ENGINEERING STEM CELL T CELLS WITH MULTIPLE T CELL RECEPTORS

TECHNICAL FIELD

This disclosure relates to methods of engineering stem cells with multiple T cell receptors.

BACKGROUND

The generation and expansion of T cells in vitro is useful for a broad range of clinical applications, including cancer treatment. For example, 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.

SUMMARY

This disclosure provides 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), 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.

In particular embodiments, this disclosure provides methods of making multi-TCR 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.

Advantages of using stem cells (e.g., HSCs) in the methods of the invention, include their abilities for regeneration and expansion. Allogenic 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.

In certain aspects, methods of the invention include producing engineered T cells from HSCs by introducing a transgene encoding at least a first TCR. The HSCs are differentiated into T cells, which express the TCR. After differentiation, the T cells can be subject to one or more selection, maturation, expansion, and/or cryopreservation steps. Before or after any of these aforementioned steps, one or more different TCRs/CARs can be introduced into the T cells (e.g., via introduction of a transgene), thereby producing an engineered T cell with multiple, different TCRs and/or TCRs and CARs.

The ability to add desired TCRs/CARs to T cells derived from HSCs confers several advantages to the methods of the invention. For example, in certain methods of the invention, HSCs are differentiated into T cells. These T cells can be matured, selected, expanded, and/or cryopreserved. Generally, the steps of this process take a total of around 2-4 weeks. Using the methods of the invention, one or more additional TCRs/CARs can be introduced into the T cells after their differentiation; and optionally after their selection, maturation, expansion, and/or cry opreservation.

Adding the additional TCR(s) after differentiation allows the T cells to be engineered with receptors that target antigens unique to a pathology, e.g., cancer, starting from an off-the- shelf engineered T cell that itself was derived from an HSC. This can reduce the lead time required to produce T cells with desired combinations of TCR(s) and/or CARs, as the additional receptors can be added after the 2-4 week time period used to produce the single receptor T cells.

Additionally, by adding the additional TCR(s) or CAR after the initial TCR, the methods of the invention may avoid the risk of transcriptional and phenotypic changes in the T cells due to a prolonged culture while expressing multiple, different introduced receptors. Further, by having differentiated T cells ready to accept the additional, the methods of the invention provide greater flexibility to produce multi-TCR T cells with minimal TCR mispairing. TCR mispairing is a phenomena in which the a or P chains of an endogenous or introduced TCR incorrectly pair with those of another introduced TCR. This causes, for example, reduced surface expression of the introduced TCR(s). By using the methods of the invention, the first introduced TCR can be engineered such that it has a reduced potential for mispairing with the second introduced TCR, e.g., through the use of a modifications, such as murine constant domains, disulfide bridges, and the like. Alternatively or additionally, the first introduced TCR can be a gamma delta (y6) TCR, while the second is an alpha beta (aP), which likewise reduces the potential for mispairing.

Thus, the present invention provides a platform for creating allogeneic, off-the-shelf, non-HLA restricted NKT or y6 T-cells, which may be cryopreserved and subsequently thawed for use. These cells may then be further engineered in a patient specific way, e.g., through the introduction of a CAR or TCR targeting a specific pathology. Therefore on stem cell cultures and compositions of the invention may be used to treat thousands of different patients across myriad pathologies.

The present invention provides methods for producing an engineered T cell. An exemplary method includes conducting a process of in vitro differentiation of a hematopoietic stem cell (HSC) to produce an allogeneic gamma delta (y6) T cell or invariant natural killer T (iNKT) cell comprising a first T cell receptor (TCR) that is not HLA restricted. The method further includes introducing at least a second TCR into the y6 T cell or iNKT cell to produce a T cell with two distinct TCRs. In certain aspects, the second or additional TCRs may be HLA restricted or patient specific.

In certain aspects, the method also includes maturing the T cell after introducing the at least second TCR into the y6 T cell or iNKT cell.

In some methods of the invention, the HSC is differentiated into a y6 T cell. In certain aspects, the second TCR is an alpha beta (aP) TCR. In some methods of the invention, the HSC is differentiated into an iNKT. In certain aspects, the second TCR is an alpha beta (aP) TCR.

In certain methods, the in vitro differentiation step includes introducing one or more nucleic acids encoding the first T cell receptor into the HSC. Introducing the second TCR may include introducing one or more nucleic acids encoding the second T cell receptor into the y6 T cell or iNKT cell.

The in vitro differentiation step may further include introducing one or more nucleic acids encoding at least one of a chimeric antigen receptor (CAR) and one or more transgene. In certain aspects, the at least one or more transgene includes at least one of a cytokine, a checkpoint inhibitor, an inhibitor of transforming growth factor beta signaling, an inhibitor of cytokine release syndrome, an inhibitor of neurotoxicity, or other payload to make the T cell more potent or less susceptible to exhaustion or rejection.

In certain methods of the invention, the first and/or second TCR is an engineered TCR. In certain aspects, the first TCR is an engineered TCR. In certain aspects, the second TCR is an engineered TCR. An engineered TCR in accordance with the invention may include one or more modifications to prevent TCR mispairing between the first and second TCRs. Exemplary modifications include one or more of murine constant domains, disulfide bridges, and other dimerizing domains.

In certain methods, the HSC is derived from a progenitor cell, such as a pluripotent stem cell. The in vitro process may thus further include gene editing of the HSC or progenitor cell to make the T cell more potent or less susceptible to exhaustion or rejection.

In an exemplary method, the second TCR is directed to a cancer germline antigen, viral antigen or tumor specific neo-antigen.

In certain aspects, the step of introducing at least a second TCR includes introducing a plurality of different TCRs. Each different TCR may be directed to a different tumor specific neo-antigen. The neoantigen reactive TCRs may be from or derived from peripheral blood T cells or tumor infiltrating lymphocytes.

In certain exemplary methods, the step of introducing the second TCR comprises inserting one or more nucleic acids into the y6 T cell or iNKT cell via retroviral transduction, lentiviral transduction, or non-viral methodologies of nucleotide transfer.

In certain aspects, the method further includes in vitro activation and expansion of the T cell using HLA matched or partially matched PBMCs loaded with peptides recognized by the second TCR.

The presently disclosed invention also provides method of treatment using the multi-TCR cells of the invention. An exemplary method of treatment includes, obtaining an HSC and conducting a process of in vitro differentiation of the HSC into a y6 T cell or iNKT cell, where the differentiated T cell includes a first TCR. The method may also include introducing at least a second TCR into the y6 T cell or iNKT cell and maturing the resulting T cell to produce a T cell with two distinct functional TCRs. Then, the method includes, introducing the T cell into a subject, wherein the at least second TCR is directed to a disease related antigen expressed on the surface of a cell in the subject. In certain aspects, the HSC is from or derived from the subject.

In an exemplary method, the method further includes, after conducting the in vitro differentiation step, obtaining data specifying one or more TCRs that target the disease related antigen and subsequently performing the step of introducing the at least second TCR.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 diagrams a method for producing multi-TCR T cells.

FIG. 2 illustrates stages of ex vivo T cell manufacturing by three different processes.

FIG. 3 provides a comparison of two processes for producing T cells in vitro.

FIG. 4 illustrates the general steps of a method of the invention to produce multi-TCR T cells from HSCs.

FIG. 5 illustrates the general steps of a method of the invention to produce multi-TCR T cells from HSCs.

FIG. 6 illustrates the general steps of a method of the invention to produce multi-TCR T cells from HSCs.

FIG. 7 shows a comparison of PBMC cell lines for expanding/activating multi-TCR T cells.

FIG. 8 shows the results of expanding multi-TCR T cells in the presence of antigen presenting PBMCs.

FIG. 9 shows the generation of antigen presenting target cell lines.

FIG. 10 shows the cytotoxic efficacy of multi-TCR T cells produced using methods of the invention.

FIG. 11 shows the cytotoxic efficacy of multi-TCR T cells produced using methods of the invention.

DETAILED DESCRIPTION

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. Allogenic 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 allogenic 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.

This disclosure provides reliable methods for manufacturing T cells from stem cells (e.g., hematopoietic stem cells) with improved phenotype and cellular function via the introduction of at least two unique t cell receptors (TCRs). The present disclosure provides methods for manufacturing large numbers of the multi-TCR t cells quickly and efficiently. Moreover, the TCRs can be added at different times during the manufacturing process. Thus, in certain aspects of the invention, t cells can be made from hematopoietic stem cells, which may then undergo expansion, maturation, and/or storage steps. These t cells can represent an off-the-shelf starting point to which one or more additional TCRs can be introduced. As needed, specific, additional TCRs can be introduced into the cells to target distinct antigens as required to treat different ailments in different patients.

Certain preferred methods of the invention include introducing a first TCR into a hematopoietic stem cell (HSC) and differentiating the cell into a gamma delta (y6) T cell or an invariant natural killer T (iNKT) cell. Subsequently, one or more different, additional TCRs are introduced into the T cell to produce a T cell with at least two distinct TCRs.

Differentiating HSCs into y6 or iNKTs provides several advantages to the methods of the disclosure.

For example, certain methods include modifying stem cells (such as HSCs) to function as invariant NKT cells that are engineered to have one or more characteristics that render the cells suitable for universal, of-the-shelf use (e.g., prepared for individuals other than the individual from which the original cells were obtained) without deleterious immune reaction in a recipient of the cells. iNKT cells are a small subpopulation of T lymphocytes, which possess several features that make them useful for off-the-shelf cellular therapies, such as cancer treatments. iNKT cells have the remarkable capacity to target multiple types of cancer independent of tumor antigen- and MHC-restrictions. iNKT cells recognize glycolipid antigens, which frees them from MHC- restriction. Although the natural ligands of iNKT cells have yet to be completely identified, it is likely that iNKT cells can recognize certain conserved glycolipid antigens derived from many tumor tissues.

Further, iNKT cells include a number of mechanisms useful in targeting and attacking tumor cells. iNKT cells remain quiescent prior to stimulation, however, when stimulated, they quickly produce large amounts of cytokines that activates the cells to kill tumor target cells. Moreover, iNKT cell-induced anti-tumor immunity can effectively target multiple types of cancer independent of tumor antigen-and MHC-restrictions, thereby effectively blocking tumor immune escape and minimizing the chance of tumor recurrence.

Concurrently, iNKT cells do not cause graft- versus-host disease (GvHD) because iNKT cells do not recognize mismatched MHC molecules and protein autoantigens. Similarly, iNKT cells can be engineered to avoid host-versus-graft (HvG) depletion. The availability of powerful gene-editing tools (e.g., the CRISPR-Cas9) system make it possible to genetically modify iNKT cells to make them resistant to host immune cell-targeted depletion. iNKT cells also seem to naturally resist allogenic NK cell killing. iNKT cells also have strong relevance to cancer. In humans, iNKT cell frequency is reduced in patients with solid tumors and blood cancers, while increased iNKT cell numbers indicate a better prognosis. Thus, iNKT cells, when created using the methods of the invention may function as effective “off-the- shelf cellular products for treatment of various diseases depending on the introduced TCRs. enabling the transfer into patients sufficient iNKT cells at multiple doses may provide patients with the best chance to exploit the full potential of iNKT cells to battle their diseases.

Similar to iNKT cells, y6 T cells form a relatively small subset of T lymphocytes in the peripheral blood of adults - y6 T cells usually account for anywhere from 1% to 10% of CD3 positive T cells in human blood. However, unlike convention conventional T cells expressing an aP TCR, which recognizes antigen-derived peptides loaded onto MHC molecules, y6 T cells typically recognize target antigens independent of antigen processing and MHC/HLA restriction. y6 T cells share traits of the adaptive immune system (e.g., expression of clonally rearranged TCR genes). Concurrently, y6 T cells are similar to innate immune cells and lack the requirement for antigen processing to activate their effector functions. Therefore, y6 T cells rapidly respond to TCR triggering.

Activated y6 T cells are capable of lysing various types of of solid tumors and other malignancies and produce an array of cytokines. In addition to TCRs, y6 T cells may also express additional activating receptors. For example, y6 T cells frequently co-express functional receptors of innate immune cells, such as activating natural killer (NK) receptors, such as NKG2D, NKp30, and/or NKp44, which trigger cytotoxic effects. Thus, y6 T cells possess two independent recognition pathways to sense stressed and malignant cells.

Further, in vitro activated cells isolated from peripheral blood have demonstrated potent and HLA-independent activity of y6 T cells against various solid tumors and leukemia/lymphoma cells.

Thus, the present disclosure provides methods for producing T cells using stem cells by introducing a first TCR into a hematopoietic stem cell (HSC) and differentiating the cell into a gamma delta (y6) T cell or an invariant natural killer T (iNKT) cell. Subsequently, one or more different, additional TCRs are introduced into the T cell to produce a T cell with at least two distinct TCRs to produce T cells with enhanced anti-tumor activities.

This disclosure further provides systems and methods for producing T cells from a stem cells (e.g., HSCs) incorporated with multiple transgenes including TCRs, chimeric antigen receptors (CAR), and/or at least one additional transgene. By initiating a production process from stem cells, systems and methods of the invention 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 and subsequently introducing at least one additional TCR. The combined expression of the multiple TCRs (and optionally any introduced CARs and additional transgene(s)) provides the T cells produced using the methods of the disclosure with specific cancer cell targeting properties useful to treat the cancer.

FIG. 1 provides a general overview of methods of the invention for producing the multi- TCR t cells of the disclosure. As shown in FIG. 1, the process starts with introduction of a first TCR into a stem cell, e.g., an HSC. The stem cell is thus differentiated into a T cell, preferably a y6 T cell iNKT cell. Surprisingly, the present inventors have discovered that the methods of the invention provide a unique flexibility in when the second TCR is introduced into the differentiated T cell. A shown, the second TCR can be introduced early in the manufacturing process, such as before maturation of the T cell. Alternatively, the second TCR can be introduced at a mid-point in the process, such as before or during expansion of the T cell. The methods disclosed herein also contemplate providing the second TCR after expansion and potentially even after cry opreservation, activation, and expansion steps. Thus, the methods of the disclosure provide the ability to produce large number of T cells from HSCs, maintain the cells in culture or cryostorage, and introduce a second, antigen-specific TCR into the cells as they are needed in response to patient requirements. As such, the methods of the disclosure provide the ability to create multi-TCR T cells with reduced manufacturing times.

The presently disclosed methods and systems may also incorporate steps that produce the initial T cells, i.e., before introduction of the second TCR, using a shortened ex vivo culture time when compared with prior methods. Certain methods of the invention reduce ex vivo culture by omitting one or more ex vivo activation steps used in traditional T cell differentiation methods, which reduces ex vivo manufacturing processes by up to 2-3 weeks. Other methods of the invention provide for a single activation step. Methods of the invention recognize lengthy activation and/or expansion processes can occur in vivo, after administration to a subject. By shortening ex vivo cell culture, methods of the invention minimize opportunities for transcriptional and/or phenotypic changes to occur during T cell production. Furthermore, shortening ex vivo culture time reduces the amount of costly cell culture consumables that are needed for T cell maintenance.

In different aspect, this disclosure provides a multi-step workflow for making a T cell product with a single activation step. Certain methods include conducting a process comprising in vitro differentiation and maturation of an HSC into a multi-TCR T cell with no more than one in vitro T cell activation step. The resulting multi-TCR T cell product can be used for a treatment or for research. Conventional methods of T cell manufacture require at least two separate in vitro T cell activation steps. Multiple activation steps generally involve multiple media types, i.e., media containing different activation factors. Advantageously, producing a T cell by a single activation step reduces time of cell culture and produces a more effective T cell product that expresses fewer markers associated with T cell exhaustion. Methods of the invention are useful for producing multi-TCR T cell products with less in vitro cell culture. By reducing in vitro cell culture, methods of the invention produce T cell products that are more effective and contain fewer exhausted/dysfunctional T cells. The T cell products may express lower levels of proteins implicated in T cell exhaustion, e.g., PD-1, CTLA- 4, LAG-3, TIM-3, 2B4/CD244/SLAMF4, CD 160, TIGIT, than a T cell produced by 2 or more T cell activation steps. The T cell products may also express higher levels of IL-2.

Certain embodiments involve a multi-step process for producing a multi-TCR T or the initial T cell (before introduction of the second TCR) wherein only one of the steps is a T cell activation step. The single activation step can be performed by culturing the T cell in activation media, for example, media comprising T cell activation reagents such as antibodies.

In some instances, the activation step involves a peripheral blood mononuclear cell (PMBC) based activation. PMBC-based activation can involve introducing the T cell to alphagalactosylceramide (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 introducing the T cell to aAPCs. Preferably, the aAPCs are 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, to the T cell, soluble antibodies including 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. In some embodiments, the media contains IL-15.

In certain aspects, the methods of the invention include a step of obtaining the stem cells into which at least the first TCR is introduced. The methods may include obtaining stem cells (e.g., CD34+ hematopoietic stem/progenitor cells); introducing into the stem cells one or more nucleic acids (e.g., encoding the first TCR and optionally one or more additional TCR, CARs, and/or additional transgenes). After introduction of the first TCR, the methods further include conducting an in vitro differentiation and maturation of the stem cells to produce T cells. As described in FIG. 1, the at least second TCR can be introduced at a variety time points during the differentiation and maturation steps. After introduction of the at least second TCR, the muti-TCR T cells can be and used (e.g., for allogenic therapy or research) or stored. In certain aspects, the methods include performing a single in vitro activation step or performing no in vitro activation step.

Certain methods of the invention require a step of obtaining 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 haematopoiesis.

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, -80 degrees Celsius until use.

In practicing methods of the disclosure, obtaining the stem cells preferably involves 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.

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. In some preferred embodiments, once the cells are thawed, the cells are plated onto tissue culture dishes pre-coated with a reagent that promotes colocalization of a virus with target cells to enhance transduction efficiency.

The CD34+ cells may be plated in a standard 6 well dish at, for example, 10,000 cells per well, 15,000 cells per well, or 20,000 cells per well, or 25,000 cells per well, or more. Preferably, the cells are plated at 15,000 cells per well.

The method may further include introducing, into the CD34+ stem cells, one or more nucleic acids encoding for the first TCR and optionally one or more of a CAR, and/or an additional transgene. Subsequently, the method may further include introducing a second nucleic acid(s) that encode at least a second, distinct TCR. In some embodiments, the introduced TCRs produce a T cells capable of targeting a specific protein expressed on a surface of cancer cells.

In preferred embodiments, one or more of the introduced TCRs are introduced by way of nucleic acid encoding 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 other preferred embodiments, one or more of the introduced TCRs are introduced by way of nucleic acid encoding a delta gamma T cell TCR. The TCR may include one of an gamma chain of a TCR, a delta chain of TCR, or both.

T cells produced by methods of the invention may be genetically modified to express at least one additional transgene, other than those encoding the introduced TCRs. 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 invention may be useful to produce multi-TCR T cells with enhanced effector function.

For example, in some instances, methods of the invention 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-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 some instances, methods of the invention 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.

Introducing 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-vial 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.

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 34+ stem cells via a lentiviral transduction. For discussion on lentiviral transduction of stem cells, see Jang, 2020, Optimizing lentiviral vector transduction of hematopoietic stem cells for gene therapy, Gene Therapy (27): 545-556, which is incorporated by reference.

Methods of the invention 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 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.

In certain aspects, the methods of the invention further involve conducting a process comprising in vitro differentiation and maturation of the stem cell (e.g., HSC) into a T cell.

One advantage of the cell manufacturing methods disclosed herein 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 invention 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 and gamma delta T cells are preferred for their allogenic cell therapy applications. In particular, the ability to activate and expand antigen-specific T cell responses to treat cancer without inducing graft versus host disease.

Accordingly, methods of the invention may include conducting an in vitro differentiation and maturation process of stem cells (e.g., HSCs) into T cells (e.g., iNKT or y6 T cells). As discussed in detail below, conducting in vitro differentiation and maturation of the stem cells may be accomplished with the proviso that the process does not involve subsequent in vitro steps of activation and expansion of the T cell.

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. Embodiments of conducting the in vitro process further 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. In some embodiments, the cells are be cultured in progenitor maturation medium for 7 days.

In certain aspects, conducting the in vitro differentiation and maturation process of CD34+ stem cells into T cells produces CD4 positive CD8 positive T cells. The CD4 positive CD8 positive T cells may be naive T cells. Naive T cells are commonly characterized by surface expression of L-selectin (CD62L) and C-C Chemokine receptor type 7 (CCR7). In some instances, 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, CD 127, and common-gamma chain, CD 132. The naive T cells may be cryopreserved for storage or introduced into a subject for during an allogenic cell therapy treatment. In certain aspects, the naive T cells include only the first TCR receptor and have the second introduced prior/sub sequent to a cry opreservation or use as an allogenic 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, costimulation, and adhesion. The expression pattern of these molecules and/or the second introduced TCR may be used to further define effector and antigen-experienced of T cell subsets.

In some embodiments, the CD4 positive CD8 positive T cells are expanded in vitro prior to cry opreservation and/or administration to an allogenic 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. In certain aspects, the T cells are expanded in vitro prior to introduction of the second TCR. Alternatively, the T cell may be expanded in vitro after introduction of the second TCR. In certain methods, the T cell may be expanded in vitro before introduction of the second TCR and after introduction of the second TCR.

Certain methods of the invention take advantage of in vivo activation mechanisms to reduce otherwise necessary in vitro culture steps used in prior methods. Once a multi-TCR T cell has been produced, without having undergone two activation steps, and is introduced into a subject’s body, the T cell is fully activated when upon encountering 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 one or more of the introduced TCRs. This interaction 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 co-stimulation 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 the differentiation of CD4+ T lymphocytes into cytokine producing effector cells, depending on environmental conditions.

FIG. 2 illustrates stages of three ex vivo T cell manufacturing processes starting from HSCs. These illustrated processes include introducing the first TCR to produce T cells, preferably iNKT or y6 T cells, from the HSCs. As was shown in FIG. 1, the flexible methods of the invention permit the second TCR to be introduced at various time points.

In FIG. 2, the topmost process 203 represents a conventional ex vivo process for making T cells from HSCs. In contrast, ex vivo processes 205 and 207 show steps used in ex vivo processes of the invention for producing T cells quicker and with fewer steps.

In the conventional process 203, matured T cells are subjected to at least two in vitro activation steps. This process 203 requires at least 35 days of ex vivo cell culture and may require longer cultures - often upwards of 42 days, and often times longer.

Conversely, process 205 of the invention omits all ex vivo T cell activation steps. Thus, this method 205 can generate T cells within as few as 21 days. By omitting the ex vivo activation steps, this process for manufacturing T cells, starting from HSCs and using ex vivo culture, takes substantially less time than the conventional process 203. Thus, rather than 35 to 42+ days required for the conventional process 203, this process 205 of the invention can produce therapeutic-ready T cells in as little as 21 days.

FIG. 2 provides another process 207 of the invention for producing T cells from HSCs. As shown, this process 207 involves a single, ex vivo activation step. Thus, this process of the invention also represents an improvement over the conventional process 203 for manufacturing T cells from HSCs. Using a single activation step, such processes of the invention for manufacturing T cells can produce allogenic T cells, including multi -TCR T cells, in about 28-31 days. These methods can achieve the benefits certain activation steps have for certain types of T cells, TCRs, multi-TCR combinations, and/or T cell treatments in less time than the conventional process can produce a single TCR T cell. For example, the single activation step may be helpful for producing effective quantities of T cells into which a second TCR is introduced, multi-TCR T cells after introduction of the second TCR, and/or to ensure the T cells are primed to treat and/or expand upon introduction into a subject as part of a therapeutic treatment.

FIG. 3 provides a comparison of two culture processes for producing T cells in vitro. A first process (Version 1.0) includes two activation steps. The two-step activation process, which is accomplished by treating double positive T cells with, for example, different activation media comprising CD3/CD28/CD2 and IL-15, increases manufacturing process by at least one week and generally more of the processes of the invention disclosed herein. The second process shown in FIG. 3 (Version 2.0) omits in vitro activation.

Methods of the invention 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 cells with antigen presenting cells, such as aAPCs. The single activation step can involve co-culturing the T cells with PBMCs.

Preferred methods of the invention include an activation step after introduction of the second TCR in a multi-TCR T cell. Certain methods of the invention include an activation step after introduction of the first TCR in a multi-TCR T cell. Certain methods of the invention include an activation step after introduction of the first and second TCRs in a multi-TCR T cell.

Methods of the invention 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.

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 allogenic 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 invention, do not cause GVHD, methods described herein provide an ideal platform for ‘off-the-shelf CAR immunotherapy.

Methods of the invention 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), which recognizes antigens bound to major histocompatibility complex molecules. A TCR may be comprised of at least two different protein chains (e.g., a heterodimer). In most (e.g., 95%) T cells, this consists of an alpha (a) and beta (P) chain, whereas in some (e.g., 5%) T cells, this consists of gamma (X) and delta (6) chains. Such T cells may have antigen-specificity in cell surface TCR molecules and differentiate in vivo into different phenotypic subsets, including, but not limited to, classical CD3 positive, alpha-beta (aP) TCR CD4 positive, CD3 negative aP TCR CD8 positive, gamma delta (yS) T cells, Natural Killer T (NTK) cells, etc. Furthermore, T cell may further include various activation states, including, but not limited to, naive, central memory, effector memory, terminal effector, etc.

In one non-limiting example, T cells can be genetically engineered to express one or more artificial TCRs that direct cytotoxicity toward tumor cells. For example, one or more of the artificial TCRs may target a specific neo-antigen. Neo-antigens are generally tumor-specific antigens caused by mutations in tumor cells. Neo-antigens may also arise from viral infections, alternative splicing, and gene rearrangement. Neoantigens are generally not expressed on normal, healthy cells. Thus, they provide an ideal target with which to specifically direct the engineered immune cells of a subject. In certain aspects, the engineered immune cells of the invention have an introduced artificial TCRs that target different neoantigens. In certain aspects, the invention provides compositions of engineered cells in which separate populations of cells have different introduced TCRs, each directed against a different neoantigen.

In certain aspects, methods of the invention provide for the manufacture of CAR T cells. CAR T cells are genetically engineered T cells with 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 stem cells with TCRs via transfer of their coding sequences facilitated 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. 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, which include one or more CARs, which recognize cancer cells in order to more effectively target and destroy them.

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

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 CD3Xi which contains 3 ITAMs. This transmits an activation signal to the T cell after antigen is bound. CARs can also include a spacer region that links the antigen binding 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.

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 Xi chain signaling domain in a single fusion molecule, independent of HLA- mediated antigen presentation. 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.

In certain aspects, methods for manufacturing multi-TCR T cells of the invention include introducing one or more additional transgene, i.e., a transgene in addition the first introduced TCR and/or second introduced TCR). For example, an additional transgene may encode one or more cytokines. Cytokines relate to substances, such as interferon, interleukin, and growth factors that are secreted by certain cells of the immune system and influence other cells. In certain aspects, transgenes encoding one or more of IL-2, IL-7, IL-15, IL-12, IL-18, or IL-21, are introduced into the cells to facilitate T cell function, efficacy, and/or antigen target specificity.

Introduction of one or more transgenes into multi-TCR 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.).

For example, IL-12 and IL-18 play a major role in augmenting certain effector functions of 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 B 16 melanoma. IL- 18 has also been used to improve the therapeutic potential of 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 T cells 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.

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 are introduced into the cells. Producing T cells with IL-2 may provide T cells with improved capabilities for responding to conditions found in a tumor environment. For example, providing IL-2 may facilitated induction and the production of proteins involved in nutrient sensing and uptake.

In some embodiments, the additional transgene introduced is an inhibitor of cytokine release syndrome. Cytokine release syndrome relates to a serious, potentially life-threatening side effect often associated with 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 invention involve producing T cells that express inhibitors of the JAK pathway to improve in vivo 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. T cell related neurotoxicity is a syndrome that often leads to severe neurologic disturbances such as seizures and coma.

In other instances, methods involve introducing one or more transgenes into HSCs, which are used to produce T cells. In certain aspects, 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-OX40 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.

In certain aspects, the introduced transgene expresses one or more inhibitor of transforming growth factor beta. Engineered cells face hostile microenvironments which limit their efficacy. Modulating the environments may convert be useful for facilitating 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 invention 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 T cells to interfere with normal functions of transforming growth factor beta. Certain methods of the invention include producing multi-TCR T cells that target solid tumor types through markers of tumor microenvironment. In certain aspects, methods of the invention include introducing a CAR after introducing the first TCR. In such methods the CAR is introduced instead of, or in addition to, depending on the requirements of the cells’ use. Thus, the present invention include methods for producing CAR T cells with a single-domain antibody (VHH)-based chimeric antigen receptor, which can be used to recognize markers of a tumor microenvironment without the need for tumor-specific targets. VHH-based CAR T cells, according to the invention, 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 invention 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 invention, 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.

As many microenvironments involve expression of inhibitory molecules such as PD-L1. Using VHHs as recognition domains, e.g., PD-L1 -specific T cells, the multi-TCR and TCR and CAR T cells produced by methods of the invention 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/TCR that recognizes PD-L1 should relieve immune inhibition and at the same time allow T cell activation in the tumor microenvironment. PD-L1 -targeted 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. According to aspects of the present disclosure, CD34+ stem cells are genetically engineered to express a first TCR, one or more different TCR/CAR, and an additional transgene (e.g., a cytokine). In certain aspects, one or more nucleic acid encoding the TCR(s), CAR(s), and/or additional transgenes are introduced using retroviral transduction. In certain aspects, the first TCR and a subsequent TCR/CAR are introduced at distinct times in the manufacturing process using retroviral 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 to introduce the first TCR and/or the second TCR/CAR. The vector may be modified with cDNA sequences containing sequences of antibodies or antibody fragments to target preferred antigens. For example, as described in Carpenito, 2008, Control of large, established tumor xenografts with genetically retargeted human T cells containing CD28 and CD137 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.

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.

Methods of the invention are useful to transform engineered HSCs into T cells for clinical application. Methods of transformation generally include differentiation of HSCs into T cells, in part, by introduction of the first TCR. 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, Notchl, IL7Ra.

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 Thl cytokine, 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.

Cell products comprising T cells, including multi-TCR T cells that have not undergone an activation and/or expansion step, can be provided systemically or directly to a subject for the treatment of a neoplasia, pathogen infection, or infectious disease. In one embodiment, multi- TCR T cells of the present invention may be directly injected into an organ of interest (e.g., an organ affected by a neoplasia). Alternatively, multi-TCR 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). In certain aspects, activation and expansion of the T cells occurs in vivo, after introduction into a subject.

T cells and compositions comprising thereof of the present invention 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.

Methods of the invention provide for compositions of cells that may be combined with pharmaceutical compositions for administration of an allogenic cell therapy. When administering a therapeutic composition of the present invention (e.g., a pharmaceutical composition comprising multi-TCR 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 antigen-binding fragment administered.

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

This disclosure provides methods for manufacture of multi-TCR T cells for cell therapies and/or research. In certain aspects, methods provide economical methods of multi-TCR T cell manufacture by reducing time of ex vivo cell culture. In certain related aspects, methods of the invention provide for the manufacture of multi-TCR 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 allogenic 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 invention are useful for consistent production of therapeutically effective T cells.

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 multi-TCR T cell, with the proviso that the process does not involve subsequent in vitro steps of activation and/or expansion of the multi-TCR T cell before or after introduction of the second TCR. In certain aspects, activation and/or expansion of the multi-TCR T cell occurs in vivo after introduction into the subject. In certain aspects, activation and/or expansion take place both in vivo and in vitro. In certain aspects, the methods for producing multi-TCR T cells include only a single in vitro activation and/or expansion step. Advantageously, omitting in vitro the multiple activation and/or T cell expansion steps of prior methods that make only single TCR T cells, the methods of the invention can produce multi- TCR T cell in less time, and may produce T cells with enhanced therapeutic efficacy.

Methods of the invention are useful for producing allogenic 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 invention involve analyzing T cells after introduction of the first and/or second TCR to identify one or more proteins expressed by the T cells. The one or more proteins may include one or more CCR7, CD62L, or CD45RA. 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. In certain methods of the invention, multiple analyzing steps are used to assure introduction of the first TCR and then introduction of the second TCR.

On demand availability of treatment is one benefit of allogenic cell therapies. Since methods may involve manufacture of cells before clinical application, some preferred methods may include cryopreserving T cells. This may include cryopreserving multi-TCR T cells. In preferred aspects, methods of the invention include producing T cells from HSCs after introduction of the first TCR. The resulting TCRs are cryopreserved until needed. When required, a second TCR, specific to an antigen in a particular patient is introduced into the preserved TCRs to produce a multi-TCR. This dramatically reduces the lead time required to produce a patient-specific multi-TCR T cell. 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. In certain aspects, the second TCR is introduced to the preserved naive T cells. After introduction of the second TCR, the cells may undergo one or more expansion, activation, maturation, and/or preservation steps. 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 engineered adoptive T cell therapy, which has been shown to effectively target certain blood cancers with 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. Allogenic 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.

Some embodiments concern an engineered iNKT cell or a population of engineered iNKT cells with multiple TCRs. In at least some cases, the engineered iNKT cells comprise at least one engineered T cell receptor. In certain aspects, the cells include 2 or more engineered TCRs. Alternatively or additionally, the cells include an endogenous TCR and one or more engineered TCR. 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) an additional transgene. 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 TCR; ii) all or part of an invariant beta TCR, and/or iii) an additional transgene. In certain methods of the invention, one or more additional TCRs are introduced to these iNKT cells.

In certain aspects, the engineered iNKT cells are engineered to express 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 some embodiments, the inhibitor receptors comprise KIR. The increase or decrease may be with respect to the levels of the same marker in non-engineered 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. 1 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.

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 a second introduced TCR, and a suicide gene, 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.

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, i.e., a TK gene from a virus. 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 certain embodiments, 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).

In additional embodiments, a the multi-TCR T cells produce by the methods of the invention include T cells lacking or with 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-P 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 an iNKT cell because the cell was manipulated by gene editing.

In some embodiments, an iNKT cell comprises one or more recombinant vector or a nucleic acid sequences from one or more recombinant vectors that was introduced into the cells. In certain aspects, the iNKT cell comprises more than one recombinant vector, introduced at different points during the manufacturing process. In certain embodiments a recombinant vector is or was a viral vector. In further embodiments, a 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 integrates into the host genome sequence.

A "vector" or "construct" (sometimes referred to as gene delivery or gene transfer "vehicle") refers to a macromolecule, complex of molecules, or viral particle, comprising a polynucleotide to be delivered to a host cell, either in vitro or in vivo. The polynucleotide can be a linear or a circular molecule. A “plasmid”, a common type of a vector, is an extra- chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently of the chromosomal DNA. In certain cases, it is circular and doublestranded.

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 doublestranded. The boundaries of a coding region are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A gene can include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and synthetic DNA sequences. A transcription termination sequence will usually be located 3' to the gene sequence.

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.).

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.

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 short-term, 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. 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 line production 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.

Methods of the invention may be used to produce iNKT cells, for example, as discussed in U.S. Pub. No. US20170283481A1, and in World Application No. 2019241400, each of which are incorporated by reference.

As an example, in some embodiments, methods of the invention produce iNKT cells that comprise one or more iNKT TCR nucleic acid sequence obtained from a subset of iNKT cells, such as the CD4/DN/CD8 subsets or the subsets that produce Thl, Th2, or Th 17 cytokines, and include double negative iNKT cells. In some embodiments, an iNKT TCR nucleic acid sequence is obtained from an iNKT cell of 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, an 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 a TCR alpha sequence is obtained and an 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 a TCR alpha sequence is obtained is different from the donor of the iNKT cell from which a TCR beta sequence is obtained. In some embodiments, a TCR alpha sequence and/or a TCR beta sequence are codon optimized for expression. In some embodiments, a TCR alpha sequence and/or a 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, an iNKT TCR nucleic acid molecule encodes a T cell receptor that recognizes alpha-galactosylceramide (alpha-GalCer) presented on CD Id. In some embodiments, an iNKT TCR nucleic acid molecule is contained in an expression vector. In some embodiments, an expression vector is a lentiviral expression vector. In some embodiments, an 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, an expression vector is phiNKT-EGFP.

In some embodiments, a nucleic acid may comprise a nucleic acid sequence encoding an a-TCR and/or a P-TCR, as discussed herein. In certain embodiments, one nucleic acid encodes both the a-TCR and the P-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 P-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.

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 or more 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.

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(+) cells and CD34(+)/ CD45RA(+)/lin(- )CD1O+ (common lymphoid progenitor cells), CD34(+)CD45RA(+)lin(- )CD10(-)CD62L(hi) (lymphoid primed multipotent 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- (megakaryocyteerythrocyte progenitor cells).

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.

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- 7), 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.

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.

Cells may be used immediately, or they may be stored for future use. In certain embodiments, cells that are used to create iNKT cells are frozen, while produced iNKT cells may be frozen in some embodiments. In certain aspects, cells are in a solution comprising dextrose, one or more electrolytes, albumin, dextran, and DMSO. In other embodiments, cells are 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 does not have cancer. In some embodiments, the cell doesn’t express an endogenous TCR.

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- IBB, 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.

In some embodiments, the present invention provides kits comprising one or more engineered cells or compositions according to the present invention 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 invention provides kits comprising one or more engineered cells or compositions according to the present invention packaged together with one or more reagents for culturing and/or storing the engineered cells. In some embodiments, the present invention provides kits comprising one or more engineered cells or compositions according to the present invention 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 invention provides kits comprising one or more engineered cells or compositions according to the present invention packaged together with OP9-DL1 stromal cells and/or MS5- DL4 stromal cells. In some embodiments, the present invention provides kits comprising one or more engineered cells or compositions according to the present invention packaged together with antigen-presenting cells or CD Id-expressing artificial antigen-presenting cells.

Methods of the invention provides methods of manufacturing engineered cells for treating any number of conditions and/or diseases. In some embodiments, the present invention provides a method of treating a subject, which comprises administering to the subject one or more engineered cells according to the present invention, one or more engineered cells made according to a method of the present invention, or one or more compositions according to the present invention. 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.

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 are fusions of single-chain variable fragments (scFv) derived from monoclonal antibodies, fused to CD3- zeta transmembrane and endodomain; CD28 or 4 IBB intracellular domains, or combinations thereof. Such molecules result in the transmission of a signal in response to recognition by the scFv of its target. An example of such a construct is 14g2a- Zeta, which is a fusion of a scFv derived from hybridoma 14g2a (which recognizes disialoganglioside GD2). When T cells express this molecule (as an example achieved by oncoretroviral vector transduction), they recognize and kill target cells that express GD2 (e.g. neuroblastoma cells). To target malignant B cells, investigators have redirected the specificity of T cells using a chimeric immunoreceptor specific for the B-lineage molecule, CD19. 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.

In certain aspects, the present invention provides methods for producing CAR T cells from HSCs. The methods may include introducing a first TCR into the HSCs to produce a T cell, preferably a gamma delta T cell or iNKT cell. Subsequently, a CAR or a second TCR and CAR are introduced into the T cell. As a result, a TCR and CAR or multi-TCR and CAR cell is produced.

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, CD 19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD44v7/8, CD70, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, ERBB3, ERBB4, ErbB3/4, EPC AM, 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, R0R1, SP17, Survivin, TAG72, TEMs, carcinoembryonic antigen, HMW-MAA, AFP, CA-125, ETA, Tyrosinase, MAGE, laminin receptor, HPV E6, E7, BING-4, Calcium-activated chloride channel 2, Cyclin-Bl, 9D7, EphA3, Telomerase, SAP-1, BAGE family, CAGE family, GAGE family, MAGE family, SAGE family, XAGE family, NY-ESO-l/L AGE-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, TGF ^A RII, 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.

In certain aspects, this disclosure provides systems and methods for producing T cells, including multi-TCR 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), second TCR and/or a chimeric antigen receptor (CAR), and at least one additional transgene. By starting with HSCs, systems and methods of the invention 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 first TCR, a second TCR and/or CAR, and at least one an additional transgene. The combined expression of TCRs and/or TCR 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 by methods of the invention are armed with cargo (e.g., cytokines) that, when in contact with the target cancer cell, is useful to treat the cancer.

For example, in preferred embodiments, methods of the invention involve introducing nucleic acids into HSCs via lentiviral transduction, which may occur at multiple time points during the manufacturing process. Introduction of the one or more nucleic acids provides for HSCs that express at least a first TCR, a second TCR and/or 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.

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. Accordingly, in some instances, methods of the invention provide for the manufacture of 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 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 invention 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 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.

Certain methods of the invention involve a single in vitro activation 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.

In some instances, the single activation step involves PMBC-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 (or multi- TCR T cell). In some instances, the activation step involves an antigen presenting cell (APC) based T cell activation step. Accordingly, the activation step can involve introducing the T cell to APC. The APC may be an engineered K562 cell expressing CD80-CD83-CD137L-CAR-antigen. The APC may be an APC+CDld, and/or APC+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, CD3/28. In some embodiments, the T cell activation step involves culturing the T cell in the presence of different cytokines added to activations expansion culture media IL-7/15, IL-2, IL-2+21, IL- 12, IL- 18. The following examples provide useful exemplary protocols for manufacture of T cells and multi -TCR T cells (e.g., iNKTs and y6 T cells) from CD34+ cells as provided by methods of the invention. For further examples and discussion, see W02019241400A1, which is incorporated by reference.

Example 1 : CD34+ HSPCs cell culture and lentiviral transduction

Day -2: Pre-stimulation

1. Coat appropriate number of wells (recommended to seed ~15xl0 ³ cells/well or 6 wells for a O.lxlO ⁶ aliquot) of a 24-well non-tissue culture treated plate with 0.5 mL/well of 20 ug/ml retronectin (RN) diluted in PBS. 1 mg/mL RN stock aliquots may be stored at -20 degrees Celsius in 60 microliter aliquots.

2. Incubate at room temperature (RT) for 2 hours (h).

3. Aspirate RN and replace with 0.5 ml/well of 2 percent BSA diluted in PBS. 30 percent BSA aliquots are stored at -20° Celsius.

4. Incubate for 30 min at RT.

5. Aspirate and replace with 0.5 ml/well of PBS.

6. Thaw a 0. IxlO ⁶ aliquot of CD34+ cells into Stem Cell Media following standard procedures and spin at 300g for 10 min.

7. Aspirate supernatant, resuspend in Stem Cell Media, and count using hemocytometer, record cell count. In some instances, applicant’s found cell counts for a O.lxlO ⁶ aliquot were too low to be reliable.

8. Dilute CD34+ cells with Stem Cell Media to 0.05xl0 ⁶ cells/ml.

9. Aspirate PBS from RN-coated wells and seed 300 ul cells/well (~15xl0 ³ cells/well).

10. Incubate at 37 degrees Celsius, 5 percent CO2 for 12-18 h.

Day -1 : Transduction

1. Thaw concentrated lentiviral vector (LVV) and pipet gently to mix (do not vortex and do not refreeze).

2. Prepare transduction tube: a. Transfer appropriate volume of LVV to 1.5 ml tube by calculating volume sufficient for a

MOI of 100-200. b. Add appropriate volume of PGE2 to same tube to achieve a final concentration of 10 nM of total culture volume. c. Add appropriate volume of poloxamer to same tube to achieve a final concentration of lug/ul of total culture volume.

3. Add contents of transduction tube to appropriate culture well and rock plate gently to mix

4. Incubate cells at 37 degrees Celsius, 5 percent CO2 for 24 h.

Day 0 Harvest

1. Collect cells by pipetting gently to remove them from the plate and transfer to conical tube.

2. Wash wells with equal volume of cold X-VIVO-15 to remove any cells still adhering to plate and transfer to conical tube.

3. Check under microscope and perform additional washes with cold X-VIVO-15 as necessary to collect all cells from plate.

4. Spin at 300g for 10 minutes and aspirate supernatant.

5. Proceed to Stage 2 (i.e., Example 2).

Example 2: Generation of iNKT-CAR Cells; Differentiation

Days 0-14: Differentiation (Duration: 2 weeks)

1. Coat appropriate number of wells (recommended to seed 1,000-2,000 cells/well or 12 wells/15,000 cells) of 12-well non-tissue culture-treated plates with 1 ml/well with a lymphoid differentiation coating material (LDCM), such as, the lymphoid differentiation material provided under the trade name StemSpan by STEMCELL, diluted 1 :200 in PBS. Incubate 12-18 h at 4 degrees Celsius.

2. Aspirate LDCM and add 2 ml/well of PBS

3. Resuspend transduced CD34+ cells collected in Stage 1 (i.e., Example 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 ³ cells/ml with LPEM

5. Aspirate PBS from LDCM-coated plates

6. Seed 0.75 ml cells/well into a LCDM-coated plate

7. Incubate cells at 37 degrees Celsius, 5 percent CO2

8. On day 3, add 0.25 ml/well of fresh LPEM and continue culture

9. On days 7 and 11, carefully remove <0.5 ml/well without disturbing cells and replenish with 0.5 ml/well of fresh LPEM

10. Continue culture to day 14

11. Proceed to Stage 3 (i.e., Example 3)

Example 3: Generation of iNKT-CAR Cells; Maturation

Day 14-21+: Maturation (Duration: 1-2 weeks)

1. Coat appropriate number of wells (recommended to seed 50-100xl0 ³ /well) of 6-well nontissue culture-treated plates with 2 ml/well of LDCM diluted 1 :200 in PBS. Incubate 12-18 h at 4 degrees Celsius if preparing a day prior to seeding or at 37 degrees Celsius for 2 h if preparing the day of seeding.

2. Aspirate LDCM and add 4 mL/well of PBS

3. On day 14, harvest and count cells using a cell viability counter, such as, the cell viability counter provided under the trade name Vi -Cell XR by Beckman Coulter. a. Collect cells by pipetting gently to remove them from the plate and transfer to conical tube. b. Wash wells with lml/4 wells of cold SFEM II to remove any cells still adhering to plate and transfer to conical tube c. Check under microscope and perform additional washes with cold SFEM II as necessary to collect cells from plate.

4. Pull an aliquot of 0.2xl0 ⁶ cells and seed into a 96-well V-bottom plate for flow staining.

5. Pull an appropriate volume of cells to seed for Stage 3 and pellet at 300 g for 10 min.

6. Aspirate supernatant. 7. Resuspend cells in T cell progenitor maturation medium (TPMM), such as, the T cell progenitor maturation medium provided under the trade name StemSpan by STEMCELL, at 2.5- 5xl0 ⁴ cells/ml.

8. Aspirate PBS from LDCM-coated plates.

9. Seed 2 ml cells/well into LDCM-coated plate.

10. Incubate cells at 37 degrees Celsius, 5 percent CO2

11. Pellet remaining cells at 300 g for 10 min.

12. Aspirate and resuspend remaining cells in appropriate volume of cryopreservation solution, such as, the cry opreservation solution sold under the trade name CryoStor CS10 by STEMCELL, to achieve 2-5xl0 ⁶ cells/mL.

13. Aliquot 1 ml/cryovial and freeze appropriately (e.g., in a negative 80 degrees Celsius freezer); move to liquid nitrogen storage within 24 hours of freezing.

14. On day 17, add 2 ml/well of fresh TPMM and continue culture.

15. On day 21, take sample for flow cytometry by pipetting 100 ul from cells in center of well and seed into a 96-well V-bottom plate for flow staining.

16. Take another lOOul from cells in center of well and count using cell viability counter.

17. If TCR expression is >50%, CD4/CD8 double positive expression is >20%, and cell size has decreased (indicative of development from HSC to T cells), cells may cryopreserved and provided for allogenic therapy, or optionally, expanded as provided by Stage 4. If these parameters are not met, continue culture with necessary feeding and splitting. Remove <2ml/well and replenish with 2ml/well of fresh TPMM every 3-4 days and splitting cultures if they approach confluence. Perform this check again on day 24/25 and day 28 until above criteria are met.

Stage 4: Generation of iNKT-CAR Cells; Optional Expansion:

Day 21-28 (assuming criteria met at day 21, adjust accordingly): Stimulation (Duration: 1 week) 1. On day 21, harvest and count cells using cell viability counter (record counts in associated excel sheet) a. Collect cells by pipetting gently to remove them from the plate and transfer to conical tube b. Wash wells with 2 ml per 4 wells of cold cell expansion medium (EM), such as, the cell expansion medium provided under the trade name OpTmizer by ThermoFisher, to remove any cells still adhering to plate and transfer to conical tube c. Check under microscope and perform additional washes with cold EM as necessary to collect cells from plate. d. Pull an aliquot of 0.2xl0 ⁶ cells and seed into a 96-well V-bottom plate for flow staining. e. Pull an appropriate volume of cells to seed for Stage 4 and pellet at 300 g for 10 min. f. Aspirate supernatant. g. Resuspend cells at 2xl0 ⁶ cells per ml in EM with IL-7 and IL-15 at 10 ng/ml

2. Follow subset of following instructions depending on desired method(s) of stimulation. iNKT-CAR cell final concentration is fixed between conditions at IxlO ⁶ ml a. No stimulation i. Dilute cells to IxlO ⁶ cells per ml with EM media with IL-7 and IL-15 at 10 ng/ml ii. Seed cells and incubate at 37 degrees Celsius, 5 percent CO2 b. Coated CD3/Soluble CD28 i. Coat plates with 1 ,23ug/ml CD3 for 2 hours at 37°C and wash with PBS before using. ii. Dilute cells to IxlO ⁶ cells per ml with EM with IL-7 and IL-15 at 10 ng/ml. iii. Add soluble CD28 to cell suspension at lug/ml. iv. Seed cells and incubate at 37 degrees Celsius, 5 percent CO2. c. Soluble CD3/Soluble CD28 with PBMCs i. Thaw human peripheral blood mononuclear cells (PBMCs) and irradiate at 6,000 rads. ii. Resuspend PBMCs EM with IL-7 and IL- 15 at 10 ng/ml. iii. Combine iNKT-CAR cells with PBMCs at a 1 :2-3 (iNKT-CAR:PBMC) ratio so that the iNKT-CAR cell final concentration is IxlO ⁶ cells per ml. iv. Add soluble CD3 and soluble CD28 to cell suspension at lug/ml. v. Seed cells and incubate at 37 degrees Celsius, 5 percent C02. d. Provide a CD3/CD28 T Cell Activator, such as, the activator sold under the trade name ImmunoCult Human CD3/CD28 T Cell Activator by StemCell Technologies. i. Dilute cells to IxlO ⁶ cells per ml with EM media with IL-7 and IL-15 at 10 ng/ml. ii. Add CD3/CD28 T Cell Activator to cell suspension at 25ul/ml. iii. Seed cells and incubate at 37 degrees Celsius, 5 percent CO2. e. Provide CD3/CD28/CD2 T Cell Activator, such as, the activator sold under the trade name ImmunoCult Human CD3/CD28/CD2 T Cell Activator by StemCell Technologies. i. Dilute cells to IxlO ⁶ cells per ml with EM with IL-7 and IL-15 at 10 ng/ml. ii. Add CD3/CD28/CD2 T Cell Activator to cell suspension at 25ul/ml. iii. Seed cells and incubate at 37 degrees Celsius, 5 percent CO2. f. aGC -loaded PBMCs i. Thaw human peripheral blood mononuclear cells (PBMCs). ii. Resuspend at 10xl0 ⁶ cells per ml in EM with 2ug/ml aGC and incubate at 37 degrees Celsius, 5 percent CO2 for 1 h. iii. Collected aGC -loaded PBMCs and irradiate at 6,000 rads. iv. Perform at least two washes of cells with EM to remove unbound aGC v. Resuspend PBMCs in EM with IL-7 and IL- 15 at 10 ng/ml. vi. Combine iNKT-CAR cells with PBMCs at a 1 :2-3 (iNKT-CAR:PBMC) ratio so that the iNKT-CAR cell final concentration is IxlO ⁶ cells per ml. vii. Seed cells and incubate at 37 degrees Celsius, 5 percent CO2. g. K562-CD80-CD83-CD137L-A2ESO artificial antigen presenting cells (aAPC). i. Collect aAPCs from in culture and irradiate at 10,000 rads. ii. Combine iNKT-CAR cells with aAPCs at a 4: 1 (iNKT-CAR: aAPC) ratio so that the iNKT-CAR cell final concentration is IxlO ⁶ cells per ml. iii. Seed cells and incubate at 37 degrees Celsius, 5 percent CO2. h. K562-CD80-CD83-CD137L-A2ESO-CDld artificial antigen presenting cells (aAPC-CDld). i. Collect aAPCs from in culture and irradiate at 10,000 rads. ii. Combine iNKT-CAR cells with aAPCs at a 4: 1 (iNKT-CAR: aAPC) ratio so that the iNKT-CAR cell final concentration is IxlO ⁶ cells per ml. iii. Seed cells and incubate at 37 degrees Celsius, 5 percent CO2. i. aGC -loaded K562-CD80-CD83-CD137L-A2ESO-CDld artificial antigen presenting cells (aAPC-CDld). i. Collect aAPCs from in culture. ii. Resuspend at 10xl0 ⁶ cells per ml in EM with 2ug/ml aGC (see a-GalCer Prep SOP) and incubate at 37°C, 5% CO2 for 1 h. iii. Collected aGC -loaded aAPCs and irradiate at 10,000 rads. iv. Combine iNKT-CAR cells with aAPCs at a 4: 1 (iNKT-CAR: aAPC) ratio so that the iNKT-CAR cell final concentration is IxlO ⁶ cells per ml. v. Seed cells and incubate at 37 degrees Celsius, 5 percent CO2.

3. On day 24 count cells using cell counter (record counts in associated excel sheet) and dilute cells to lxl0 ⁶ cells per ml using EM with IL-7 and IL-15 at lOng/ml, transfer culture vessels as necessary. If utilizing PBMCs or aAPCs the cell counts taken at this timepoint may be harder to interpret, in this case carefully remove half of the culture media without disturbing the cells and add an equivalent volume of fresh EM with IL-7 and IL-15 at 10 ng/ml. If conditions without PBMCs or aAPCs do not require dilution also perform this half-media change.

4. On day 26 count cells using cell viability counter (record counts in associated excel sheet) and dilute cells to IxlO ⁶ cells per ml using EM with IL-7 and IL-15 at lOng/ml, transfer culture vessels as necessary.

5. On day 28 harvest cells and count using Vi-Cell (record counts in associated excel sheet) a. Pull an aliquot of 0.2xl0 ⁶ cells and seed into a 96-well V-bottom plate for flow staining. b. Pull aliquots of 0.2xl0 ⁶ cells and seed into a 96-well V-bottom plate for additional flow staining characterization. c. Pull aliquot of IxlO ⁶ cells, dilute to IxlO ⁶ cells per ml using EM with IL-7 and IL- 15 at lOng/ml, and seed for longitudinal tracking (optional step, prioritize after cell freezing is completed). d. Pellet remaining cells at 300 g for 10 min and aspirate supernatant. e. Resuspend in appropriate volume of cry opreservation media to achieve 25-50 xlO ⁶ cells per mL. f. Aliquot 1 ml/cryovial and freeze appropriately, move to liquid nitrogen storage within 24 hours of freezing.

Example 5: Generating multi-TCR T cells from HSCs

This example provides methods used to produce multi-TCR T cells from HSCs. These methods differ in the timing of when the second TCR is introduced to produce a multi-TCR T cell. However, despite this difference in protocol, each method of this example produces a multi- TCR T cell, starting from an HSC in about 30 days. Moreover, all methods of this example include a cryopreservation step, which allows the multi-TCR T cells to be produced quickly on demand and/or be stored for use when needed.

Version 1

In first version of the method, an HSC is differentiated into a T cell, in this particular example an iNKT or y6 T cell, into which an antigen-directed TCR is introduced. The general steps for this version of the method are provided FIG. 4.

Initially, the method begins the same or similar to those outlined in examples 1-4 to produce a TCR/CAR T cell. Thus, the method may include an initial phase 403 including a prestimulation step, a transduction step, and/or a harvesting step. The result of this initial transduction step is to introduce a nucleic acid encoding a first TCR into the HSCs. Again, similar to the methods in examples 1-4, the method further includes a step 405 to differentiate the HSCs with the introduced first TCR into T cells (e.g., iNKT or y6 T cells). Similar to examples 1-4, the T cells are subject to a maturation step 407 and an expansion and/or activation step 409. In this example, after expansion, the single TCR T cells are cryopreserved. After cryopreservation, and a subsequent thaw, the T cells undergo a second round of transduction, during which a second TCR is introduced into the T cells. In this case, the second TCR was introduced via lentiviral transduction. This second TCR specifically targets the ESO cancer-testis antigen. The result of this method is the transformation of an HSC into an iNKT cell or y6 T cell, which in addition to the iNKT/ y6 TCR also expresses a second TCR that specifically targets a cancer antigen.

Version 2

The steps of a second version of the method are provided in FIG. 5. The steps of this method proceed in the same manner as in version 1. However, owing to the flexibility of the method, the final steps of expansion, activation, and/or cryopreservation occur after introduction of the second, antigen-specific TCR, i.e., at between day 21 and day 34.

Version 3

The steps of a third version of the method are provided in FIG. 6. The steps mirror thse of the prior two methods. Thus, the method includes an initial phase 603 including a pre-stimulation step, a transduction step, and/or a harvesting step. The method further includes one or more steps 605 to differentiate the HSCs with the introduced first TCR into T cells (e.g., iNKT or y6 T cells). Similar to examples 1-4, the T cells are subject to a maturation step 607 and an expansion and/or activation step 609. However, in this version of the method, the second, target-specific TCR is introduced at around day 14, i.e., prior to, or at the start of, T cell maturation.

Example 6: Generating ~/5 Multi-TCR Antigen-Specific T Cells

HSCs were used to produce y6 T cells in accordance with the steps outlined in the method provided in FIG. 6. Briefly, the HSCs underwent lentiviral transduction to introduce a y6 TCR into the cells. The cells underwent a differentiation step 605. After differentiation, the resulting y6 (“GD-T”) T cells underwent a second lentiviral transduction, which introduced a TCR that targets the ESO cancer-testis antigen.

After maturation, the resulting multi-TCR T cells (with a y6 TCR and ESO-targeting TCR) were expanded/activated via contact with ESO antigen presenting (ESOp-loaded) HLA-A2 PBMCs.

In certain variations, PBMCs can be screened to determine whether they express an activating/expanding antigen at sufficiently high levels. FIG. 7 shows the results of such a screening. In the figure, PBMC line D4105 had the highest HLA-A2 expression. Thus, the line was chosen to expand the multi-TCR T cells.

As shown in FIG. 8 in the right panel, the multi-TCR T cells (GD-TCRZESO-TCR) showed marked expansion from contact with the ESOp-PBMCs. In contrast, cells without the ESO-TCR (“NTD”), showed little expansion from contact with the PBCMs.

In order to test the efficacy of the multi-TCR T cells, target cells expressing HLA-A2- ESO were produced. Three such lines were produced, two derived from human ovarian cancer cells line (SKOV3-luc and OVCAR3-luc) and one derived from the peripheral blood of a multiple myeloma patient (MM. 1 S-luc). The lines were transduced with a transgene that caused the cells to express the ESO antigen. Data regarding these test cells lines is presented in FIG. 9.

These cells were then used to measure the cytotoxic efficiency and specificity of the multi-TCR T cells. Briefly, multi-TCR T cells were exposed to each of the test cell lines as well as to the target cell parental lines, which did not have the ESO antigen transduced. Likewise, y6 T cells produce using the methods of the invention, but without introduction of the second, ESO- specific TCR were exposed to the test cell lines and control lines. All experiments were repeated, but additionally included contacting the T cells with the aminobisphosphonate zoledronic acid (ZOL), which is known to contribute to activation of y6 T cells. FIGS. 10 and 11 provide the results of these in vitro killing assays.

Across all target cell lines, the multi-TCR T cells (which included the ESO-specific TCR), provided a larger cytotoxic effect on the ESO-antigen expressing target cells when compared with the single-TCR T cells. This confirms that the second TCR was properly introduced into the y6 T cells, and that it possessed antigen-specific cytotoxic effects. Further, across all T cells (i.e., y6 T cells and multi-TCR T cells), contact with ZOL enhanced the killing capacity of the T cells. This is expected as ZOL is known to activate y6 T cells, and the multi- TCR T cells include the y6 TCR.

Incorporation by Reference

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

Various modifications of the invention 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. The subject matter herein contains important information, exemplification and guidance that can be adapted to the practice of this invention in its various embodiments and equivalents thereof.