into T cells; (iii) maturation II (equivalent to maturation II step above); and (iv) activation. Thus, the methods of the disclosure for producing engineered immune cells “turbo” embodiments are able to produce engineered immune cells without the separate maturation step.

[0047] Both variations of these methods of the disclosure recognize that the lengthy activation and / or expansion processes can occur in vivo, after administration to a subject. By shortening ex vivo cell culture, methods of the disclosure minimize opportunities for transcriptional and / or phenotypic changes to occur during T cell production, thereby reducing cell-to-cell variability. Furthermore, shortening ex vivo culture time reduces the amount of costly cell culture consumables that are needed for T cell maintenance.

[0048] The present disclosure also includes multi-step methods or workflows for making a T cell product with using the deep maturation and turbo deep maturation concepts described herein. Surprisingly, these methods / workflows may include conducting a process comprising in vitro differentiation, maturation, and activation of an HSC into a T cell with no more than one in vitro T cell activation step. The resulting T cell product may be used for a treatment or for research.

[0049] Conventional methods of T cell manufacture require at least two separate in vitro T cell activation steps and do not incorporate a maturation step followed by an activation step, as with the presently disclosed methods. The multiple activation steps of prior methods generally involve multiple media types, i.e., different medias containing different activation factors. Methods of thedisclosure may be used to produce T cell products with less in vitro cell culture(s) or culture time. By reducing in vitro cell culture, the methods of the disclosure produce T cell products faster and that are more effective and contain fewer exhausted / dysfunctional T cells.

[0050] Certain embodiments involve a multi-step process for producing a T cell wherein only one of the steps is a T cell activation step. The single activation step may be performed, for example, by culturing the T cell in activation media, e.g., media comprising T cell activation reagents such as antibodies.

[0051] In certain methods of the disclosure, the activation step includes a peripheral blood mononuclear cell (PMBC) based activation. PMBC-based activation may involve introducing the T cell to alpha-galactosylceramide (aGC)-loaded PBMCs, anti-CD3 / 28 antibodies or beads and PBMCs, and anti-CD2 / 3 / 28 antibodies or beads and PBMCs.

[0052] In certain aspects, the activation step includes the use of an antigen presenting cell (aAPC) based T cell activation step. Accordingly, the activation step may include introducing the T cell to aAPCs. Preferably, when used, the aAPCs are irradiated. In preferred aspects, the aAPC may be an engineered K562 cell expressing CD80-CD83-CD137L-CAR-antigen. The aAPC may be an aAPC+CDld, and / or aAPC+CDld+ / -aGC.

[0053] In certain aspects, the activation step comprises a feeder free -based T cell activation step. The feeder free based T cell activation step may include introducing, to the T cell, soluble antibodies including anti-CD3, anti-CD28, anti-CD2 / 3 / 28, CD3 / 28. In some embodiments, the method uses a culture media comprising one or more of IL-7 / 15, IL-2, IL-2+21, IL-12, or IL-18. In some embodiments, the media does not contain exogenous IL- 15. In certain aspects, engineered immune cells of the disclosure are modified to express one or more cytokine. In preferred aspects, the engineered immune cells of the disclosure are modified to express IL-15.

[0054] The presently disclosed methods may be further improved by the addition of a P13K inhibitor (e.g., Duvelisib) and / or IL-21 to the cells during the manufacturing method, and particularly in the final 1-14 days of the method. Adding the P13K inhibitor and IL-21 was found to improve yields of harvestable cells, help create a pure final product, and improve potency of the harvested cells.

[0055] Thus, preferred aspects of the disclosure, include adding to the methods for producing engineered immune cells herein, a step of contacting the T cells with a P13K inhibitor. In certain aspects, the T cells are contacted with the P13K inhibitor during the maturing step. In certainaspects, the T cells are contacted with the P13K inhibitor during the inducing step. In certain aspects, the T cells arc contacted with the P13K inhibitor during the activating step. In certain aspects, the T cells are contacted with the P13K after the activating step. In pre I erred aspects, the T cells are contacted with duvelisib only during and / or after the activating step. In more preferred aspects, the T cells are contacted with duvelisib only during the activating step.

[0056] In preferred aspects, the method is performed in less than 40 days. In certain aspects, the method is performed in less than 38 days. In certain aspects, the method is performed in less than 37 days. In certain aspects, the method is performed in less than 36 days. In certain aspects, the T cells are contacted with the P13k inhibitor during the final 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day(s).

[0057] In preferred aspects, the P13K inhibitor is selected from alpelisib (BYL719), Buparlisib (BKM120), Duvelisib, CH5132799 / PA-79, Copanlisib (BAY 80-6946), Idelalisib (GS-1101), Pictilisib (GDC-0941), Pilaralisib (XL-147), Serabelisib (MLN1117), Taselisib (GDC-0032), and Umbralisib. In more preferred aspects, the P13K inhibitor is Duvelisib. Alternatively or additionally to a P13K inhibitor, in some embodiments, the T cells are contacted with an inhibitor of Bruton's tyrosine kinase (BTK), e.g., Ibrutinib (PCI 32765).

[0058] In preferred embodiments, the T cells are contacted with IL-21 concurrent with the P13K inhibitor.

[0059] Methods of the disclosure may be used to produce T cells with enhanced anti-tumor activities. This disclosure provides systems and methods for producing T cells from a stem cells (e.g., HSCs) incorporated with multiple transgenes including one or more TCRs, CARs, and / or at least one additional transgene. By initiating a production process from stem cells, systems and methods of the disclosure take advantage of self-renewal and cellular differentiation capabilities for manufacture of T cells with “younger” phenotypes and enhanced anti-tumor activities. In particular, this disclosure provides for introduction of nucleic acids, into CD34 positive stem cells, which encode for at least one TCR, CAR, and at least one an additional transgene. The combined expression of TCRs and CARs and the additional transgene is useful for providing T cells with specific cancer cell targeting properties useful to treat the cancer.

[0060] FIG. 1 diagrams a method 101 for producing T cells. In particular, illustrated is a simple flow diagram to provide a general overview of methods for producing T cells according to aspects of the disclosure. The method 101 includes obtaining 105 stem cells (e.g., CD34+ hematopoieticstem / progenitor cells); introducing 109 into the stem cells one or more nucleic acids (e.g., encoding TCRs, CARs, and additional transgcncs); conducting 111 an in vitro differentiation of the stem cells to produce T cells and in the deep maturation methods (as opposed to turbo deep maturation methods) a step of maturing the T cells; inducing 113 the T cells; and activating 115 the T cells. The resulting engineered cells may be used, for example, in allogeneic therapy or research.

[0061] The method 101 involves obtaining 105 stem cells. Preferably, the cells are CD34+ cells. In one non-limiting example the CD34+ stem cells are hemopoietic stem / progenitor cells. Hematopoietic stem or progenitor cells are stem cells that give rise to other blood cells in a process referred to as hematopoiesis.

[0062] The hematopoietic stem / progenitor cells may be obtained from a healthy donor. The hematopoietic stem / progenitor cells may be obtained from, for example, bone marrow, peripheral blood, amniotic fluid, or umbilical cord blood. The hematopoietic stem / progenitor cell may be obtained from umbilical cord blood by clamping ends of an umbilical cord and aspirating blood from between the clamped ends with a needle. The hematopoietic stem / progenitor cells may be isolated from cord blood using positive immunomagnetic separation techniques, and citrate- phosphate-dextrose (CPD) may be added to the cord blood as an anticoagulant. The cells from the cord blood may be cryopreserved and stored at a temperature of, for example, - 150 degrees Celsius until use.

[0063] In practicing methods of the disclosure, obtaining 105 the stem cells preferably includes receiving a vial of cryopreserved CD34+ cord blood cells including hemopoietic stem / progenitor cells. The vial of cryopreserved cord blood cells may be received from a cell bank in an insulated container on dry ice, for example.

[0064] The vial of cryopreserved cord blood cells may be thawed according to methods known in the art. For example, the vial of cells may be thawed by placing the vial into a 37-degree water bath for approximately 1 to 2 minutes or in a thawing device such as a Plasmatherm thawer. In certain aspects, thawing is completed when only an ice chunk remains in the CD34+ cells. In some preferred embodiments, once the cells are thawed, the cells are transferred to tissue culture dishes / plates / vials that are pre-coated with a reagent that promotes colocalization of a virus with target cells to enhance transduction efficiency.

[0065] The CD34+ cells may be incubated in a cell culture bag (e.g., a PL30 bag), for example, at about lc5 cells to about lc6 cells per bag, or more. Preferably, the cells arc cultured at lc6 cells per bag.

[0066] The method 101 further includes introducing 109, into the CD34+ stem cells, one or more nucleic acids encoding for one or more of a TCR, a CAR, and / or an additional transgene. Preferably, the one or more nucleic acids encode at least two chains of a TCR, a CAR, or an additional transgene. For example, in some embodiments, the one or more nucleic acids introduced 109 into stem cells encode at least a TCR and a CAR, to produce T cells capable of targeting a specific protein expressed on a surface of cancer cells. In other embodiments, the one or more nucleic acids introduced 109 into the stem cells encode for each chain of a TCR, a CAR, and an additional transgene.

[0067] In preferred embodiments, the TCR introduced by way of nucleic acid is an iNKT TCR. The iNKT TCR may include one of an alpha chain of an iNKT cell receptor, a beta chain of an iNKT cell receptor, or both. Preferably, the iNKT cell receptor is expressed by the stem cells such that the stem cells recognizes alpha-galactosylceramide. In addition, preferred embodiments may include introducing nucleic acids encoding at least one CAR. The CAR, as discussed below, may be of a first generation, a second generation, or a third generation CAR. The CAR may provide cells with a receptor specific to an antigen associated with cancer. For example, in some embodiments the antigen comprises one of Mesothelin, Glypican 3, CD19, or BCMA.

[0068] T cells produced by methods of the disclosure may be genetically modified to express at least one additional transgene. The transgene may be, for example, one of a cytokine, a checkpoint inhibitor, an inhibitor of transforming growth factor beta signaling, an inhibitor of cytokine release syndrome, or an inhibitor of neurotoxicity. Accordingly, methods of the disclosure may be useful to produce T cell with enhanced effector function.

[0069] For example, in some instances, methods of the disclosure are useful for the manufacture of CAR T cells with improved expansion and persistence capabilities, which is provided by introduction of transgenes encoding one or more of IL-2, IL-7, IL- 15. In some instances, methods may provide CAR T cells with increased IFN-g production and thus improved T cell potency by, for example, introduction of transgenes encoding one or more of IL- 12, IL- 18. In some instances, methods of the disclosure are useful for enhancing naive T cell production by introducing transgenes including IL-21. In some instances, methods described herein provide for theproduction of CAR T cells with improved safety properties by, for example, introducing inhibitors of IL-6, GM-CSF, or other mediators of cytokine release syndrome and neurotoxicity. Methods may provide for CAR T cells with improved efficacy by providing payloads useful for combating tumor microenvironment, e.g., via inhibitors of TGF-B, checkpoints.

[0070] Introducing 109 the one or more nucleic acids into the stem cells may be accomplished by viral transduction method or a non-viral transfection. In some instances, for example, methods for introducing the one or more nucleic acids involve non-viral methods, for example, using a Sleeping Beauty transposon / transposase system. The Sleeping Beauty transposon system involves a synthetic DNA transposon designed to introduce precisely defined DNA sequences into the chromosomes of cells. The system uses a Tcl / mariner-type system, with the transposase resurrected from multiple inactive fish sequences. Advantageously, non-viral methods may provide for cost-savings benefit and reduce risks associated with use of certain virus. However, non-viral methods may be associated with reduced efficiency. As such, preferred embodiments introduce nucleic acids into stem cells by viral transduction, e.g., via a retrovirus.

[0071] Viral transduction methods are well recognized for their versatility and involve the use of lentiviral vectors, which are useful to transduce both dividing and nondividing cells with significant amounts of nucleic acid. The use of lentiviral vectors is considered safe and often provides long-term transgene expression. Accordingly, the method 101 preferably introduces 109 the one or more nucleic acids into the CD34+ stem cells via a lentiviral transduction. For discussion on lentiviral transduction of stem cells, see lang, 2020, Optimizing lentiviral vector transduction of hematopoietic stem cells for gene therapy, Gene Therapy (27): 545-556, which is incorporated by reference. In some embodiments, an expression vector (e.g., a lentiviral vector) may be used to introduce the two nucleic acids encoding the TCR(s) / CAR(s) / additional transgenes.

[0072] A “vector” is a composition of matter that includes one or more isolated nucleic acid and is used to deliver the isolated nucleic acid(s) to the interior of a cell, where introduced nucleic acids may be expressed by the cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” includes an autonomously replicating plasmid or a virus. The term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like.

[0073] Certain viral vectors are able to autonomously replicate in a host cell into which they are introduced. In alternative aspects, the vectors integrate into the genome of a host cell upon introduction into the host cell, and are thus replicated along with the genome of the host. Viral vectors used in the disclosure may include one or more sequences that direct expression of introduced transgenes genes to which they are operatively linked, i.e., expression vectors.

[0074] A variety of delivery vectors and expression vehicles may be used to introduce into cells the nucleic acids encoding the endogenous gene products as described herein. Exemplary such vectors comprise lentiviral vectors, oncoretro viral vectors, expression plasmids, adenoviral vectors, adeno-associated virus vectors, herpes simplex virus vectors, transposons, vaccinia virus vectors, human papilloma virus vectors, simian immunodeficiency virus vectors, HTLV, human foamy virus vectors, spumavirus vectors, mammalian type B retrovirus vectors, mammalian type C retrovirus vectors, avian type C retrovirus vectors, mammalian type D retrovirus vectors, and / or variants and combinations thereof.

[0075] In preferred aspects, introduced nucleic acids encoding one or more TCR / CAR / transgene are delivered by a viral vector that is a lentiviral vector. Lentiviral vectors (LVs), a subset of retroviruses, are known and modified to transduce a wide range of dividing and non-dividing cell types with high efficiency, conferring stable, long-term expression of the gene products of the introduced nucleic acids.

[0076] Methods of the disclosure are not limited by any one process or laboratory procedure for introducing nucleic acids into stem cells. In some instances, a single lentiviral vector is used. The single lentiviral vector may encode each of a TCR, a CAR, and an additional transgene. In other instances, at least two distinct lentiviral vectors are used, wherein each one of the at least two lentiviral vectors encode at least one of a TCR, a CAR, and an additional transgene, such that, upon transduction, each of the TCR, the CAR, and the additional transgene are transduced into the stem cells. Moreover, in instances wherein more than one lentiviral vector is used, the method 101 is not limited by the temporal sequence of introducing the two (or more) lentiviral vectors the stem cells. The vectors may be introduced concurrently, in the same transduction, or sequentially.

[0077] In some embodiments, nucleic acids or vectors comprising the nucleic acid(s) used to introduce the TCR / CAR / transgenes into the engineered immune cells of the disclosure mayinclude one or more transcriptional control element, including, but not limited to, promoters and enhancer elements.

[0078] Suitable promoters include, but are not limited to: lad; lacZ; T3; T7; gpt; lambda P; tre; light and / or heavy chain immunoglobulin gene promoters and enhancer elements; cytomegalovirus immediate early promoter; herpes simplex virus thymidine kinase promoter; early and late SV40 promoters; promoter present in long terminal repeats from a retrovirus; mouse metallothionein-I promoter; and various art-known tissue specific promoters. Suitable reversible promoters, including reversible inducible promoters may also be used. Reversible promoters, and systems based on reversible promoters may include additional control proteins, and include for example: alcohol regulated promoters (e.g., alcohol dehydrogenase I (ale A) gene promoter, promoters responsive to alcohol transactivator proteins (AlcR), etc.), tetracycline regulated promoters, (e.g., promoter systems including TetActivators, TetON, TetOFF, etc.), steroid regulated promoters (e.g., rat glucocorticoid receptor promoter systems, human estrogen receptor promoter systems, retinoid promoter systems, thyroid promoter systems, ecdysone promoter systems, mifepristone promoter systems, etc.), metal regulated promoters (e.g., metallothionein promoter systems, etc.), pathogenesis-related regulated promoters (e.g., salicylic acid regulated promoters, ethylene regulated promoters, benzothiadiazole regulated promoters, etc.), temperature regulated promoters (e.g., heat shock inducible promoters (e.g., HSP- 70, HSP-90, soybean heat shock promoter, etc.), light regulated promoters, synthetic inducible promoters, and the like. In certain aspects, one or more of the promoters is a CD8 cellspecific promoter, a CD4 cell-specific promoter, a neutrophil-specific promoter, or an NK- specific promoter.

[0079] Suitable promoters may include one or more of a bacteriophage T7 RNA polymerase promoter; a trp promoter; a lac operon promoter; a hybrid promoter, e.g., a lac / tac hybrid promoter, a tac / trc hybrid promoter, a trp / lac promoter, a T7 / lac promoter; a tre promoter; a tac promoter, and the like; an araBAD promoter; in vivo regulated promoters, such as an ssaG promoter or a related promoter, a pagC promoter, a nirB promoter; a sigma70 promoter, e.g., a consensus sigma70 promoter; a stationary phase promoter, a promoter derived from the pathogenicity island SPI-2; an actA promoter; an rpsM promoter; a tet promoter; an SP6 promoter; lactose promoter operator (Lad repressor protein changes conformation when contacted with lactose, thereby preventing the Lad repressor protein from binding to the operator), atryptophan promoter operator (when complexed with tryptophan, TrpR repressor protein has a conformation that binds the operator; in the absence of tryptophan, the TrpR repressor protein has a conformation that does not bind to the operator), and a tac promoter operator.

[0080] Other examples of suitable promoters include the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto. Other constitutive promoter sequences may also be used, including, but not limited to a simian virus 40 (SV40) early promoter, a mouse mammary tumor virus (MMTV) or human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, a MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma vims promoter, the EF-1 alpha promoter, as well as human gene promoters such as, but not limited to, an actin promoter, a myosin promoter, a hemoglobin promoter, and a creatine kinase promoter. Inducible promoters may also be used. Examples of inducible promoters include metallothionine promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0081] In some embodiments, a nucleic acid of the present disclosure further comprises a nucleic acid sequence encoding a TCR / CAR / transgene as part inducible expression cassette. In one embodiment, the inducible expression cassette is for the production of a transgenic polypeptide product that is released upon TCR and / or CAR signaling. In some embodiments, the nucleic acid o further comprises a nucleic acid sequence encoding a cytokine operably linked to a T-cell activation responsive promoter.

[0082] In embodiments of the invention, a TCR / CAR may be provided in combination with a regulatory element capable of modulating the immune activity of a host cell expressing the CAR. For example, the regulatory element may act as an “on” or “off’ switch targeted and directed killing activity of the engineered cells.

[0083] In certain aspects, the regulatory element downregulates the activity of the cell. For example, the regulatory element may kill a cell expressing an introduced immune cell receptor. In this regard, the regulatory element is a suicide gene. In an embodiment of the invention, the regulatory element is an inducible dimerization kill switch. An example of an inducible dimerization kill switch is the IC9 suicide gene. Another example of an inducible dimerization kill switch is an element which provides for small-molecule-induced dimerization of the intracellularsignaling domain of Fas, which induces apoptosis via a caspase-8-dependent pathway. This approach may be used to induce apoptosis using a small molecule made by fusing two molecules of the drug calcineurin or the FKBP / AP1903 dimerizer system.

[0084] In an embodiment of the invention, the regulatory element is a cell surface marker. In certain aspects, cell surface markers may be used to deplete CAR-positive cells in vivo.

[0085] In an embodiment of the invention, the regulatory element upregulates the anti-cytotoxic activity of the host cell, e.g., an “on” switch to control expression or activity of an introduced immune receptor (e.g., a TCR / CAR). For example, the regulatory element may be an element conferring dependence on a ligand for cell survival, expansion, or activity. An example of such an element may be a drug-responsive, ribozyme-based regulatory device linked to growth cytokine targets to control cell (e.g., T cell) proliferation. Another example may be to design the antigenbinding and intracellular signaling components of the CAR to assemble only in the presence of a heterodimerizing small molecule.

[0086] Regulatory elements may alternatively or additionally include those that control the location of transgene integration or a genetic deletion which produces an auxotrophic cell (e.g., T cell).

[0087] As used herein, the term “suicide gene” refers to a gene that causes the cell expressing the suicide gene to die. The suicide gene can be a gene that confers sensitivity to an agent, e.g., a drug, upon the cell in which the gene is expressed, and causes the cell to die when the cell is contacted with or exposed to the agent. Suicide genes are known in the art and include, for example, the Herpes Simplex Virus (HSV) thymidine kinase (TK) gene, cytosine deaminase, inducible caspase 9 (IC9) gene, purine nucleoside phosphorylase, and nitroreductase.

[0088] The suicide gene may be the IC9 gene. The product of the IC9 gene contains part of the proapoptotic protein human caspase 9 (“caspase 9 component”) fused to a binding domain derived from human FK-506 binding protein (FKBP12 component). Activation of the caspase 9 domain of IC9 is dependent on dimerization of IC9 proteins that occurs when a small molecule drug, rimiducid (AP1903), binds to the FKBP12 moiety of IC9. After caspase 9 is activated, the cells carrying the IC9 gene undergo apoptosis.

[0089] The two nucleic acids used to produce the cells of the disclosure may include a nucleotide sequence encoding any of the leader domains, hinge domains, antigen binding domains, cleavage sequences, TM domains, and intracellular T cell signaling domains described herein. Accordingly,an embodiment of the invention provides one or more nucleic acid comprising a nucleotide sequence encoding a cleavage sequence disposed between sequences that encode different immune cell receptors (TCR(s) / CAR(s)).

[0090] In an embodiment, the cleavable cleavage sequence comprises a self-cleaving sequence. In an embodiment, the self-cleaving sequence is a self-cleaving 2A peptide. 2A peptides are viral oligopeptides, which mediate polypeptide cleavage during translation in eukaryotic cells. “2A” refers to a specific region of the viral genome. The mechanism of 2A-mediated self-cleavage is ribosome skipping of the formation of a glycyl-prolyl peptide bond at the C-terminus of the 2A peptide. In certain aspects, the cleavable ribosomal skip sequence is a porcine teschovirus-1 2A (P2A) amino acid sequence, equine rhinitis A virus (E2A) amino acid sequence, thosea asigna vims 2A (T2A) amino acid sequence, or foot-and-mouth disease vims (F2A) amino acid sequence. In an embodiment of the invention, the ribosomal skip sequence is a 2A peptide amino acid sequence comprising, consisting, or consisting essentially of, the amino acid sequence of (F2A). In an embodiment, the cleavable cleavage sequence comprises an enzyme-cleavable sequence. In an embodiment, the enzyme-cleavable sequence is a furin-cleavable sequence.

[0091] In certain aspects, one or more nucleic acids comprises a cleavage sequence that includes an enzyme-cleavable sequence and a self-cleaving sequence. For example, the cleavage sequence may comprise an enzyme-cleavable sequence (e.g., a furin cleavable sequence), a spacer, and a self-cleaving sequence (e.g., F2A).

[0092] The present disclosure also provides nucleic acid constructs, which are inserted within expression vectors and / or cloning vectors. An expression vector can include a selectable marker, an origin of replication, and other features that provide for replication and / or maintenance of the vector. Suitable expression vectors include, e.g., plasmids, viral vectors, and the like. Large numbers of suitable vectors and promoters are known to those of skill in the art; many are commercially available for generating a subject recombinant construct. Exemplary vectors include - prokaryotic: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a; pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5; Eukaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG) pSVK3, pBPV, pMSG and pSVL.

[0093] Expression vectors may include restriction sites located near a promoter sequence to provide for the insertion of nucleic acid sequences encoding heterologous proteins. A selectablemarker operative in the expression host may be present. Tn preferred aspects, the two nucleic acids arc inserted into viral vectors.

[0094] In certain embodiments, the one or more nucleic acids and / or vectors used to introduce the one or more nucleic acids comprises one or more additional elements, e.g., one or more elements selected from the following: 5' LTR, 3'LTR, cPPT, CTS, RRE, enhancer sequences, and packaging signals.

[0095] In some embodiments, the nucleic acids and / or vectors comprising said nucleic acids (e.g., lentiviral vectors) further comprises a non-requisite cis acting sequence that may improve titers and gene expression. A non-limiting example of a non-requisite cis acting sequence is the central polypurine tract and central termination sequence (cPPT / CTS), which has been shown to provide efficient reverse transcription and nuclear import. Other non-requisite cis acting sequences are known to those of skill in the art and may be incorporated into the nucleic acid(s) and / or vector(s) used to produce the 19CAR / 20CAR iNKT cells of the disclosure. The cPPT, which facilitates nuclear translocation of the pre-integration complexes, together with the CTS involved in the separation of reverse transcriptase, has been seen to improve viral titer (Zennou, et al. 2000; Follenzi et al. 2000). In particular embodiments of any of the two nucleic acids and / or vectors described herein, comprise the central polypurine tract and central termination sequence of HIV- 1 (cPPT / CTS)

[0096] In some embodiments, the nucleic acids and / or vectors comprising said nucleic acids (e.g., lentiviral vectors) further comprise one or more posttranscriptional regulatory elements, posttranscriptional regulatory elements, may for example, improve RNA translation and transgene expression and stabilize RNA transcripts. One example of a posttranscriptional regulatory element is the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). Accordingly, in some embodiments one or more of the two nucleic acids and / or vectors further comprises a WPRE sequence. WPRE significantly increases transgene expression in target cells, by increasing RNA stability in a transgene, promoter and vector-independent manner (Zuffrey et al, 1999). However, it can express a truncated 60- amino acid protein derived from the WHV X gene involved in liver cancer (Kingsman et al, 2005). Therefore, most pre-clinical protocols and clinical trials include a mutated version of the WPRE element (Zanta-Boussif et al, 2009). On the other hand, the use of two SV40-USE elements in SIN-LV vectors has been seen to be more efficient than the WPRE sequence in supressing transcriptional read through (Schambach et al, 2007). Moreprecisely, the WPRE disclosed herein is a chimeric wPRE that carries 589 nucleotides from the modified WPRE performed by Axel Schambach (nucleotides 1-589) (WO 2008136670 A2) and 88 from a former WPRE (nucleotide 590-677) (Zuffrey et al, 1999). Data disclosed herein shows this chimeric WPRE works better than the former wPRE

[0097] Various posttranscriptional regulator elements are known to those of skill in the art and may be incorporated.

[0098] A vector comprising the one or more nucleic acids may further comprise additional elements such as a rev response element (RRE) for RNA transport, packaging sequences, and 5' and 3' long terminal repeats (LTRs). The term “long terminal repeat” or “LTR” refers to domains of base pairs located at the ends of retroviral DNAs which comprise U3, R and U5 regions. LTRs generally provide functions required for the expression of retroviral genes (e.g., promotion, initiation and polyadenylation of gene transcripts) and to viral replication. In one embodiment, a vector (e.g., lentiviral vector) of the present invention includes a 3' U3 deleted LTR. Consequently, a self-inactivating vector may be capable of infecting and then integrating into a host genome (e.g., a mammalian genome) only once, and cannot be passed further. Accordingly, self-inactivating vectors may reduce the risk of creating a replication-competent virus. The RRE sequence improves the efficiency of gene transfer of any of the two nucleic acids and / or vectors comprising the nucleic acids disclosed herein.

[0099] The retroviral leader region contains the packaging signal (T), which is involved in packaging the retroviral genome into the viral capsid. LV vectors have been thought to require approximately 40-300 bp of the Gag gene in this region. In particular embodiments of any of the expression cassettes and gene therapy vectors described herein, the T sequence is an HIV-1 \| / sequence.

[0100] In order to assess the expression of transgenes introduced by the one or more nucleic acids, the nucleic acid(s) may encode either a selectable marker gene or a reporter gene, or both, to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. A selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers include, without limitation, antibiotic -resistance genes.

[0101] Reporter genes are used to identify potentially transfected cells and for evaluating the functionality of regulatory sequences. Suitable reporter genes may include, without limitation, genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene.

[0102] Returning to FIG. 1, as shown, the method 101 further involves conducting I ll a process comprising in vitro differentiation, and in some embodiments maturation 112, of the stem cell (e.g., HSC) into a T cell.

[0103] One advantage of the cell manufacturing method 101 lies in the ability to produce a broad array of T cell subtypes from a single starting material, i.e., stem cells. T cells produced by methods of the disclosure may include, for example, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, invariant natural killer T cells, alpha beta T cells, gamma delta T cells. In preferred embodiments, the method 101 involves producing invariant natural killer T cells. Production of invariant natural killer T (iNKT) cells are preferred for their allogeneic cell therapy applications. In particular, its ability to activate and expand antigen- specific T cell responses to treat cancer without inducing graft versus host disease.

[0104] Accordingly, the method 101 includes conducting 111 an in vitro differentiation and in certain methods, a maturation process, of stem cells (e.g., HSCs) into T cells (e.g., iNKT).

[0105] Differentiation of CD34 positive cells into T cells may occur in stages. A first stage may involve in vitro differentiation of CD34 positive stem cells into CD4 and CD8 double negative T cells. Differentiation of the CD34 positive stem cells generally involves introducing CD34 positive stem cells to a combination of cytokines and / or chemokines in culture, e.g., 1-2 weeks. In some instances, the cytokines and / or chemokines may be provided by commercially available progenitor expansion supplements, such as, the supplement sold under the trade name StemSpan by STEMCELL.

[0106] Embodiments of conducting 111 the in vitro process may involve maturation of CD4 and CD8 double negative T cells into CD 4 and CD 8 double positive cells. In some instances, maturating the double negative cells involves culturing the cells in a commercially available progenitor maturation medium, such as, the progenitor maturation medium provided under the trade name StemSpan by STEMCELL.

[0107] According to certain embodiments of the method 101, conducting 111 the in vitro differentiation and 112 maturation process of CD34+ stem cells into T cells produces CD4 positiveCD8 positive T cells. The CD4 positive CD8 positive T cells may be naive T cells. Naive T cells arc commonly characterized by surface expression of L-sclcctin (CD62L) and C-C Chcmokinc receptor type 7 (CCR7). In some instances, the cells are characterized by the absence of the activation markers CD25, CD44 or CD69, and the absence of memory CD45RO isoform. Naive T cells may also express functional IL-7 receptors, consisting of subunits IL-7 receptor-alpha, CD127, and common-gamma chain, CD132. The naive T cells may be cryopreserved for storage or introduced into a subject for during an allogeneic cell therapy treatment. Inside the subject, the naive T cells may circulate through peripheral lymphatics awaiting initial antigenic stimulation. Upon initial stimulation through the naive cells’ TCRs, the cells begin to modulate expression of surface molecules associated with activation, co-stimulation, and adhesion. The expression pattern of these molecules may be used to further define effector and antigen-experienced of T cell subsets.

[0108] In some embodiments, the CD4 positive CD8 positive T cells are expanded in vitro prior to cryopreservation and / or administration to an allogeneic cell therapy recipient. Expansion of the CD4 positive CD8 positive T cells may involve culturing the cells in the presence of one or more of IL-7, IL-15, CD3, CD28, CD2, alpha-galactosylceramide.

[0109] Methods of the disclosure take advantage of in vivo activation mechanisms to reduce in vitro culture steps. Once a T cell has been produced, without having undergone two activations steps, and is introduced into a subject’s body, the T cell is fully activated when the T cell encounters a properly activated antigen presenting cell (APC), such as a dendritic cell, for example, at secondary lymphoid organ. If the APC displays an appropriate peptide ligand through the major histocompatibility complex (MHC) class II molecule, it is recognized by the TCR. This is important for activating the T cell. Two other stimulatory signals delivered by the APC may also be required. These signals can be provided by two different ligands on the APC surface, such as CD80 and CD86, to a surface molecule on the T cell, e.g., CD28. Other factors important for activation include those factors involved in directing T cell differentiation into different subsets of effector T cells, e.g., cytokines, such as IL-6, IL- 12 and TGF-p. The CD28-dependent costimulation of activated T cells can lead to production of IL-2 by the activated T cell themselves. Following expression of IL-2, there can also be an upregulation of the third component (called a- chain) of the IL-2 receptor, also known as CD25, in addition to other regulatory molecules such as ICOS and CD40L. Binding of IL-2 to its high affinity receptor promotes cell growth, whilst APCs, mainly dendritic cells generate various cytokines or express surface proteins that induce thedifferentiation of CD4+ T lymphocytes into cytokine producing effector cells, depending on environmental conditions.

[0110] FIG. 2 illustrates generalized stages of ex vivo T cell manufacturing. Specifically, this illustrates a previously discovered process by the present Inventors for making engineered T cells without using the deep maturation and turbo deep maturation concepts of the disclosure. Advantageously, the present inventors found that, relative to previous methods, this process, which used a single activation step produced effective quantities of T cells. By using only one activation - and not two activation steps - such methods were able to generate T cells in at least 14 days less than prior art T cell manufacturing processes.

[0111] Methods for performing the “deep maturation” embodiments of the disclosure include, in order, the steps of: (i) transducing HSPCs Day -2 (e.g., the first day); (ii) differentiation of HSPC into T cells (Days 0-14); (iii) maturation (Days 14-21); (iv) maturation II (Days 21-28); and (v) activation (after Day 28). As the name implies, the “turbo deep maturation” embodiments of the disclosure involve fewer steps than do the “deep maturation” embodiments. Methods for performing the “turbo deep maturation” embodiments of the disclosure include, in order, the steps of: (i) transducing HSPCs; (ii) differentiation of HSPC into T cells; (iii) maturation II (Days 14- 21); and (iv) activation (after day 21). Thus, the methods of the disclosure for producing engineered immune cells “turbo” embodiments are able to produce engineered immune cells without the separate maturation step. As shown, in certain aspects, maturation II occurs with or without the use of a lymphoid differentiation coating material. In certain aspects, maturation II includes diluting or removing PBMCs / SCTs at day 3 of maturation II.

[0112] After maturation II, the deep maturation and turbo deep maturation methods of the disclosure may involve a single activation step. A single activation step can involve culturing T cells with activation reagents for a period of time no longer than 7 days. In other embodiments, a single activation step involves culturing a T cell in activation media for no longer than 6 days or 5 days or 4 days or 3 days or 2 days or 1 day. In other embodiments, a single activation step comprises not culturing a T cell in activation media for longer than 8 days, or 9 days, or 10 days or 11 days, or 12 days or 13 days or 14 days. A single activation step can involve culturing T cells with a single type of activation media. The single type of activation media can include activation reagents, such as soluble antibodies. The single activation step can involve co-culturing the T cellswith antigen presenting cells, such as aAPCs. The single activation step can involve co-culturing the T cells with PBMCs.

[0113] Methods of the disclosure involve production of T cells from hemopoietic stem / progenitor cells. The hemopoietic stem / progenitor cells generally related to CD34+ cells that may be found in cord blood. In some instances, the cells may be derived from a progenitor cell. In some instances, the cell is a pluripotent stem cell, such as, an embryonic stem cell.

[0114] Allogeneic CAR T cells produced from HSCs may provide a curative therapeutic approach for certain pathologies. However, some limitations include GVHD, a donor T-cell-mediated alloreactive process responsible for much of the morbidity and mortality associated with allogeneic cell therapies. Some clinical research show that donor iNKT cells can prevent GVHD without increasing the risk of disease relapse. Adoptive transfer of donor CAR iNKT cells followed by in vivo activation and / or expansion, may prevent or alleviate symptoms of GVHD. This protective effect may be mediated through Th2 polarization of alloreactive T-cells and expansion of donor regulatory T-cells (Tregs). Since allogeneic iNKT-cells, as produced by methods of the disclosure, do not cause GVHD, methods described herein provide an ideal platform for ‘off-the-shelf’ CAR immunotherapy .

[0115] Methods of the disclosure are useful to manufacture therapeutically active T cells that acquire antigen- specificity via functional rearrangements of antigen recognition regions of TCRs. The TCR is a molecule found on the surface of T cells (or T lymphocytes) that is responsible for recognizing antigens bound to major histocompatibility complex molecules. The TCR may be composed of at least two different protein chains (e.g., a heterodimer). In most (e.g., 95%) T cells, this consists of an alpha and beta chain, whereas in some (e.g., 5%) T cells, this consists of gamma and delta chains. Such T cells may have antigen-specificity in cell surface TCR molecules differentiate in vivo into different phenotypic subsets, including, but not limited to, classical CD3 positive, alpha-beta TCR CD4 positive, CD3 negative alpha-beta TCR CD8 positive, gamma delta T cells, Natural Killer T cells, etc. Furthermore, T cell may further include various activation states, including, but not limited to, naive, central memory, effector memory, terminal effector, etc.

[0116] In preferred embodiments, methods of the disclosure provide for the manufacture of CAR T cells. CAR T cells are T cells that have been genetically engineered to produce an artificial T- cell receptor for use in immunotherapy. CARs (i.e., chimeric antigen receptors) can be used to graft the specificity of a monoclonal antibody onto cells via transfer of their coding sequencesfacilitated by, for example, retroviral vectors. The CARs are receptor proteins that have been engineered to give T cells the new ability to target a specific protein, c.g., CD19 and / or CD20. The receptors are chimeric because they combine both antigen-binding and T-cell activating functions into a single receptor. Accordingly, methods of the invention provide products for immunotherapy by producing modified T cells that recognize cancer cells in order to more effectively target and destroy them. CARs are used to redirect T-cell target specificity and / or reactivity toward a target in a non-MHC-restricted manner. In this way, CARs are able to exploit the antigen-binding properties, specificity, and design of monoclonal antibodies with the immune function of a T cell. The non-MHC-restricted antigen binding allows T-cells expressing CARs the ability to recognize an antigen in a way that is independent of antigen processing, which may help alleviate tumor escape. Moreover, when expressed in T-cells, CARs advantageously do not dimerize with endogenous T-cell receptor (TCR) alpha and beta chains.

[0117] The phrases “has antigenic specificity” and “elicit antigen- specific response,” as used herein, means that the CAR can specifically bind to and immunologically recognize an antigen, such that binding of the CAR to the antigen elicits an immune response.

[0118] In practicing methods of the disclosure, any CAR suitable for engineering effector cells (e.g., T cells) as used in adoptive immunotherapy therapy, may be used in the present disclosure. CARs that can be used in the present disclosure include those described in Kim and Cho, 2020, Recent Advances in Allogeneic CAR-T Cells, Biomolecules, 10(2):263, which is incorporated by reference.

[0119] CARs generally include an extracellular domain, a transmembrane domain and an intracellular domain. The extracellular domain may include an antigen binding / recognition region / domain. The antigen binding domain of the CAR is useful to bind to a specific antigen, e.g., a tumor antigen, a pathogen antigen (e.g., viral antigen), a CD (cluster of differentiation) antigen. The extracellular domain may also include a signal peptide that directs nascent protein into the endoplasmic reticulum. Signal peptide may be essential if the CAR is to be glycosylated and anchored in the cell membrane. The transmembrane domain is a hydrophobic alpha helix that spans the membrane. Different transmembrane domains result in different receptor stability. After antigen recognition, receptors cluster and a signal is transmitted to the cell. The most commonly used intracellular component is CD3^ which contains 3 IT AMs. This transmits an activation signal to the T cell after antigen is bound. CARs can also include a spacer region that links the antigenbinding domain to the transmembrane domain. The spacer region should be flexible enough to allow the antigen binding domain to orient in different directions to facilitate antigen recognition. The spacer can be the hinge region from IgGl, or the CH2CH3 region of immunoglobulin and portions of CD3.

[0120] Presently, there are three generations of CARs. First generation CARs typically comprise an antibody derived antigen recognition domain (e.g., a single-chain variable fragments (scFv)) fused to a transmembrane domain, fused to cytoplasmic signaling domain of the T cell receptor chain. First generation CARs typically have the intracellular domain from the CD3 Xi-chain, which is the primary transmitter of signals from endogenous TCRs. First generation CARs can provide de novo antigen recognition and cause activation of both CD4+ and CD8+ T cells through their CD3(^ chain signaling domain in a single fusion molecule, independent of HLA-mediated antigen presentation. In one non-limiting example, T cells can be genetically engineered to express artificial TCRs that direct cytotoxicity toward tumor cells, for discussion, see Eshhar 1993, Specific activation and targeting of cytotoxic lymphocytes through chimeric single chains consisting of antibody-binding domains and the gamma or zeta subunits of the immunoglobulin and T-cell receptors, Proc Natl Acad Sci, 90, 720-724, incorporated by reference. Second generation CARs are similar to first generation CARs but include two co- stimulatory domains, such as, CD28 or 4- IBB. The involvement of these intracellular signaling domains improve T cell proliferation, cytokine secretion, resistance to apoptosis, and in vivo persistence. Third generation CARs combine multiple co-stimulatory domains, such as CD28-41BB or CD28-OX40, to further augment T cell activity.

[0121] One potential way to imbue greater target cell specificity to CAR-expressing T cells is to use combinatorial CAR approaches. In one system, the CD3-(^ and / or CD28 signal units are split between two different CAR constructs expressed in the same cell.

[0122] In certain aspects, methods of the disclosure produced engineered immune cells expressing one or more introduced TCR / CAR with an antigen binding domain selected from a Fab fragment (Fab), F(ab')2 fragment, diabody, triabody, tetrabody, single-chain variable region fragment (scFv), or a disulfide- stabilized variable region fragment (dsFv). In a preferred embodiment, the antigen binding domain is an scFv. An scFv is a truncated Fab fragment including the variable (V) domain of an antibody heavy chain linked to a V domain of an antibody light chain via a synthetic peptide, which can be generated using recombinant and / or directed mutagenesis methods.

[0123] As used herein, the term “single-chain variable fragment” or “scFv” is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of an immunoglobulin covalently linked to form a VH-VL heterodimer. The heavy (VH) and light chains (VL) are either joined directly or joined by a peptide-encoding linker, which connects the N-terminus of the VH with the C-terminus of the VL, or the C-terminus of the VH with the N-terminus of the VL. In some embodiments, the antigen binding domain comprises an scFv having the configuration from N- terminus to C-terminus, VH-linker-VL. In some embodiments, the antigen binding domain comprises an scFv having the configuration from N-terminus to C-terminus, VL-linker-VH. Those of skill in the art would be able to select the appropriate configuration for use in the present invention.

[0124] As used herein, “Fab” refers to a fragment of an antibody structure that binds to an antigen but is monovalent and does not have a Fc portion, for example, an antibody digested by the enzyme papain yields two Fab fragments and an Fc fragment (e.g., a heavy (H) chain constant region; Fc region that does not bind to an antigen).

[0125] As used herein, “F(ab')2” refers to an antibody fragment generated by pepsin digestion of whole IgG antibodies, wherein this fragment has two antigen binding (ab') (bivalent) regions, wherein each (ab') region comprises two separate amino acid chains, a part of a H chain and a light (L) chain linked by an S — S bond for binding an antigen and where the remaining H chain portions are linked together. A “F(ab')2” fragment can be split into two individual Fab' fragments.

[0126] In some embodiments, the antigen binding domain may be derived from the same species in which the CAR will ultimately be used. For example, for use in humans, the antigen binding domain of the CAR may comprise a human antibody or a fragment thereof. In some embodiments, the antigen binding domain may be derived from a different species in which the CAR will ultimately be used. For example, for use in humans, the antigen binding domain of the CAR may comprise a murine antibody or a fragment thereof.

[0127] An introduced TCR / CAR may comprise a light chain variable region and the heavy chain variable region may be joined by an antigen binding domain linker peptide. The linker peptide may be of any length and many comprise any amino acid sequence. For example, the antigen binding domain linker peptide may comprise or consist of any one or more of glycine, serine, lysine, proline, glutamic acid, and threonine, with or without other amino acid residues. In certain aspects, the linker peptide may have a length of about 5 to about 100 amino acid residues, about 8to about 90 amino acid residues, about 10 to about 70 amino acid residues, about 12 to about 50 amino acid residues, or about 8 to about 25 amino acid residues. An antigen binding domain linker peptide may be of the foregoing lengths and consist of amino acid residues selected, independently, from the group consisting of glycine and serine, several of such linkers are known in the art. In certain aspects, one or more of the antigen binding domains has the light chain and heavy chain variable regions linked via a Whitlow linker. In certain aspects, the antigen binding domain an introduced CAR may have a sequence from N-terminus to C-terminus of heavy-chain variable domain, linker, light-chain variable domain. In certain aspects, embodiment, the antigen binding domain has a sequence from N-terminus to C-terminus of light-chain variable domain, linker, heavy-chain variable domain.

[0128] In another embodiment, an introduced CAR / TCR comprises a leader or signal sequence. A leader sequence may be positioned at the amino terminus of an antigen binding domain. The leader sequence may be a human leader sequence. The leader sequence may comprise any suitable amino acid sequence. In one embodiment, the leader sequence is a human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor leader sequence or a human CD8a leader sequence. In an embodiment of the invention, while the leader sequence may facilitate expression of the introduced CAR on the surface of the cell, the presence of the leader sequence in the expressed CARs may not be necessary in order for the CAR to function. In an embodiment of the invention, upon expression of the introduced CARs on the cell surface, all or a portion of the leader sequence may be cleaved off the one or both of the CARs. Accordingly, in an embodiment of the invention, one or more introduced CARs lack a leader sequence.

[0129] Introduced TCRs / CARs of the present invention may include a transmembrane domain that connects the antigen binding domain of the CAR to an intracellular domain of the CAR. The transmembrane domain is a region spanning the membrane of a cell. The transmembrane domain may be disposed between the antigen binding domain and the intracellular domain of a CAR. In some embodiments, the transmembrane domain can be selected or modified from another sequence (e.g., a naturally occurring sequence) by one or more amino acid substitutions to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins, thereby minimizing interactions with other members of the receptor complex.

[0130] Transmembrane domains may be derived either from a natural or a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembraneprotein, e.g., a Type T transmembrane protein. Where the source is synthetic, the transmembrane domain may be any artificial sequence that facilitates insertion of the CAR into a cell membrane, e.g., an artificial hydrophobic sequence. Examples of the transmembrane domain of particular use in this invention include, without limitation, transmembrane domains derived from (i.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD7, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134 (OX-40), CD137 (4-1BB), CD154 (CD40L), Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR9. In some embodiments, the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine.

[0131] The transmembrane domain may include a hinge region. The hinge region of the CAR is a hydrophilic region which is located between the antigen binding domain and the transmembrane domain. The hinge may facilitate proper protein folding for the CAR. The hinge region is an optional component for the CAR. The hinge region may include a domain selected from Fc fragments of antibodies, hinge regions of antibodies, CH2 regions of antibodies, CH3 regions of antibodies, artificial hinge sequences or combinations thereof. Examples of hinge regions include, without limitation, a CD8a hinge, a CD28 hinge, artificial hinges made of polypeptides which may be as small as, three glycines (Gly), as well as CHI and CH3 domains of IgGs (such as human IgG4).

[0132] In some embodiments, a subject CAR of the present disclosure includes a hinge region that connects the antigen binding domain with the transmembrane domain, which, connects to the intracellular domain. The hinge is preferably flexible to facilitate the antigen binding domain to be positioned in order to recognize the specific structure and density of the target antigens on a cell.

[0133] In some embodiments, the hinge region is an immunoglobulin heavy chain hinge region. In some embodiments, the hinge region is a hinge region polypeptide derived from a receptor (e.g., a CD 8 -derived hinge region).

[0134] The hinge region can have a length of from about 4 amino acids to about 50 amino acids, e.g., from about 4 aa to about 10 aa, from about 10 aa to about 15 aa, from about 15 aa to about 20 aa, from about 20 aa to about 25 aa, from about 25 aa to about 30 aa, from about 30 aa to about 40 aa, or from about 40 aa to about 50 aa. In some embodiments, the hinge region can have a length of greater than 5 aa, greater than 10 aa, greater than 15 aa, greater than 20 aa, greater than 25 aa,greater than 30 aa, greater than 35 aa, greater than 40 aa, greater than 45 aa, greater than 50 aa, greater than 55 aa, or more.

[0135] Suitable hinge regions can be readily selected and can be of any of a number of suitable lengths, such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids. Suitable hinge regions can have a length of greater than 20 amino acids (e.g., 30, 40, 50, 60 or more amino acids).

[0136] Preferably the hinge is a short sequence of amino acids to facilitate antibody or receptor flexibility. The hinge domain may be positioned between the antigen binding domain and the transmembrane (TM). The hinge domain may comprise the hinge domain of human CD8a or human CD28. For example, the human hinge domain may comprise a sequence comprising, consisting of, or consisting essentially of the hinge domain of human CD8a.

[0137] A CAR / TCR may also include an intracellular signaling domain, which activates at least one of the effector functions of the cell in which the CAR is expressed. The intracellular signaling domain transduces the effector function signal and directs the cell (e.g., immune cell) to perform its specialized function, e.g., harming and / or destroying a target cell expressing a target antigen.

[0138] The intracellular domain may include the cytoplasmic portion of a surface receptor, a costimulatory molecule, and / or any molecule that acts in concert to initiate signal transduction in the T cell.

[0139] Examples of the intracellular signaling domain include, without limitation, the chain of the T cell receptor complex or any of its homologs, e.g., q chain, FcsRIy and chains, MB 1 (Iga) chain, B29 (Ig) chain, etc., human CD3 zeta chain, CD3 polypeptides (A, 5 and s), syk family tyrosine kinases (Syk, ZAP 70, etc.), sre family tyrosine kinases (Lek, Fyn, Lyn, etc.), and other molecules involved in T cell transduction, such as CD2, CD5 and CD28. In one embodiment, the intracellular signaling domain may be human CD3^ chain, FcyRIII, FcsRI, cytoplasmic tails of Fc receptors, an immunoreceptor tyrosine-based activation motif (ITAM) bearing cytoplasmic receptors, and combinations thereof.

[0140] In one embodiment, the intracellular signaling domain of the CAR includes any portion of one or more co-stimulatory molecules, such as at least one signaling domain from CD2, CD3,CD8, CD27, CD28, ICOS, 4- IBB, PD-1 , any derivative or variant thereof, any synthetic sequence thereof that has the same functional capability, and any combination thereof.

[0141] Other examples of the intracellular domain include a fragment or domain from one or more molecules or receptors including, but not limited to, TCR, CD3, CD3 gamma, CD3 delta, CD3 epsilon, CD86, common FcR gamma, FcR beta (Fc Epsilon Rib), CD79a, CD79b, Fcgamma Rlla, DAP10, DAP 12, T cell receptor (TCR), CD8, CD27, CD28, 4- IBB (CD137), 0X9, 0X40, CD30, CD40, PD-1, ICOS, a KIR family protein, lymphocyte function-associated antigen- 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CD5, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD127, CD160, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAL, CDl la, LFA-1, ITGAM, CDl lb, ITGAX, CDl lc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD 96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, LylO8), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, Tolllike receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, other costimulatory molecules described herein, any derivative, variant, or fragment thereof, any synthetic sequence of a co- stimulatory molecule that has the same functional capability, and any combination thereof.

[0142] Intracellular signaling domains as used herein may be derived from among several types of various other immune signaling receptors, including, but not limited to, first, second, and third generation T cell signaling proteins including CD3, B7 family costimulatory, and Tumor Necrosis Factor Receptor (TNFR) superfamily receptors. Intracellular signaling domains may include signaling domains used by NK and NKT cells such as signaling domains of NKp30, DAP 12, NKG2D, NKp44, NKp46, DAP 10, and CD3z.

[0143] Intracellular’ signaling domains suitable for use in a subject CAR of the present invention include any desired signaling domain that provides a distinct and detectable signal (e.g., increased production of one or more cytokines by the cell; change in transcription of a target gene; change in activity of a protein; change in cell behavior, e.g., cell death; cellular proliferation; cellular differentiation; cell survival; modulation of cellular signaling responses; etc.) in response toactivation of the CAR (i.e., activated by antigen and dimerizing agent). In some embodiments, the intracellular signaling domain includes at least one (c.g., one, two, three, four, five, six, etc.) IT AM motifs as described below. In some embodiments, the intracellular signaling domain includes DAP10 / CD28 type signaling chains.

[0144] Intracellular signaling domains suitable for use in a CAR include immunoreceptor tyrosinebased activation motif (ITAM)-containing intracellular signaling polypeptides. An IT AM motif may be repeated twice in an intracellular signaling domain, where the first and second instances of the IT AM motif are separated from one another by 6 to 8 amino acids.

[0145] In certain aspects, the intracellular signaling domains include the signaling domains of human immunoglobulin receptors that contain immunoreceptor tyrosine-based activation motifs (ITAMs) such as, but not limited to, FcgammaRI, FcgammaRIIA, FcgammaRIIC, FcgammaRIIIA, FcRL5.

[0146] A suitable intracellular signaling domain may be an IT AM motif-containing portion that is derived from a polypeptide that contains an ITAM motif. For example, a suitable intracellular signaling domain can be an ITAM motif-containing domain from any ITAM motif-containing protein. A suitable intracellular signaling may include an ITAM motif-containing polypeptides such as: DAP12, FCER1G (Fc epsilon receptor I gamma chain), CD3D (CD3 delta), CD3E (CD3 epsilon), CD3G (CD3 gamma), CD3L) and CD79A (antigen receptor complex-associated protein alpha chain).

[0147] In certain aspects, the intracellular signaling domain is derived from DAP 12 (also known as TYROBP; TYRO protein tyrosine kinase binding protein; KARAP; PLOSL; DNAX-activation protein 12; KAR-associated protein; TYRO protein tyrosine kinase-binding protein; killer activating receptor associated protein; killer-activating receptor-associated protein; etc.); derived from FCER1G (also known as FCRG; Fc epsilon receptor I gamma chain; Fc receptor gammachain; fc-epsilon Rl-gamma; fcRgamma; fceRl gamma; high affinity immunoglobulin epsilon receptor subunit gamma; immunoglobulin E receptor, high affinity, gamma chain; etc.); derived from T-cell surface glycoprotein CD3 delta chain (also known as CD3D; CD3-DELTA; T3D; CD3 antigen, delta subunit; CD3 delta; CD3d antigen, delta polypeptide (TiT3 complex); OKT3, delta chain; T-cell receptor T3 delta chain; T-cell surface glycoprotein CD3 delta chain; etc.); derived from T-cell surface glycoprotein CD3 epsilon chain (also known as CD3e, T-cell surface antigen T3 / Leu-4 epsilon chain, T-cell surface glycoprotein CD3 epsilon chain, AI504783,CD3, CD3epsilon, T3e, etc.); derived from T-cell surface glycoprotein CD3 gamma chain (also known as CD3G, T-cell receptor T3 gamma chain, CD3-GAMMA, T3G, gamma polypeptide (TiT3 complex), etc.); derived from T-cell surface glycoprotein CD3 zeta chain (also known as CD3Z, T-cell receptor T3 zeta chain, CD247, CD3-ZETA, CD3H, CD3Q, T3Z, TCRZ, etc.); derived from CD79A (also known as B-cell antigen receptor complex-associated protein alpha chain; CD79a antigen (immunoglobulin-associated alpha); MB-1 membrane glycoprotein; ig- alpha; membrane-bound immunoglobulin-associated protein; surface IgM-associated protein; etc.). In certain aspects, the intracellular signaling domain includes a cytoplasmic signaling domain of TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, or CD66d. In one embodiment, the intracellular signaling domain in the CAR includes a cytoplasmic signaling domain of human CD3 zeta.

[0148] The CAR may comprise a transmembrane (TM) domain. Preferably, the TM domain is a human TM domain. For example, the TM domain may comprise the TM domain of a human CD8a molecule or a human CD28 molecule. CD8 is a TM glycoprotein that serves as a co-receptor for the TCR, and is expressed primarily on the surface of cytotoxic T-cells. The most common form of CD8 exists as a dimer composed of a CD8a and CD80 chain. CD28 is expressed on T-cells and provides co-stimulatory signals for T-cell activation. CD28 is the receptor for CD80 (B7.1) and CD86 (B7.2).

[0149] A CAR may include an intracellular (i.e., cytoplasmic) T-cell signaling domain. In preferred aspects, a CAR includes an intracellular T-cell signaling domain obtained or derived from a CD28, CD3^ molecule, an Fc receptor gamma (FcRy) chain, CD27, 0X40, a 4- IBB molecule, an inducible T-cell costimulatory protein (ICOS), and / or other intracellular signaling molecules known in the art, or modified versions of any of the foregoing.

[0150] CD28 is a T-cell marker which is involved in T-cell co-stimulation. CD3^ associates with TCRs to produce a signal and contains immunoreceptor tyrosine-based activation motifs (IT AMs). 4-1BB, also known as CD137, transmits a potent costimulatory signal to T-cells, promoting differentiation and enhancing long-term survival of T lymphocytes. ICOS is a CD28-superfamily costimulatory molecule that is expressed on activated T cells.

[0151] The CARs of embodiments of the invention (including functional portions and functional variants thereof) can be obtained by methods known in the art. The CARs may be made by anysuitable method of making polypeptides or proteins. For example, CARs can be recombinantly produced using the nucleic acids described herein using standard recombinant methods.

[0152] In some instances, methods of the disclosure involve performing genetic modification of HSCs to provide for an additional transgene (i.e., a transgene in addition to one of a CAR or TCR). For example, in some embodiments, the additional transgene encodes one or more cytokines. Cytokines relate to substances, such as interferon, interleukin, and growth factors, which are secreted by certain cells of the immune system and influence other cells. According to embodiments of the disclosure, transgenes encoding one or more of IL-2, IL-7, IL-15, IL-12, IL- 18, or IL-21, may be provided to facilitate T cell function or tumor efficacy.

[0153] Introduction of one or more transgenes into CAR T cells may improve properties such as T cell expansion and persistence, (e.g., using IL-2, IL-7 / 15), IFN-g production and T-cell potency (e.g., with IL-12, IL-18), enhancing naive subsets (e.g., IL-21), improve safety (e.g., via inhibitors of IL-6, GM-CSF or other mediators of CRS and neurotoxicity), or improve efficacy by combating the tumor microenvironment (TGF-B, checkpoints, etc.).

[0154] For example, IL- 12 and IL- 18 play a major role in augmenting certain effector functions of CAR T cells. IL- 12 is known to activate certain NK cells and T lymphocytes, induce Th-1 type responses, and increase IFN-gamma secretion. The inducible expression of IL- 12 may augment antitumor capabilities of CAR T cells against certain pathologies, such as, lymphoma, hepatocellular carcinoma, ovarian tumors, and B16 melanoma. IL- 18 has also been used to improve the therapeutic potential of CAR T cells. Initially identified as a potent inducer of IFN- gamma, IL- 18 may contribute to T and NK cell activation and Th-1 cell polarization. For example, Meso-targeted CAR T cell may be provided with transgenes encoding IL- 18 to augment the secretion of IFN-gamma and to eradicate cancer cells. For further discussion, see, Tian, 2020, Gene modification strategies for next-generation CAR T cells against solid cancers, Journal of Hematology & Oncology, volume 13(5), incorporated by reference.

[0155] IL-7, IL- 15, and IL-21 are useful for promoting generation of stem cell- like memory T cell phenotype. This phenotype may provide for increased expansion and persistence of T cells in vivo. In some instances, transgenes encoding IL-2 may be provided. Producing T cells with IL-2 may provide T cells with improved capacities for responding to tumor environments by, for example, facilitating maturation II and the production of proteins involved in nutrient sensing and uptake.

[0156] In some embodiments, the transgene is an inhibitor of cytokine release syndrome. Cytokine release syndrome relates to a serious, potentially life-threatening side effect often associated with CAR T-cell therapy. Cytokine release syndrome manifests as a rapid (hyper)immune reaction driven by excessive inflammatory cytokine release, including, for example, IFN-gamma and IL-6. Many cytokines implicated in cytokine release syndrome are known to operate through a JAK- STAT pathway. Accordingly, in some embodiments, methods of the disclosure involve producing CAR-T cells that express inhibitors of the JAK pathway to improve in vivo CAR T-cell proliferation, antitumor activity, and cytokine levels. For example, transgenes may be provided that inhibit function of IL-6, JAK-STAT, or BTK. Moreover, the inhibitors may further be useful for inhibition of neurotoxicity. CAR T cell related neurotoxicity is a syndrome that often leads to severe neurologic disturbances such as seizures and coma.

[0157] In other instances, methods involve introducing a transgene into HSCs, wherein the transgene is a checkpoint inhibitor, e.g., an immune checkpoint inhibitor. Immune checkpoints are regulators of certain aspects of immune systems. In normal physiological conditions, checkpoints enable the immune system to respond to host antigens preserving healthy tissues. In cancer, these molecules facilitate tumor cell evasion. In some instances, transgenes may encode antibodies or antibody fragments, such as, anti-cathepsin antibodies, galectin-1 blockade and anti- 0X40 agonistic antibodies. The antibodies may be secreted or expressed on surfaces of cells. The antibodies may be secreted that, for example, target PD1 or PDL1.

[0158] In embodiments, CAR T cells expressing an inhibitor of transforming growth factor beta are produced. Engineered cells face hostile microenvironments which limit their efficacy. Modulating the environments may convert be useful for facilitating CAR T cells ability to proliferate, survive and / or kill cancer cells. One of the main inhibitory mechanisms within the tumor environment is transforming growth factor beta. Accordingly, some aspects of the disclosure involve introducing transgenes encoding inhibitors of transforming growth factor beta. The inhibitors may be, for example, antibodies or fragments thereof. The antibody or antibody fragments may be secreted from CAR T cells to interfere with normal functions of transforming growth factor beta.

[0159] In some embodiments, methods of include making CAR T cells to target solid tumor types through markers of tumor microenvironment. In other embodiments, methods make CAR T cells with a single-domain antibody (VHH)-based chimeric antigen receptor, which can be used torecognize markers of a tumor microenvironment without the need for tumor-specific targets. VHH- bascd CAR T cells, according to the disclosure, may target the tumor microenvironment through immune checkpoint receptors or through stroma and extra cellular matrix markers, which effective against solid tumors in syngeneic, immunocompetent animal models. Accordingly, methods of the disclosure are useful to make CAR T cells that target tumors which may lack tumor- specific antigen expression. The variable regions of heavy-chain-only antibodies (VHHs or nanobodies) are small, stable, camelid-derived single-domain antibody fragments with affinities comparable to traditional short chain variable fragments (scFvs). VHHs are generally less immunogenic than scFvs and, owing to their small size, can access epitopes different from those seen by scFvs. VHHs, as provided by the disclosure, can therefore serve as suitable antigen recognition domains in CAR T cells. Unlike scFvs, VHHs do not require the additional folding and assembly steps that come with V-region pairing. They allow surface display without the requirement for extensive linker optimization or other types of reformatting. The ability to switch out various VHH-based recognition domains yields a highly modular platform, accessible without having to reformat each new conventional antibody into an scFv.

[0160] Moreover, many microenvironments involve expression of inhibitory molecules such as PD-L1. Using VHHs as recognition domains, e.g., PD-L1 -specific CAR T cells, CAR T cells produced by methods of the disclosure can target the tumor microenvironment. PD-L1 is widely expressed on tumor cells, as well as on the infiltrating myeloid cells and lymphocytes. A CAR that recognizes PD-L1 should relieve immune inhibition and at the same time allow CAR T cell activation in the tumor microenvironment. PD-L1 -targeted CAR T cells might thus reprogram the tumor microenvironment, dampening immunosuppressive signals and promoting inflammation. For example, as discussed in Xie, 2019, Nanobody -based CAR T cells that target the tumor microenvironment inhibit the growth of solid tumors in immunocompetent mice, PNAS April 16, 2019 116 (16) 7624-7631, which is incorporated by reference.

[0161] According to embodiments of the present disclosure, CD34+ stem cells are genetically engineered to express one or more of a CAR, a TCR, and an additional transgene (e.g., a cytokine). For initial genetic modification of the cells to provide for tumor or viral antigen- specific cells, a retroviral vector may be used for viral transduction. Combinations of retroviral vector and an appropriate packaging infecting human cells in culture are known in the art. In preferred embodiments, a third-generation lentiviral vector may be used. The vector may be modified withcDNA sequences containing sequences of antibodies or antibody fragments to target preferred antigens. For example, as described in Carpcnito, 2008, Control of large, established tumor xenografts with genetically retargeted human T cells containing CD28 and CD 137 domains, PNAS, 106(9) 3360-3365; Li, 2017, Redirecting T Cells to Glypican-3 with 4-1BB Zeta Chimeric Antigen Receptors Results in Thl Polarization and Potent Antitumor Activity, Human Gene Therapy, 28(5): 437-448; Adusumilli, 2014, Regional delivery of mesothelin-targeted CAR T cell therapy generates potent and long-lasting CD4-dependent tumor immunity, Science Translational Medicine, 261(6): 1-14; each of which are incorporated by reference.

[0162] Some aspects of the disclosure involve introducing and expressing multiple transgenes in HSCs. To facilitate the expression of multiple genes, it may be useful to separate the transgenes, on nucleic acids, with 2A sequences, i.e., coding domains of 2A peptides. 2A self-cleaving peptides, or 2A peptides, is a class of 18-22 aa-long peptides, which can induce ribosomal skipping during translation of a protein. Inside the cell, when the coding domains of a 2A peptide is inserted between two coding domains of two proteins (e.g., TCR and CAR), the peptide will be translated into two proteins folding independently due to ribosome skipping.

[0163] Methods of the disclosure are useful to transform engineered HSCs into T cells for clinical application. Methods of transformation generally include differentiation of HSCs into T cells. Cellular differentiation is the process in which a cell changes from one cell type to another. Usually, the cell changes to a more specialized type. Differentiation of HSCs into T cells may involve multiple stages of differentiation. As a first stage, CD34+ cells may be differentiated into CD4 CD8 double negative T cells. Generation of double negative T cells can be achieved by culture of CD34+ cells in the presence of a cocktail of cell factors including hematopoietic cytokines. The cocktail may include SCF (e.g., hSCF), Flt3L (e.g., hFlt3L), and at least one cytokine, and bFGF for hematopoietic specification. The cytokine can be a Thl cytokine, which includes, but is not limited to IL-3, IL-15, IL-7, IL-12 and IL-21. The cells may be immunophenotypically analyzed by FACS for expression of CD34, CD31, CD43, CD45, CD41a, ckit, Notch 1, IL7Ra.

[0164] Double negative T cells may be further differentiated via an antigen-independent maturation process to produce functional, inactivated, T cells. This process may involve culturing double negative T cells in a lymphoid progenitor expansion medium. The media may include, for example, a feeder cell and SCF, Flt3L and at least one cytokine. The cytokine may be a Thlcytokine, which includes, but is not limited to, IL-3, IL-15, IL-7, IL-12 and IL-21. In some embodiments, the cytokine may enhance survival and / or functional potential of the cells.

[0165] Cell products comprising T cells, including engineered T cells can be provided systemically or directly to a subject for the treatment of a neoplasia, pathogen infection, or infectious disease. In one embodiment, T cells of the present disclosure may be directly injected into an organ of interest (e.g., an organ affected by a neoplasia). Alternatively, T cells and compositions comprising thereof can be provided indirectly to the organ of interest, for example, by administration into the circulatory system (e.g., the tumor vasculature). Preferably, activation and expansion of the T cells occurs in vivo, after introduction into a subject. T cells and compositions comprising thereof of the present disclosure may be administered in any physiologically acceptable vehicle, normally intravascularly, although they may also be introduced into bone or other convenient site where the cells may find an appropriate site for regeneration and differentiation (e.g., thymus). Usually, at least 100,000 cells will be administered, and sometimes 10,000,000,000 cells, or more.

[0166] Methods of the disclosure provide for compositions of cells that may be combined with pharmaceutical compositions for administration of an allogeneic cell therapy. When administering a therapeutic composition of the present disclosure (e.g., a pharmaceutical composition comprising CAR T cells derived from HSCs), it will generally be formulated in a unit dosage injectable form (solution, suspension, emulsion). The compositions may be provided in a therapeutically effective concentration. The therapeutically effective concentration is an amount sufficient to affect a beneficial or desired clinical result upon treatment. An effective amount can be administered to a subject in one or more doses. In terms of treatment, an effective amount is an amount that is sufficient to palliate, ameliorate, stabilize, reverse or slow the progression of the disease, or otherwise reduce the pathological consequences of the disease. The effective amount is generally determined by the physician on a case-by-case basis and is within the skill of one in the art. Several factors are typically taken into account when determining an appropriate dosage to achieve an effective amount. These factors include age, sex and weight of the subject, the condition being treated, the severity of the condition and the form and effective concentration of the antigenbinding fragment administered.

[0167] For adoptive immunotherapy using antigen-specific T cells of the disclosure, cell doses in the range of 10,000,000-10,000,000,000 may be infused. Upon administration of the T cells into the subject T cells may undergo an antigen-dependent activation process.

[0168] This disclosure provides methods for manufacture of T cells for cell therapies and / or research. In some aspects, methods provide economical methods of T cell manufacture by reducing time of ex vivo cell culture. In some related aspects, methods of the disclosure provide for the manufacture of T cells with enhanced cytotoxic efficacy. Prolonged cell culture has previously been associated with transcriptional and phenotypic changes of certain cell types. Although transcriptional and phenotypic changes of T cells in culture are poorly characterized, this disclosure recognizes that unintended changes of cells during prolonged culture may account for observed reductions in therapeutic efficacy and product batch variability identified in allogeneic cells. For example, prolonged cell culture of T cells may give rise to elevated levels of exhaustion markers, which reflect loss of effector function. For example, prolonged culture may be associated with increased expression of PD1, LAG3. CD244, CD160, for further discussion, see Wherry, 2016, Molecular and cellular insights into T cell exhaustion, Nat Rev Immunol, 15(8): 486-499, which is incorporated by reference. By shortening ex vivo manufacture, methods of the disclosure are useful for consistent production of therapeutically effective T cells.

[0169] Accordingly, in one aspect, this disclosure provides a method of producing a T cell. The method involves conducting a process involving in vitro differentiation and maturation of a hematopoietic stem cell (HSC) into a T cell, with the proviso that the process does not involve subsequent in vitro steps of activation and / or expansion of the T cell. Rather, activation and / or expansion of the T cell preferably occurs in vivo after introduction into the subject. Advantageously, omitting in vitro activation and / or T cell expansion saves weeks (e.g., at least two weeks) off conventional T cell manufacturing processes, which may be useful for producing a T cell with enhanced cytotoxic efficacy.

[0170] Methods of the disclosure are useful for producing allogeneic therapies that are safe and effective. In some instances, methods may involve characterizing cell products at one or more points during manufacture to ensure product quality. In some embodiments, methods of the disclosure involve analyzing T cells to identify one or more proteins expressed by the T cells. The one or more proteins may include one or more of CCR7, CD62L, CD45RA, CXCR3, CD56, NKp44, and NKG2D. The proteins may include markers associated with naive stem cells.Analyzing preferably includes high throughput methods of analyzing cell surface proteins, e.g., methods based on fluorescent signals of individual cells in bulk, such as, FACS.

[0171] On demand availability of treatment is one benefit of allogeneic cell therapies. Since methods may involve manufacture of cells before clinical application, some preferred methods may include cryopreserving T cells. Cryopreserving T cells is useful for safe and effective storage of cells until they are needed by a patient. Cryopreserving is also useful for transportation of cell products to clinical facilities where they can be administered to patients. In some preferred embodiments, double positive T cells (e.g., naive T cells) are cryopreserved without preforming an in vitro activation step.

[0172] 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 CAR-engineered adoptive T cells therapy, which targets certain blood cancers at impressive efficacy. 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. Allogeneic immune cellular products, by methods described herein, can be manufactured at a large-scale and can be readily distributed to treat a higher number of patients therefore are in great demand.

[0173] Some embodiments concern an engineered iNKT cell or a population of engineered iNKT cells. In at least some cases, the engineered iNKT cells comprise CAR and / or engineered T cell receptor. Any embodiment discussed in the context of a cell can be applied to a population of such cells. In particular embodiments, an engineered iNKT cell comprises a nucleic acid comprising 1, 2, and / or 3 of the following: i) all or part of an invariant alpha T-cell receptor coding sequence; ii) all or part of an invariant beta T-cell receptor coding sequence, or iii) a suicide gene. In further embodiments, there is an engineered iNKT cell comprising a nucleic acid having a sequence encoding: i) all or part of an invariant alpha T- cell receptor; ii) all or part of an invariant beta T- cell receptor, and / or iii) a suicide gene product.

[0174] Further aspects relate to engineered iNKT cells with increased levels of NK activation receptors, decreased levels of NK inhibitory receptors, and / or increased levels of cytotoxic molecules. In some embodiments, the NK activation receptors comprise NKG2D and / or DNAM- 1. In some embodiments, cytotoxic molecules comprise Perforin and / or Granzyme B. In someembodiments, the inhibitor receptors comprise KIR. The increase or decrease may be with respect to the levels of the same marker in non-cnginccrcd iNKTs isolated from a healthy individual. Further aspects relate to a population of engineered iNKT cells, wherein the population of cells has increased levels of NK activation receptors, decreased levels of NK inhibitory receptors, and / or increased levels of cytotoxic molecules.

[0175] In some embodiments, the engineered iNKT cell comprises a nucleic acid under the control of a heterologous promoter, which means the promoter is not the same genomic promoter that controls the transcription of the nucleic acid. It is contemplated that the engineered iNKT cell comprises an exogenous nucleic acid comprising one or more coding sequences, some or all of which are under the control of a heterologous promoter in many embodiments described herein.

[0176] In a particular embodiment, there is an engineered invariant natural killer T (iNKT) cell that expresses at least one invariant natural killer T-cell receptor (iNKT TCR) and an exogenous suicide gene product, wherein the at least one iNKT TCR is expressed from an exogenous nucleic acid and / or from an endogenous invariant TCR gene that is under the transcriptional control of a recombinantly modified promoter region. An iNKT TCR refers to a TCR that recognizes lipid antigen presented by a CD Id molecule. It may include an alpha- TCR, a beta-TCR, or both. In some cases, the TCR utilized can belong to a broader group of “invariant TCR”, such as a MAIT cell TCR, GEM cell TCR, or gamma / delta TCR, resulting in HSC -engineered MAIT cells, GEM cells, or gamma / delta T cells, respectively.

[0177] In certain embodiments, a suicide gene is enzyme-based, meaning the gene product of the suicide gene is an enzyme and the suicide function depends on enzymatic activity. One or more suicide genes may be utilized in a single cell or clonal population. In some embodiments, the suicide gene encodes herpes simplex virus thymidine kinase (HSV-TK), purine nucleoside phosphorylase (PNP), cytosine deaminase (CD), carboxypetidase G2, cytochrome P450, linamarase, beta-lactamase, nitroreductase (NTR), carboxypeptidase A, or inducible caspase 9. Methods in the art for suicide gene usage may be employed, such as in U.S. Patent No. 8628767, U.S. Patent Application Publication 20140369979, U.S. 20140242033, and U.S. 20040014191, all of which are incorporated by reference in their entirety. In further embodiments, a TK gene is a viral TK gene. In particular embodiments, the TK gene is a herpes simplex virus TK gene. In some embodiments, the suicide gene product is activated by a substrate. Thymidine kinase is a suicide gene product that is activated by ganciclovir, penciclovir, or a derivative thereof. In certainembodiments, the substrate activating the suicide gene product is labeled in order to be detected. In some instances, the substrate that may be labeled for imaging. In some embodiments, the suicide gene product may be encoded by the same or a different nucleic acid molecule encoding one or both of TCR-alpha or TCR-beta. In certain embodiments, the suicide gene is sr39TK or inducible caspase 9. In alternative embodiments, the cell does not express an exogenous suicide gene. In some embodiments, the engineered iNKT cell specifically binds to alpha-galactosylceramide (a- GC).

[0178] In additional embodiments, a cell is lacking or has reduced surface expression of at least one HLA-I or HLA-II molecule. In some embodiments, the lack of surface expression of HLA-I and / or HLA-II molecules is achieved by disrupting the genes encoding individual HLA- I / II molecules, or by disrupting the gene encoding B2M (beta 2 microglobulin) that is a common component of all HLA-I complex molecules, or by disrupting the genes encoding CIITA (the class II major histocompatibility complex transactivator) that is a critical transcription factor controlling the expression of all HLA-II genes. In specific embodiments, the cell lacks the surface expression of one or more HLA-I and / or HLA-II molecules, or expresses reduced levels of such molecules by (or by at least) 50, 60, 70, 80, 90, 100% (or any range derivable therein). In some embodiments, the HLA-I or HLA-II are not expressed in the iNKT cell because the cell was manipulated by gene editing.

[0179] In some embodiments, an iNKT cell comprises a recombinant vector or a nucleic acid sequence from a recombinant vector that was introduced into the cells. In certain embodiments the recombinant vector is or was a viral vector. In further embodiments, the viral vector is or was a lentivirus, a retrovirus, an adeno-associated virus (AAV), a herpesvirus, or adenovirus. It is understood that the nucleic acid of certain viral vectors integrate into the host genome sequence.

[0180] A "gene," “transgene”, "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 (i.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, cDNAfrom 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.

[0181] 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 self-replicating, 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.).

[0182] INKT cells are a small population of alpha beta T lymphocytes highly conserved from mice to humans. iNKT cells have been suggested to play important roles in regulating many diseases, including cancer, infections, allergies, and autoimmunity. When stimulated, iNKT cells rapidly release a large amount of effector cytokines, e.g., like IFN-gamma and IL-4, both as a cell population and at the single-cell level. These cytokines then activate various immune effector cells, such as natural killer cells and dendritic cells (DCs) of the innate immune system, as well as CD4 helper and CD8 cytotoxic conventional alpha beta T cells of the adaptive immune system via activated DCs. Because of their unique activation mechanism, iNKT cells can attack multiple diseases independent of antigen, and MHC, restrictions, making them attractive universal therapeutic agents.

[0183] Previously, a series of iNKT cell-based clinical trials have been conducted, mainly targeting cancer. A recent trial reported encouraging anti-tumor immunity in patients with head and neck squamous cell carcinoma, attesting to the potential of iNKT cell-based immunotherapies. However, most clinical trials to date have yielded unsatisfactory results since they are based on the direct activation or ex vivo expansion of endogenous iNKT cells, thereby yielding only shortterm, limited clinical benefits to a small number of patients. The low frequency and high variability of iNKT cells in humans (about 0.01-1% in blood), as well as the rapid depletion of these cells post-activation, are considered to be the major stumbling blocks limiting the success of these trials.

[0184] iNKT cells have been engineered from induced pluripotent stem (iPS) cells. See U.S. Pat. No. 8,945,922, incorporated by reference. iPS cells are produced by transducing a somatic cell with exogenous nuclear reprogramming factors, Oct4, Sox2, Klf4, and c-Myc, or the like. Unfortunately, since the transcription level of the exogenous nuclear reprogramming factors decreases with cell transition into the pluripotent state, the efficiency of stable iPS cell lineproduction can decrease. Additionally, transcription of the exogenous nuclear reprogramming factors can resume in iPS cells and cause neoplastic development from cells derived from iPS cells since Oct4, Sox2, Klf4, and c-Myc are oncogenes that lead to oncogenesis.

[0185] As an example, in some embodiments, methods of the disclosure produce iNKT cells in which the iNKT TCR nucleic acid sequence is obtained from a subset of iNKT cells, such as the CD4 / DN / CD8 subsets or the subsets that produce Thl, Th2, or Thl7 cytokines, and includes double negative iNKT cells. In some embodiments, the iNKT TCR nucleic acid sequence is obtained from an iNKT cell from a donor who had or has a cancer such as melanoma, kidney cancer, lung cancer, prostate cancer, breast cancer, lymphoma, leukemia, a hematological malignancy, and the like. In some embodiments, the iNKT TCR nucleic acid molecule has a TCR alpha sequence from one iNKT cell and a TCR beta sequence from a different iNKT cell. In some embodiments, the iNKT cell from which the TCR alpha sequence is obtained and the iNKT cell from which the TCR beta sequence is obtained are from the same donor. In some embodiments, the donor of the iNKT cell from which the TCR alpha sequence is obtained is different from the donor of the iNKT cell from which the TCR beta sequence is obtained. In some embodiments, the TCR alpha sequence and / or the TCR beta sequence are codon optimized for expression. In some embodiments, the TCR alpha sequence and / or the TCR beta sequence are modified to encode a polypeptide having one or more amino acid substitutions, deletions, and / or truncations compared to the polypeptide encoded by the unmodified sequence. In some embodiments, the iNKT TCR nucleic acid molecule encodes a T cell receptor that recognizes alpha-galactosylceramide (alpha- GalCer) presented on CD Id. In some embodiments, the iNKT TCR nucleic acid molecule is contained in an expression vector. In some embodiments, the expression vector is a lentiviral expression vector. In some embodiments, the expression vector is a MIG vector in which the iNKT TCR nucleic acid molecule replaces the IRES-EGFP segment of the MIG vector. In some embodiments, the expression vector is phiNKT-EGFP.

[0186] 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; with transfer 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 arc fusions of singlechain variable fragments (scFv) derived from monoclonal antibodies, fused to CD3-zetatransmembrane and endodomain; CD28 or 41 BB 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.

[0187] Preferably, the CAR is directed to a particular tumor antigen. Examples of tumor cell antigens to which a CAR may be directed include at least 5T4, 8H9, anbb integrin, BCMA, B7- H3, B7-H6, CAIX, CA9, CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD 123, CD 138, CD 171, CEA, CSPG4, EGFR, EGFR family including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, ERBB3, ERBB4, ErbB3 / 4, EPCAM, EphA2, EpCAM, folate receptor- a, FAP, FBP, fetal AchR, FRcc, GD2, G250 / CAIX, GD3, Glypican-3 (GPC3), Her2, IL-13Rcx2, Lambda, Lewis-Y, Kappa, KDR, MAGE, MCSP, Mesothelin, Mucl, Mucl6, NCAM, NKG2D Ligands, NY-ESO-1, PRAME, PSC1, PSCA, PSMA, ROR1, 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-ESO-l / LAGE-1, PAME, SSX-2, Melan- A / MART- 1 , GP100 / pmell7, TRP-1 / - 2, P. polypeptide, MC1R, Prostate-specific antigen, b-catenin, BRCA1 / 2, CML66, Fibronectin, MART-2, TGFARII, 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.

[0188] In some embodiments, a nucleic acid may comprise a nucleic acid sequence encoding an a-TCR and / or a b-TCR, as discussed herein. In certain embodiments, one nucleic acid encodes both the a-TCR and the b-TCR. In additional embodiments, a nucleic acid further comprises a nucleic acid sequence encoding a suicide gene product. In some embodiments, a nucleic acid molecule that is introduced into a selected CD34+ cell encodes the a-TCR, the b- TCR, and the suicide gene product. In other embodiments, a method also involves introducing into the selected CD34+ cells a nucleic acid encoding a suicide gene product, in which case a different nucleic acid molecule encodes the suicide gene product than a nucleic acid encoding at least one of the TCR genes.

[0189] Methods for preparing, making, manufacturing, and using engineered iNKT cells and iNKT cell populations are provided. Methods include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more of the following steps in embodiments: obtaining hematopoietic cells; obtaining hematopoietic progenitor cells; obtaining progenitor cells capable of becoming one or more hematopoietic cells; obtaining progenitor cells capable of becoming iNKT cells; selecting cells from a population of mixed cells using one or more cell surface markers; selecting CD34+ cells from a population of cells; isolating CD34+ cells from a population of cells; separating CD34+ and CD34- cells from each other; selecting cells based on a cell surface marker other than or in addition to CD34; introducing into cells one or more nucleic acids encoding an iNKT T-cell receptor (TCR); infecting cells with a viral vector encoding an iNKT T-cell receptor (TCR); transfecting cells with one or more nucleic acids encoding an iNKT T-cell receptor (TCR); transfecting cells with an expression construct encoding an iNKT T-cell receptor (TCR); integrating an exogenous nucleic acid encoding an iNKT T-cell receptor (TCR) into the genome of a cell; introducing into cells one or more nucleic acids encoding a suicide gene product; infecting cells with a viral vector encoding a suicide gene product; transfecting cells with one or more nucleic acids encoding a suicide gene product; transfecting cells with an expression construct encoding a suicide gene product; integrating an exogenous nucleic acid encoding a suicide gene product into the genome of a cell; introducing into cells one or more nucleic acids encoding one or more polypeptides and / or nucleic acid molecules for gene editing; infecting cells with a viral vector encoding one or more polypeptides and / or nucleic acid molecules for gene editing; transfecting cells with one or more nucleic acids encoding one or more polypeptides and / or nucleic acid molecules for gene editing; transfecting cells with an expression construct encoding one ormore polypeptides and / or nucleic acid molecules for gene editing; integrating an exogenous nucleic acid encoding one or more polypeptides and / or nucleic acid molecules for gene editing; editing the genome of a cell; editing the promoter region of a cell; editing the promoter and / or enhancer region for an iNKT TCR gene; eliminating the expression one or more genes; eliminating expression of one or more HLA-I / II genes in the isolated human CD34+ cells; transfecting into a cell one or more nucleic acids for gene editing; culturing isolated or selected cells; expanding isolated or selected cells; culturing cells selected for one or more cell surface markers; culturing isolated CD34+ cells expressing iNKT TCR; expanding isolated CD34+ cells; culturing cells under conditions to produce or expand iNKT cells; culturing cells in an artificial thymic organoid (ATO) system to produce iNKT cells; culturing cells in serum-free medium; culturing cells in an ATO system, wherein the ATO system comprises a 3D cell aggregate comprising a selected population of stromal cells that express a Notch ligand and a serum-free medium. It is specifically contemplated that one or more steps may be excluded in an embodiment.

[0190] Cells that may be used to create engineered iNKT cells are hematopoietic progenitor stem cells. Cells may be from peripheral blood mononuclear cells (PBMCs), bone marrow cells, fetal liver cells, embryonic stem cells, cord blood cells, or a combination thereof. The present disclosure encompasses "HSC-iNKT cells", invariant natural killer T (iNKT) cells engineered from hematopoietic stem cells (HSCs) and / or hematopoietic progenitor cells (HPCs), and methods of making and using thereof. As used herein, "HSCs" is used to refer to HSCs, HPCs, or both HSCs and HPCs. “Hematopoietic stem and progenitor cells” or “hematopoietic precursor cells” refers to cells that are committed to a hematopoietic lineage but are capable of further hematopoietic differentiation and include hematopoietic stem cells, multipotential hematopoietic stem cells (hematoblasts), myeloid progenitors, megakaryocyte progenitors, erythrocyte progenitors, and lymphoid progenitors. “Hematopoietic stem cells (HSCs)” are multipotent stem cells that give rise to all the blood cell types including myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells), and lymphoid lineages (T- cells, B-cells, NK-cells). In this disclosure, HSCs refer to both “hematopoietic stem and progenitor cells” and “hematopoietic precursor cells”. The hematopoietic stem and progenitor cells may or may not express CD34. The hematopoietic stem cells may co-express CD 133 and be negative for CD38 expression, positive for CD90, negative for CD45RA, negative for lineage markers, or combinations thereof. Hematopoietic progenitor / precursor cells include CD34(+) / CD38(+) cellsand CD34(+) / CD45RA(+) / lin(-)CD10+ (common lymphoid progenitor cells),CD34(+)CD45RA(+)lin(- )CD10(-)CD62L(hi) (lymphoid primed multipotcnt progenitor cells), CD34(+)CD45RA(+)lin(-)CD10(-)CD123+ (granulocyte-monocyte progenitor cells), CD34(+)CD45RA(-)lin(-)CD10(-)CD123+ (common myeloid progenitor cells), or CD34(+)CD45RA(-)lin(-)CD10(-)CD123- (megakaryocyte-erythrocyte progenitor cells).

[0191] Certain methods involve culturing selected CD34+ cells in media prior to introducing one or more nucleic acids into the cells. Culturing the cells can include incubating the selected CD34+ cells with media comprising one or more growth factors. In some embodiments, one or more growth factors comprise c-kit ligand, flt-3 ligand, and / or human thrombopoietin (TPO). In further embodiments, the media includes c-kit ligand, flt-3 ligand, and TPO. In some embodiments, the concentration of the one or more growth factors is between about 5 ng / ml to about 500 ng / ml with respect to either each growth factor or the total of any and all of these particular growth factors. The concentration of a single growth factor or the combination of growth factors in media can be about, at least about, or at most about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 410, 420, 425, 430, 440, 441, 450, 460, 470, 475, 480, 490, 500 (or any range derivable) ng / ml or mg / ml or more.

[0192] In some embodiments, cells are cultured in cell-free medium. In certain embodiments, the serum-free medium further comprises externally added ascorbic acid. In particular embodiments, methods involve adding ascorbic acid medium. In further embodiments, the serum- free medium further comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or all 16 (or a range derivable therein) of the following externally added components: FLT3 ligand (FLT3L), interleukin 7 (IL- 71, stem cell factor (SCF), thrombopoietin (TPO), stem cell factor (SCF), IL-2, IL-4, IL-6, IL-15, IL-21, TNF-alpha, TGF-beta, interferon-gamma, interferon-lambda, TSLP, thymopentin, pleotrophin, or midkine. In additional embodiments, the serum-free medium comprises one or more vitamins. In some cases, the serum-free medium includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of the following vitamins (or any range derivable therein): comprise biotin, DL alpha tocopherol acetate, DL alpha-tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, folic acid nicotinamide, pyridoxine, riboflavin, thiamine, inositol, vitamin B 12, or a salt thereof.In certain embodiments, medium comprises or comprise at least biotin, DL alpha tocopherol acetate, DL alpha-tocopherol, vitamin A, or combinations or salts thereof. In additional embodiments, serum-free medium comprises one or more proteins. In some embodiments, serum- free medium comprises 1, 2, 3, 4, 5, 6 or more (or any range derivable therein) of the following proteins: albumin or bovine serum albumin (BSA), a fraction of BSA, catalase, insulin, transferrin, superoxide dismutase, or combinations thereof. In other embodiments, serum-free medium comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 of the following compounds: corticosterone, D- Galactose, ethanolamine, glutathione, L-camitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, or triodo-I-thyronine, or combinations thereof. In further embodiments, serum-free medium comprises a B-27 supplement, xeno-free B-27 supplement, GS21TM supplement, or combinations thereof. In additional embodiments, serum- free medium comprises or further comprises amino acids, monosaccharides, and / or inorganic ions. In some aspects, serum-free medium comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 of the following amino acids: arginine, cysteine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine, or combinations thereof. In other aspects, serum-free medium comprises 1, 2, 3, 4, 5, or 6 of the following inorganic ions: sodium, potassium, calcium, magnesium, nitrogen, or phosphorus, or combinations or salts thereof. In additional aspects, serum-free medium comprises 1, 2, 3, 4, 5, 6 or 7 of the following elements: molybdenum, vanadium, iron, zinc, selenium, copper, or manganese, or combinations thereof.

[0193] In some methods, cells are cultured in an artificial thymic organoid (ATO) system. The ATO system involves a three-dimensional (3D) cell aggregate, which is an aggregate of cells. In certain embodiments, the 3D cell aggregate comprises a selected population of stromal cells that express a Notch ligand. In some embodiments, a 3D cell aggregate is created by mixing CD34+ transduced cells with the selected population of stromal cells on a physical matrix or scaffold. In further embodiments, methods comprise centrifuging the CD34+ transduced cells and stromal cells to form a cell pellet that is placed on the physical matrix or scaffold. In certain embodiments, stromal cells express a Notch ligand that is an intact, partial, or modified DLL1, DLL4, JAG1, JAG2, or a combination thereof. In further embodiments, the Notch ligand is a human Notch ligand. In other embodiments, the Notch ligand is human DLL1.

[0194] Cells may be used immediately, or they may be stored for future use. hi certain embodiments, cells that are used to create iNKT cells are frozen, while produced iNKT cells maybe frozen in some embodiments. In some aspects, cells are in a solution comprising dextrose, one or more electrolytes, albumin, dextran, and DMSO. In other embodiments, cells arc in a solution that is sterile, nonpyrogenic, and isotonic. In some embodiments, the engineered iNKT cell is derived from a hematopoietic stem cell. In some embodiments, the engineered iNKT cell is derived from a G-CSF mobilized CD34+ cells. In some embodiments, the cell is derived from a cell from a human patient that doesn’t have cancer. In some embodiments, the cell doesn’t express an endogenous TCR.

[0195] Engineered iNKT cells may be used to treat a patient. In some embodiments, methods include introducing one or more additional nucleic acids into the cell population, which may or may not have been previously frozen and thawed. This use provides one of the advantages of creating an off-the-shelf iNKT cell. In particular embodiments, the one or more additional nucleic acids encode one or more therapeutic gene products. Examples of therapeutic gene products include at least the following: 1. Antigen recognition molecules, e.g. CAR (chimeric antigen receptor) and / or TCR (T cell receptor); 2. Co-stimulatory molecules, e.g. CD28, 4-1BB, 4-1BBL, CD40, CD40L, ICOS; and / or 3. Cytokines, e.g. IL-lcc, IL-Ib, IL- 2, IL-4, IL-6, IL-7, IL-9, IL- 15, IL-12, IL-17, IL-21, IL-23, IFN-g, TNF-a, TGF-b, G-CSF, GM-CSF; 4. Transcription factors, e.g. T-bet, GATA-3, RORyt, FOXP3, and Bcl-6. Therapeutic antibodies are included, as are chimeric antigen receptors, single chain antibodies, monobodies, humanized, antibodies, bi-specific antibodies, single chain FV antibodies or combinations thereof.

[0196] In some embodiments, the present disclosure provides kits comprising one or more engineered cells or compositions according to the present disclosure packaged together with a drug delivery device, e.g., a syringe, for delivering the engineered cells or compositions to a subject. In some embodiments, the present disclosure provides kits comprising one or more engineered cells or compositions according to the present disclosure packaged together with one or more reagents for culturing and / or storing the engineered cells. In some embodiments, the present disclosure provides kits comprising one or more engineered cells or compositions according to the present disclosure packaged together with one or more agents that activate cells, e.g., iNKT cells comprising a CAR and at least one additional transgene. In some embodiments, the present disclosure provides kits comprising one or more engineered cells or compositions according to the present disclosure packaged together with OP9-DL1 stromal cells and / or MS5-DL4 stromal cells. In some embodiments, the present disclosure provides kits comprising one or more engineeredcells or compositions according to the present disclosure packaged together with antigen- presenting cells or CDld-cxprcssing artificial antigen-presenting cells.

[0197] Methods of the disclosure provides methods of manufacturing engineered cells for treating any number of conditions and / or diseases. In some embodiments, the present disclosure provides a method of treating a subject, which comprises administering to the subject one or more engineered cells according to the present disclosure, one or more engineered cells made according to a method of the present disclosure, or one or more compositions according to the present disclosure. In some embodiments, the subject is an animal such as a mouse or a test animal. In some embodiments, the subject is a human. In some embodiments, the subject has a cancer, a bacterial infection, a viral infection, an allergy, or an autoimmune disease. In some embodiments, the cancer is melanoma, kidney cancer, lung cancer, prostate cancer, breast cancer, lymphoma, leukemia, or a hematological malignancy. In some embodiments, the subject has tuberculosis, HIV, or hepatitis. In some embodiments, the subject has asthma or eczema. In some embodiments, the subject has Type I diabetes, multiple sclerosis, or arthritis. In some embodiments, the subject is administered a therapeutically effective amount of the one or more engineered cells. In some embodiments, the therapeutically effective amount of the one or more engineered cells is about 10x107 to about 10x109 cells per kg body weight of the subject being treated. In some embodiments, the method further comprises administering an agent that activates iNKT cells, e.g., a-GalCer or salts or esters thereof, a-GalCer-presenting dendritic cells or artificial APCs, before, during, and / or after administration of the one or more engineered cells.

[0198] In certain aspects, this disclosure provides systems and methods for producing T cells with enhanced anti-tumor phenotypes. In particular, this disclosure provides methods of making T cells from stem cells (e.g., HSCs) engineered with multiple transgenes including a T cell receptor (TCR), a chimeric antigen receptor (CAR), and at least one additional transgene. By starting with HSCs, systems and methods of the disclosure take advantage of self-regeneration and cellular differentiation capabilities of stem cells for the manufacture of T cells with improved anti-tumor phenotypes. In particular’, this disclosure provides for introduction of nucleic acids, into CD34+ stem cells, which encode a TCR, a CAR, and at least one an additional transgene. The combined expression of TCRs and CARs is useful for providing T cells with specific cancer cell targeting properties. Moreover, endowed with at least one additional transgene, the T cells produced bymethods of the disclosure are armed with cargo (e.g., cytokines) that, when in contact with the target cancer cell, is useful to treat the cancer.

[0199] For example, in preferred embodiments, methods of the disclosure involve introducing nucleic acids into HSCs via lentiviral transduction. Introduction of the one or more nucleic acids provides for HSCs that express at least one TCR, CAR, and an additional transgene. Incorporation of the additional transgene is useful for providing therapeutic T cells with improved functional properties, such as, improved cell expansion, persistence, safety, and / or antitumor activities.

[0200] The one or more additional transgenes may include any one or more of a cytokine, a checkpoint inhibitor, an inhibitor of transforming growth factor beta signaling, an inhibitor of cytokine release syndrome, or an inhibitor of neurotoxicity. For example, transgenes may be provided that encode one or more of IL-2, IL-7, IL-15, IL-12, IL-18, or IL-21.

[0201] Accordingly, in some instances, methods of the disclosure provide for the manufacture of CAR T cells with improved expansion and persistence capabilities by, for example, introducing nucleic acids encoding one or more of IL-2, IL-7, IL- 1-15. In some instances, methods may provide CAR T cells with increased IFN-g production and thus improved T cell potency by, for example, introduction of transgenes encoding one or more of IL- 12, IL- 18. In other instances, methods of the disclosure are useful for enhancing naive T cell production by introducing transgenes including IL-21. In some instances, methods described herein provide for the production of CAR T cells with improved safety properties by, for example, introducing inhibitors of IL-6, GM-CSF, or other mediators of cytokine release syndrome and neurotoxicity. Methods may provide for CAR T cells with improved efficacy by providing payloads useful for combating tumor microenvironment, e.g., via inhibitors of TGF-B, checkpoints.

[0202] Certain methods of the disclosure involve a single in vitro activation step after a maturation II step. In some instances, the T cells are briefly activated with reagents, e.g., for 1-3 days and following this, activation reagents are often removed from the media so as to not continuously stimulate cells and thus exhaust the cell. Following activation, an activated T cell population may expand rapidly, e.g., double in number every 24 hours. Some reagents may be added to facilitate the expansion. In some embodiments, the method involves a culture media may be supplemented with one or more of IL-7 / 15, IL-2, IL-2+21, IL-12, or IL-18.

[0203] In some instances, the single activation step involves PBMC -based T cell activation. Accordingly, the activation step may involve introducing aGC-loaded PBMCs, soluble anti-CD3 / 28 positive PBMCs, and soluble anti-CD2 / 3 / 28 positive PBMCs to the T cell. In some instances, the activation step involves an antigen presenting cell (aAPC) based T cell activation step. Accordingly, the activation step can involve introducing the T cell to aAPC. The aAPC may be an engineered K562 cell expressing CD80-CD83-CD137L-CAR-antigen. The aAPC may be an aAPC+CDld, and / or aAPC+CDld+ / -aGC. In other instances, the activation step comprises a feeder free-based T cell activation step. The feeder free based T cell activation step can involve introducing, to the T cell, soluble antibodies including anti-CD3, anti-CD28, anti-CD2 / 3 / 28, or anti-CD3 / 28. In some embodiments, the T cell activation step involves culturing the T cell in the presence of different cytokines added to activation expansion culture media, including IL-7 / 15, IL-2, IL-2+21, IL-12, IL-18.

[0204] The methods described herein can further include formulating the engineered immune cells in a pharmaceutical composition. Pharmaceutical compositions may comprise engineered immune cells, as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. Compositions can be formulated, e.g., for intravenous administration.

[0205] Preferably, the pharmaceutical composition is substantially free of, e.g., there are no detectable levels of a contaminant, e.g., selected from the group consisting of endotoxin, mycoplasma, replication competent lentivirus (RCL), p24, VSV-G nucleic acid, HIV gag, residual anti-CD3 / anti-CD28 coated beads, mouse antibodies, pooled human serum, bovine serum albumin, bovine serum, culture media components, vector packaging cell or plasmid components, a bacterium and a fungus. In one embodiment, the bacterium is at least one selected from the group consisting of Alcaligenes faecalis, Candida albicans, Escherichia coli, Haemophilus influenza, Neisseria meningitides, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumonia, and Streptococcus pyogenes group A.

[0206] When “an immunologically effective amount,” “an anti-cancer effective amount,” “a cancer-inhibiting effective amount,” or “therapeutic amount” is indicated, the precise amount of the compositions to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and conditionof the patient (subject). It can generally be stated that a pharmaceutical composition comprising the cells described herein may be administered at a dosage of 104to 109cclls / kg body weight, in some instances 105to 106cells / kg body weight, including all integer values within those ranges. T cell compositions may also be administered multiple times at these dosages.

[0207] In some embodiments, a dose of the engineered immune cells of the disclosure comprises about IxlO6, l.lxlO6, 2xl06, 3.6xl06, 5xl06, IxlO7, 1.8xl07, 2xl07, 5xl07, IxlO8, 2xl08, or 5xl08cells / kg. In some embodiments, a dose of the engineered immune cells of the disclosure comprises at least about IxlO6, l.lxlO6, 2xl06, 3.6xl06, 5xl06, IxlO7, 1.8xl07, 2xl07, 5xl07, IxlO8, 2xl08, or 5xl08cells / kg. In some embodiments, a dose of the engineered immune cells of the disclosure comprises up to about IxlO6, l.lxlO6, 2xl06, 3.6xl06, 5xl06, IxlO7, 1.8xl07, 2xl07, 5xl07, IxlO8, 2xl08, or 5xl08cells / kg. In some embodiments, a dose of the engineered immune cells of the disclosure comprises about l.lxl06-1.8xl07cells / kg. In some embodiments, a dose of the engineered immune cells of the disclosure comprises about IxlO7, 2xl07, 5xl07, IxlO8, 2xl08, 5xl08, IxlO9, 2xl09, or 5xl09cells. In some embodiments, a dose of the engineered immune cells of the disclosure comprises at least about IxlO7, 2xl07, 5xl07, IxlO8, 2xl08, 5xl08, IxlO9, 2xl09, or 5xl09cells. In some embodiments, a dose of the engineered immune cells of the disclosure comprises up to about IxlO7, 2xl07, 5xl07, IxlO8, 2xl08, 5xl08, IxlO9, 2xl09, or 5xl09cells.

[0208] The administration of the subject compositions may be carried out in any convenient manner. The engineered immune cells of the disclosure may be used and / or formulated into compositions described herein may be administered to a patient arterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (IV) injection, or intraperitoneally, e.g., by intradermal or subcutaneous injection. The compositions of cells may be injected directly into a tumor, lymph node, or site of infection.

[0209] The following examples provide useful exemplary protocols for manufacture of T cells (e.g., iNKTs) from CD34+ cells as provided by methods of the disclosure. For further examples and discussion, see W02019241400A1, which is incorporated by reference.EXAMPLESExample 1: Exemplary method for preparing engineered immune cells

[0210] This example provides an exemplary portion of the deep maturation and turbo deep maturation methods of the disclosure in which frozen CD34+ cells arc thawed and transduced with exogenous genes to be expressed, e.g., CARs / TCRs. Briefly, a sample containing CD34+ cells is collected from a donor, purified, tested for purity and cryopreserved. The CD34+ cells are shipped to a laboratory. At the laboratory, the following steps are taken to culture and transduce the cells with LVV containing one or more nucleic acids to be expressed. An overview of this process is provided as part of FIGS. 3A-3B.Thaw and Seeding of CD34+ Cells

[0211] A vial of approximately le6 selected CD34+cells from the vendor are removed from the vapor phase LN2 storage at < -150°C and thawed using a Plasmatherm dry thawer at approximately 37°C. All content in the vial is removed into X-vivo 15 media complete with 50 ng / pL of fetal liver tyrosine kinase (FLT) 3 ligand, 10 ng / pL of IL3, 50 pg / pL of stem cell factor (SCF), and 50 ng / pL of thrombopoietin (TPO) cytokines and transferred into a conical tube. Cells are pelleted in a centrifuge at room temperature. Supernatant is removed and cells are resuspended in X-vivo 15 media with cytokines. Cells are counted using the NC200 NucleoCounter, an automated cell counter that utilizes fluorescence microscopy principles with DNA intercalating dyes to count total cells while determining cell viability. Cells are then transferred into a RetroNectin-coated culture bags. X-vivo 15 media with cytokines is added into the Origen Permalife PL07 or PL30 culture bags with 7.5e5 to l.le6 total viable cells. Cells are cultured at 37 ± 2°C with 5 + 1% CO2 for approximately 18 to 24 hours.Lentiviral Transduction

[0212] After incubation, the CD34+cells are transduced with one or more lentiviral vectors. The vectors are added at a desired total multiplicity of infection (MOI), equal for each vector, and vector volume is calculated based on the total viable cell number seeded on Day 0. Transduction enhancers, for example, Poloxamer 407 or 338 and prostaglandin E2 (PGE2), are mixed with the vector. The mixture of enhancers and vectors is then added into the culture bag and incubated at 37 + 2°C with 5 + 1% CO2 for 24 hours.Differentiation

[0213] On Day 2 of the process, at approximately 24 hours after transduction, culture bags are removed from the incubator and placed in an ISO 5 biological safety cabinet (BSC). Culture bags are flushed with media to lift cells, bags are additionally rinsed and pooled cells arc placed into a conical tube and centrifuged at room temperature. Supernatant is then removed, and cells are resuspended with lymphocyte progenitor expansion media (LPEM). Washed cells are added into lymphocyte differentiation coating media (LDCM)-coated bag. The cells arc then cultured at 37+ 2°C with 5 + 1% CO2 in LPEM to begin differentiation of CD34+cells to the lymphoid progenitor pathway. On Day 9, cells are replenished with the addition of LPEM media into the culture vessel and continued to be cultured at 37 + 2°C with 5 + 1% CO2

[0214] On Day 13, cells are transferred into a conical tube followed by addition of a 1:1 ratio of Dulbecco’s phosphate-buffered saline (DPBS) and gentle cell dissociation (GCD) reagent to the flasks. The culture bags are then briefly incubated at room temperature to detach the remaining adherent cells. Detached cells are pooled into the conical tube with suspension cells. The cells are then pelleted in the centrifuge and resuspended in LPEM media. All cells are seeded into a newly LDCM-coated cell culture bag or alternatively a 1 -layer CellSTACK and incubated at 37 + 2°C with 5 ± 1% CO2 until Day 15. On Day 15, cells are replenished with LPEM media and incubated at 37 + 2°C with 5 + 1% CO2 until Day 16.Maturation

[0215] On Day 16, the maturation phase begins by culturing the cells with T cell progenitor maturation media (TPMM). The suspension cells are drained from the cell culture vessel into a conical tube or transfer bag followed by the addition of approximately a 1 : 1 ratio of GCD and DPBS solution to the culture vessel and a brief incubation at room temperature. The detached cells are combined with the suspension cells, pelleted in the centrifuge and resuspended in TPMM media. Alternatively, lifted cells can be washed on the Gibco CTS Rotea Counterflow Centrifugation System and eluted into TPMM media. Cells are then seeded into an LDCM-coated HYPERStack-12 (HS-12) cell culture vessel and incubated at 37 ± 2°C with 5 ± 1% CO2 until Day 23.

[0216] Maturation II On process Day 23, suspension cells are transferred from the HS-12 into a transfer bag, washed on the CTS Rotea Counterflow Centrifugation System and eluted into TPMMsupplemented with IL- 15. All cells are seeded into newly LDCM-coated HS-12 containing TPMM + IL15 media. Approximately 12.5 pL per lc6 cells of CD3 / CD28 / CD2 ImmunoCult is added to the cells and incubated at 37 ± 2°C with 5 ± 1% CO2 until Day 30.Activation

[0217] Activation of cells occurs by stimulation with irradiated aAPC, an engineered K562 cell expressing CD80-CD83-CD137L-CAR antigen (CD19 in some exemplified aspects of the disclosure). On Day 30, suspension cells are transferred from the HS- 12 into a transfer bag, washed using CTS Rotea Counterflow Centrifugation System and eluted into OpTmizer basal media containing OpTmizer supplement, immune cell serum replacement (ICSR), L-glutamine, Interleukin 7 (IL-7), IL-15, IL-21, and Duvelisib. Washed cells are seeded into a 10 L Xuri bag and cultured with irradiated aAPC cells at a 4 cells to 1 aAPC cell in a Xuri Cell Expansion System bioreactor. Activation in the Xuri provides incubation of the cells at 37 ± 0.5°C with 5 ± 1% CO2. Cell counts for the Xuri are taken from Days 33 to 37 to monitor cell growth and viability. Dilution and perfusion occur from Day 33 to 37 to provide nutrients to the activated cells and remove metabolic waste such as lactate.Harvest and Formulation

[0218] Harvesting of the final product begins on Day 37. The cells are then split into 2 bags; each bag is washed on a separate CTS Rotea Counterflow Centrifugation System and eluted into a buffer containing a 10% dextran 40 in saline solution and HSA. When the wash is complete, the cell bags are combined and a cell count is used to calculate the amount of formulation buffer needed to dilute the cells to the final concentration. The final formulation buffer contains 4.5% w / v dextran 40, 2.5% w / v HSA, and 45% v / v Cryostor CS10.Example 2: Fill Finish Protocol

[0219] An overview of this process is provided in Fig. 3B, which continues the process set forth in Example 1.Fill Finish and Cryopreservation

[0220] Formulated cells are fdled into 6, 10, 20, or 50 mL AT-Closed vials for cryopreservation. After formulation, the cells arc counted to confirm the concentration of cells being added into AT- Closed vials. The culture bag containing cells is attached to the Ml filling kit in the BSC and placed on a rocker to ensure constant mixing of the formulated cells. The Ml kit is connected to the Crystal Ml filling station. Volume checks and recalibrations are conducted to ensure vial homogeneity during the filling process. All filled AT-Closed vials are loaded in custom AT-nest cryopreservation racks with a “chessboard pattern” and transferred to the controlled rate freezer (CRF). AT-Closed vials are frozen in a CRF with a customized profile to ensure accurate nucleation and controlled cooling of the cells. An exemplary CRF profile is provided in the table below.Customized CRF profile

[0221] Cells prepared using the deep maturation methods and turbo deep maturation methods revealed distinct and favorable characteristics. Advantageously and surprisingly, the present inventors discovered that using the combined maturation II / activation steps of the deep and turbodeep maturation methods of the disclosure produced engineered immune cells that exhibit, relative to methods that incorporate solely activation or maturation: (a) improved cell viability; (b) improved cell purity; (c) improved expression of introduced immune cell receptors (e.g., introduced T cell receptors [TCRs] and / or chimeric antigen receptors [CARs]); increased expansion of the engineered immune cells; and (d) improvements in cell profile shown in increases in expression of CCR7, CXCR3, NKp44, and NKG2D. Furthermore, despite using both maturation II and activation steps, the presently disclosed deep maturation and turbo deep maturation methods of the disclosure, the methods are able to produce high numbers of engineered immune cells, with a high purity, within 30-40 days of initially transducing donor cells to produce the resulting engineered immune cells.Example 3: Addition of P13K Inhibitor and / or IL-21

[0222] This example provides experimental results and data for producing T cells using the methods of the present disclosure, in which cells were produced in media containing added IL-21 and / or the P13K inhibitor duvelisib.

[0223] In this example, blood was obtained from donors from which, CD34+ HSPCs were obtained. The following Sample IDs correlate the gene products encoded by two nucleic acids introduced into the donor cells by vector transduction:Sample ID ConstructFull Scale Platform Introduced Nucleic Acid 1Invariant natural killer T-cell receptor beta (iTCRb) gene product- anti-CD19 CAR (19CAR)- anti-CD20 CAR (20CAR)Introduced Nucleic Acid 2- IL-15Invariant natural killer T-Cell receptor alpha (iTCRa) gene productGd 2+2 Introduced Nucleic Acid 1- 19CART-cell receptor delta (TCR6) gene productIntroduced Nucleic Acid 2T-cell receptor gamma (TCRy) gene productTethered IL- 15NKT 2+2 Introduced Nucleic Acid 1- 19CAR- iTCRbIntroduced Nucleic Acid 2- iTCRaTethered IL- 15

[0224] To assess the quantitative and qualitative effects caused by the addition of IL-21 and a P13K inhibitor (duvelisib) during the manufacturing process, the Full Scale Platform, Gd 2+2, and NKT 2+2 cells were produced. Data from these manufacturing runs was compared relative to prior results using the manufacturing process to produce anti-CD19 / anti-CD20 CAR-T cells in the absence of IL-21 and duvelisib. The Gd 2+2 uses a 19CAR containing a CD28 costimulatory domain.

[0225] The following is the deep maturation “Research” protocol used to produce cells in this example. A related, scaled-up process, which is referenced throughout this disclosure, is provided in Fig. 3A.

[0226] Briefly, for the “Research” protocol, a sample containing CD34+ cells was collected from a donor, purified, tested for purity and cryopreserved. The following steps are taken to culture and transduce the cells with LVVs containing the two nucleic acids that each encode one of the introduced nucleic acids. Across the cells lines described above, this protocol was used to produce all cell lines with cell lines Gd 2+2 and NKT 2+2 cells receiving a spike of IL-21 and duvelisib during the final week of manufacture, at around day 30. Duvelisib is added to media to reach afinal concentration of 1 micromolar, while, IL-21 is added to media to reach a final concentration of 20 nanograms per milliliter.

[0227] Day 0: Pre- stimulation (i.e., first day of “Research” protocol)

[0228] General steps:1. Thaw CD34+ cells using a water bath.2. Wash cells in conical tubes using a centrifuge.3. Seed the CD34+ cells onto RetroNectin coated well, plate, vial or bag.Variations in certain early steps in the protocols disclosed herein may be used to control the density, and ultimately total number, of cells produced. Examples of these variations are provided herein, and are referred to, in order of increasing number of cells produced: “Normal Density”, “Medium Density”, and “High Density”.Normal Density: Coat appropriate number of wells of a 24-well non-TC plate with 0.5 mL / well retronectin (RN) at 20 ug / ml, diluted in IX PBS. Coat wells with 0.5 mL / well of 2% BSA, diluted in IX PBS. Replace with 0.5 mL PBS / well until cells are ready to seed. Thaw CD34+ 200,000 cell (e.g., -180,000 viable cells) aliquots, which are divided among 12 wells. Spin cells in X-VIVO- 15 with thawed cells, and resuspend cells at -15,000 cells / 300uL in stem cell media. Seed 300uL of resuspended cells on retronectin coated 24 well plate (-625 cells per well).Medium Density: Coat appropriate number of wells of a 24-well non-TC plate with 0.5 mL / well retronectin (RN) at 20 ug / ml, diluted in IX PBS. Coat wells with 0.5 mL / well of 2% BSA, diluted in IX PBS. Replace with 0.5 mL PBS / well until cells are ready to seed. Thaw CD34+ 200,000 cell (e.g., -180,000 viable cells) aliquots, which are divided among 12 wells. Spin cells in X-VIVO- 15 with thawed cells, and resuspend cells at -15,000 cells / 50uL in stem cell media. Seed 50uL of the resuspended cells in each well of a retronectin coated 96 well plate (-15,000 cells per well).High Density: Coat appropriate number of wells of a 24-well non-TC plate with 0.5 mL / well retronectin (RN) at 20 ug / ml, diluted in IX PBS. Coat wells with 0.5 mL / well of 2% BSA, diluted in IX PBS. Replace with 0.5 mL PBS / well until cells are ready to seed. Thaw CD34+ 200,000 cell (e.g., -180,000 viable cells) aliquots, which are divided among 12 wells. Spin cells in X-VIVO- 15 with thawed cells, and resuspend cells at -50,000-60,000 cells / 50uL in stem cell media. Seed the 50uL of the resuspended cells in each well of a retronectin coated 96 well plate (-50,000- 60,000 cells per well).

[0229] Day2: Transduction

[0230] General steps:1. Thaw concentrated lentiviral vectors (LVV) and pipet gently to mix (do not vortex and do not refreeze). Each transduction includes two LVV (“LVV 1” and “LVV 2”), each encoding one of two nucleic acids used to produce cells.2. Prepare transduction tube - transfer appropriate volume of LVV 1 and LVV 2 to tube by calculating volume sufficient for a desired MOI.3. Add contents of transduction tube to CD34+ cell culture(s) in well(s) and rock plate gently to mix4. Incubate cells.Normal Density: Thaw concentrated lentiviral vector (LVV) and pipet gently to mix (do not vortex and do not refreeze). Transfer appropriate volume of LVV to 1.5 ml tube by calculating volume sufficient for the MOI desired (-500 in Normal Density variation of protocol). Add appropriate volume of PGE2 to same tube to achieve a final concentration of 10 nM of total culture volume. Add appropriate volume of poloxamer (aka P338) to same tube to achieve a final concentration of lug / ul of total culture volume. Bring up total volume to 100 uL by adding appropriate volume of Stem Cell media. Add contents of transduction tube to appropriate culture well and rock plate gently to mix.Medium & High Density: Thaw concentrated lentiviral vector (LVV) and pipet gently to mix (do not vortex and do not refreeze). Dilute PGE2 1:10 in Stem Cell media (typically 10 uL PGE2 in 90 uL Stem Cell media is more than sufficient). Transfer appropriate volume of LVV to 1.5 mltube by calculating volume sufficient for the MOI desired (-200). Add appropriate volume of PGE2 to same tube to achieve a final concentration of 10 nM of total culture volume. Add appropriate volume of poloxamer (aka P338) to same tube to achieve a final concentration of lug / ul of total culture volume. Add the correct volume of transduction mix per well per condition (final column of table below).

[0231] Day 2 through 16: Differentiation (Duration: 2 weeks)

[0232] General Steps:1. Coat appropriate number of wells of differentiation plate with differentiation agent or coating, for example, coating the wells with 1 ml / well of a lymphoid differentiation coating material (LDCM), such as, the lymphoid differentiation material provided under the trade name StemSpan by STEMCELL. Incubate 12-18 h.3. Resuspend transduced CD34+ cells collected in Stage 1 in a lymphoid progenitor expansion medium (LPEM), such as, the lymphoid progenitor expansion medium sold under the trade name StemSpan by STEMCELL.4. Adjust cell density to l-2xl0 cells / ml with LPEM5. Aspirate PBS from LDCM-coated plates6. Seed 0.75 ml cells / well into a LCDM-coated plate7. Incubate cells at 37 degrees Celsius, 5 percent CO28. On day 53, add 0.25 ml / well of fresh LPEM and continue culture9. On days 9 and 13, carefully remove <0.5 ml / well without disturbing cells and replenish with 0.5 ml / well of fresh LPEM10. Continue culture to day 16Normal Density: Each well of the normal density cells from Day Obecomes two wells at day 0 (third day). Coat appropriate number of wells of 12-well non-tissue culture-treated plates with 1 ml / well of StemSpan lymphoid differentiation coating material (LDCM) diluted 1:100 in PBS. Incubate at RT for 2 hours. Aspirate LDCM and add 2 ml / well of PBS. Collect and pool cells by pipetting gently to remove them from the plate and transfer to conical tube. Add 300 uL Gentle Cell Dissociation reagent per well, incubate at RT for 1-2 minutes. Collect and pool cells by pipetting gently to remove them from the plate and transfer to conical tube. Spin at 300g for 10 minutes and carefully aspirate supernatant. Aspirate PBS from LDCM-coated wells right before adding cells. Resuspend cells (per donor) at appropriate volume to seed 1 mL / well with IX LPEM diluted in Stem Span SFEM II basal media. Incubate cells at 37 °C, 5% CO2.Medium / High Density: Coat appropriate number of wells of 12-well non-tissue culture-treated plates with 1 ml / well of StemSpan lymphoid differentiation coating material (LDCM) diluted 1 : 100 in PBS. Incubate at RT for 2 hours. Aspirate LDCM and add 2 ml / well of PBS. Add 100 uL LPEM to each well of cells in 96 well plate and pipette up and down gently to resuspend cells. Move cells into appropriate volume of LPEM per condition (7,500 cells / mL). Add 100 uL Gentle Cell Dissociation reagent per well, incubate at RT for 1-2 minutes. Collect cells, add to previously collected cells in LPEM. Aspirate PBS from LDCM-coated wells right before adding cells. Add 1 mL of cells in LPEM (7,500 cells per well) into each well of the 12-well plate. Incubate cells at 37°C, 5% CO2.In certain preferred aspects, a split may occur at some point between days the 13 through 16 day, in which the cells are divided and either expanded in parallel or one portion discarded if not needed.

[0233] Maturation

[0234] Days 16 through 23(Duration: 1 week)1. Cells are washed in conical tubes using a centrifuge.2. Cells are matured on plates.3. The cells are incubated in TPMM mediaIn some preferred aspects, a split may occur at some point between days 19 and 22, in which the cells are divided and either expanded in parallel or one portion discarded if not needed.Maturation TI Day 23 (24th1. At Day 23, the cells arc washed in conical tubes using a centrifuge.2. The cells undergo a second maturation with Immunocult™ human CD3 / CD28 / CD2 T cell activator and IL- 15.Generally there is 3-10 fold expansion in the first week of Maturation II.ActivationDay 301. At Day 30 cells are washed in conical tubes using a centrifuge.2. CD19-expressing K562 cells are added at a 4:1 ratio (cells:K562 cells).3. Cells are seeded in T225 Flask at a density of le6 cells / mL in complete Optimizer + IL-7 and IL- 15.Harvest and FormulationDay 371. At Day 37, the cells are washed in conical tubes using a centrifuge.2. An aliquot of cells is pulled and used to produce a formulate with dextran 40 in saline, 2.5% human serum albumin and Cryostor CS10.Fill Finish and CryopreservationDay 371. Cryovials are manually filled using a pipette.2. The cells are cryopreserved using Coolcell (Corning).In the case of Gd 2+2 and NKT 2+2 cells, the cells were exposed to added IL-21 and a P13K inhibitor (duvelisib) during activation, specifically on days days 30, 33, and 35. In the exemplary protocol above and described with reference to the cells produced in the examples, on the listed days Duvelisib was added at luM (a 1:10,000 dilution) and IL-21 at 20 ng / mL (a 1:1000 dilution from stock).

[0235] Additionally, as set forth in the protocol, after the frozen cells were obtained from a donor and thawed, the total viable cell counts were measured at thaw and at days 2, 16, 23, and 30. As shown in Figs. 4A-4B, the donor (PD50) that provided cells used to produce Gd 2+2 and NKT 2+2 anti-CD19 cells exhibited approximately a 50% lower total number of viable CD34+ HSPC recovered than donors from whom cells were obtained to produce other cells lines describedherein. Consequently, those cell lines were produced using only half the target number of donor cells.

[0236] During the day 30 wash, there was an instrument failure that caused the loss of some cells. Nevertheless, as shown in Fig. 5, by harvest, both cell types produced with the addition of IL-21 and a P13K inhibitor (duvelisib) during the final week of manufactured, produced an equivalent number of total viable cells, percent of viable cells, and cells of a similar size to methods that did not employ IL-21 and duvelisib, but otherwise started with at least twice the number of viable cells.

[0237] Surprisingly, as shown in Fig. 5, the Gd 2+2 and NKT 2+2 cells had the highest total viable cell (TVC) recovery at harvest and produced the highest vial number (e.g., potential therapeutic dose) calculated using the post-harvest TVC. As shown in Fig. 7, (upper panel) the Gd 2+2 and NKT 2+2 cells expressed the introduced TCR and CAR at over 80% when produced using the presently disclosed method incorporating IL-21 and a P13K inhibitor (duvelisib) during the final week.

[0238] Thus, as demonstrated herein, using IL-21 and duvelisib appeared to improve expansion of transduced cells in both the “Research” protocol and the high-throughput full-scale protocol. Both the “Research” and full-scale protocols produced a cellular product in which comparatively high proportions of the T cells were successfully transduced and thereby express the introduced TCR and CAR. This effective transduction was consistent using this high-throughput method as were rates measured using a low-throughput, research-grade method for producing the same cells. As shown in Fig. 8, the cells were produced with an acceptable vector copy number (VCN) of around 7.4 per cell, which was consistent with the VCN using a lower-throughput research-grade production. The cells were also produced with low levels of residual K562 cells, with measurements below LOQ (0.5%) in the final product.

[0239] To assess the cytotoxicity of the cells, the cells underwent a potency, short-term cytotoxicity and serial killing assays using CD19+ expressing target cells. Cytotoxicity was measured using CD107a+ expression. As shown in Fig. 9A, the potency assay indicated that the cells manufactured using IL-21 and duvelisib exceeded the potency of the CD19 / CD20 cells produced in the absence of IL-21 and duvelisib. Moreover, as a control, a Gd cell line was also produced using the method outline above, but without adding IL-21 and duvelisib. Surprisingly, it appears that the addition of IL-21 and duvelisib during the manufacture process, not only helpsproduce higher number of viable cells, but also cells that exhibit a higher potency. As shown in Fig. 9B, short-term cytotoxicity and serial killing results indicate, relative to the ACUA cells described above, that the cells produced using IL-21 and duvelisib achieved a durable cytotoxic response.

[0240] As shown in Fig. 10, the final product was pure and free of residual, endogenous TCR cells and K562 cells. The final product met standards for osmolality and pH.

[0241] Accordingly, as shown, use of duvelisib and IL-21 in the methods disclosed herein produces a higher number of viable engineered CAR-T cells, they exhibit a highly potent and durable cytotoxic response relative to cells manufactured in the absence of P13K inhibitor and IL- 21.Incorporation by Reference

[0242] References and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, have been made throughout this disclosure. All such documents are hereby incorporated herein by reference in their entirety for all purposes.Equivalents

[0243] Various modifications of the disclosure and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the full contents of this document, including references to the scientific and patent literature cited herein.

Claims

CL IMSWhat is claimed is:

1. A method of producing engineered T cells, the method comprising: conducting a process comprising in vitro differentiation of a hematopoietic stem cells(HSC) into engineered T cells expressing one or more introduced genes; inducing the engineered T cells; and activating the induced T cells.

2. The method of claim 1, wherein prior to the inducing step, the method further comprises a step of maturating the engineered T cells prior to the inducing step.

3. The method of claim 1 or 2, further comprising formulating the cells into a pharmaceutical composition.

4. The method of claim 3, wherein formulating the cells comprises combining the activated t cells with one or more of HAS, Dextran40 and a cryoprotectant to formulate the pharmaceutical composition.

5. The method of claim 4, further comprising cry opreserving the pharmaceutical composition.

6. The method of claim 5, wherein the cryoprotectant is an animal protein-free, serum-free cryopreservation medium.

7. The method of any one of claims 3-6, further comprising providing the pharmaceutical composition for use in a treatment.

8. The method of any one of claims 1-7, wherein the T cell is purified from a TCR negative cell.

9. The method of any one of claims 1 -8, wherein the method is performed without a cell purification step.

10. The method of any one of claims 1-9, wherein the at least one introduced gene causes the T cell to express at least one TCR or CAR.

11. The method of claim 10, wherein the at least one introduced gene comprises a plurality of genes, and said plurality of genes causes the T cell to express at least one introduced TCR and at least one CAR.

12. The method of claim 11, wherein the plurality of introduced genes further comprises one or more additional transgenes.

13. The method of claim 12, wherein the one or more additional transgenes comprise at least one of a cytokine, a checkpoint inhibitor, an inhibitor of transforming growth factor beta signaling, an inhibitor of cytokine release syndrome, or an inhibitor of neurotoxicity.

14. The method of claim 13, wherein the one or more additional transgenes comprise a gene causing the T cell to express one or more cytokine selected from IL-2, IL-7, IL-15, IL-12, IL-18, IL-21, or any combination thereof.

15. The method of any one of claims 1-14, wherein the HSC is derived from a progenitor cell.

16. The method of any one of claims 1-15, wherein the progenitor cell is a pluripotent stem cell.

17. The method of claim 16, wherein the pluripotent stem cell is obtained from a body fluid.

18. The method of any one of claims 1-17, wherein differentiating the stem cells comprises generating double negative progenitor T cells.

19. The method of claim 18, further comprising treating the double negative progenitor cells with a cocktail of cytokines and / or chemokines, and growth factors to thereby produce the T cells.

20. The method of any one of claims 1-19, wherein the method is performed in less than 40 days.

21. The method of any one of claims 1-20, wherein the method is performed in less than 38 days.

22. The method of any one of claims 1-21, wherein the method is performed in less than 37 days.

23. The method of any one of claims 1-22, wherein the method is performed in less than 36 days.

24. The method of any one of claims 1-23, further comprising one or more steps of analyzing the T cells to identify one or more proteins expressed by the T cells.

25. The method of claim 13, wherein the one or more proteins include CCR7, CD62L, CD45RA, CXCR3, CD56, NKp44, and NKG2D.

26. The method of any one of claims 1-25, wherein the T cell is an invariant natural killer T (iNKT) cell.

27. The method of claim 26, wherein the iNKT cell is an alpha / beta iNKT cell.

28. The method of any one of claims 1-27, wherein the method involves a single in vitro T cell activation step.

29. The method of any one of claims 1 -28, wherein the activation step involves culturing the T cell in activation media comprising activation antibodies.

30. The method of any one of claims 1-28, during the T cell activation step, the method does not involve introducing different types of activation antibodies to the T cell.

31. The method of any one of claims 1-30, wherein the T cell activation step lasts no longer than 7 days.

32. The method of any one of claims 1-31, wherein the activation step comprises a PBMC- based T cell activation step.

33. The method of any one of claims 1-32, wherein the activation step involves alpha- galactosylceramide-loaded PBMCs, soluble anti-CD3 / CD28+ PBMCs, and soluble anti- CD2 / 3 / 28+PBMCs.

34. The method any one of claims 1-31, wherein the activation step comprises an aAPC- based T cell activation step.

35. The method of claim 34, wherein the activation step involves aAPCs comprising an engineered K562 cell expressing a CD80-CD83-CD137L-CAR-antigen, an aAPC+CDld, and / or an aAPC+CDld+ / -aGC.

36. The method of any one of claims 1-29, wherein the activation step involves soluble antibodies comprising anti-CD3 +, anti-CD28, anti-CD2 / 3 / 28, and anti-CD3 / 28.

37. The method of any one of claims 1-36, wherein the activation step involves a culture media comprising one or more of IL-7 / 15, IL-2, IL-2+21, IL-12, IL-18, or IL-15.

38. The method of any one of claims 1-36, wherein the activation step involves a culture media comprising one or more of IL-2, IL-2+21, IL-12, IL-18, IL-7 and no exogenous IL-15.

39. A method of producing engineered T cells, the method comprising: conducting a process comprising in vitro differentiation of a hematopoietic stem cells (HSC) into engineered T cells expressing one or more introduced genes; maturing the engineered T cells; inducing the matured engineered T cells; and activating the induced T cells, wherein the activated T cells are contacted with a P13K inhibitor.

40. The method of claim 39, wherein the T cells are contacted with the P13K inhibitor during the maturing step.

41. The method of claim 39 or claim 40, wherein the T cells are contacted with the P13K inhibitor during the inducing step.

42. The method of any one of claims 39-41, wherein the T cells are contacted with the P13K inhibitor during the activating step.

43. The method of any one of claims 39-42, wherein the P13K inhibitor is selected from alpelisib (BYL719), Buparlisib (BKM120), Duvelisib, CH5132799 / PA-79, Copanlisib (BAY SO- 6946), Idelalisib (GS-1101), Pictilisib (GDC-0941), Pilaralisib (XL-147), Serabelisib (MLN1117), Taselisib (GDC-0032), and Umbralisib.

44. The method of claim 43, wherein the P13K inhibitor is Duvelisib.

45. The method of anyone of claims 39-40 claim 44, wherein the T cells are contacted with a Bruton's tyrosine kinase (BTK) inhibitor.

46. The method of any one of claims 39-45, wherein the T cells are contacted with IL-21 with the P13K inhibitor or BTK inhibitor.

47. The method of any one of claims 39-46, further comprising formulating the cells into a pharmaceutical composition.

48. The method of claim 48, wherein formulating the cells comprises combining the activated T cells with one or more of HSA, Dextran40 and a cryoprotectant to formulate the pharmaceutical composition.

49. The method of claim 48, further comprising cryopreserving the pharmaceutical composition.

50. The method of claim 49, wherein the cryoprotectant is an animal protein-free, serum-free cryopreservation medium.

51. The method of any one of claims 39-50, wherein the T cell is purified from a TCR negative cell.

52. The method of any one of claims 39-51, wherein the method is performed without a cell purification step.

53. The method of any one of claims 39-52, wherein the at least one introduced gene causes the T cell to express at least one TCR or CAR.

54. The method of claim 53, wherein the at least one introduced gene comprises a plurality of genes, and said plurality of genes causes the T cell to express at least one introduced TCR and at least one CAR.

55. The method of claim 54, wherein the plurality of introduced genes further comprises one or more additional transgenes.

56. The method of claim 55, wherein the one or more additional transgenes comprise at least one of a cytokine, a checkpoint inhibitor, an inhibitor of transforming growth factor beta signaling, an inhibitor of cytokine release syndrome, or an inhibitor of neurotoxicity.

57. The method of claim 56, wherein the one or more additional transgenes comprise a gene causing the T cell to express one or more cytokine selected from IL-2, IL-7, IL-15, IL-12, IL-18, or any combination thereof.

58. The method of claim 57, wherein the cytokine is a tethered IL- 15.

59. The method of any one of claims 39-58, wherein the HSC is derived from a progenitor cell.

60. The method of claim 59, wherein the progenitor cell is a pluripotent stem cell.

61. The method of any one of claims 39-60, wherein differentiating the stem cells comprises generating double negative progenitor T cells.

62. The method of claim 62, further comprising treating the double negative progenitor cells with a cocktail of cytokines and / or chemokines, and growth factors to thereby produce the T cells.

63. The method of any one of claims 39-62, wherein the method is performed in less than 40 days.

64. The method of any one of claims 39-63, wherein the method is performed in less than 38 days.

65. The method of any one of claims 39-64, wherein the method is performed in less than 37 days.

66. The method of any one of claims 39-65, wherein the method is performed in less than 36 days.

67. The method of any one of claims 63-66, wherein the T cells are contacted with the Pl 3k inhibitor during the final 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 days.

67. The method of any one of claims 63-66, wherein the T cells are contacted with the Pl 3k inhibitor during the final 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 days.

67. The method of any one of claims 63-66, wherein the T cells are contacted with the Pl 3k inhibitor on three separate days during the final 7 days of the method.

68. The method of claim 67, wherein the T cells are contacted with IL-21 on the three separate days during the final 7 days of the method.

69. The method of claim 68, wherein the T cells are contacted with the PI3k inhibitor at a concentration of between about .OluM and 2uM.

70. The method of claim 69, wherein the T cells are contacted with IL-21 at a concentration of 20 ng / mL.

71. The method of any one of claims 39-70, wherein the activation step involves a culture media comprising one or more of IL-2, IL-2, IL-21, IL-12, IL-18, IL-7 and no exogenous IL-15.

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