WO2023230440A1

WO2023230440A1 - Batf3 overexpression in lymphocytes

Info

Publication number: WO2023230440A1
Application number: PCT/US2023/067297
Authority: WO
Inventors: Andras HECZEY; Leonid METELITSA
Original assignee: Baylor College Of Medicine
Priority date: 2022-05-23
Filing date: 2023-05-22
Publication date: 2023-11-30

Abstract

The present disclosure relates to methods and compositions related to Natural Killer T cells (NKT cells) that are engineered to harbor an expression construct that encodes an exogenous BATF polypeptide. Activation by of the IL-15 pathway through an exogenous BATF transcription factor promotes NKT expansion over the course of multiple tumor cell challenges and improves long term tumor control in vitro. The present disclosure further includes NKT cells, populations, and methods to prepare them, that are engineered to express exogenous BATF polypeptides either alone, or in combination with chimeric antigen receptors (CARs), Wnt activators and MHC suppressors for therapeutic use.

Description

BATF3 OVEREXPRESSION IN LYMPHOCYTES

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from U.S. Provisional Patent Application No. 63/344,831, filed May 23, 2022, which is incorporated herein by reference in its entirety.

INCORPORATION OF SEQUENCE LISTING

[0002] The sequence listing that is contained in the file named “P35193WOOO_SL.xml,” which is 4,212 bytes (measured in operating system MS-Windows), created on May 22, 2023, is filed herewith and incorporated herein by reference.

FIELD

[0003] The present disclosure relates the field of adoptive immunotherapy and methods to enhance adoptive immunotherapy by the expression of Basic leucine zipper ATF-like transcription factor 3 (BATF3), a transcription factor belonging to the Activator Protein-1 (API) family.

BACKGROUND

[0004] Adoptive immunotherapy employs tumor redirected white blood cells, most commonly lymphocytes, to eradicate tumors and requires the sustained functionality of these tumor specific effectors. The present application provides for methods to enhance adoptive immunotherapy by the transgenic expression of Basic leucine zipper ATF-like transcription factor 3 (BATF3), a transcription factor belonging to the Activator Protein-1 (API) family. See Wu et al., “AP-1 family transcription factors: a diverse family of proteins that regulate varied cellular activities in classical hodgkin lymphoma and ALK+ ALCL,” Experimental Hematology & Oncology. 10( 1 ): 4 (2021); Seo et al., “ BATF and IRF4 cooperate to counter exhaustion in tumor-infiltrating CAR T cells,” Nature Immunology 22(8):983-95 (2021); Qiu et al., “Cutting Edge: Batf3 Expression by CD8 T Cells Critically Regulates the Development of Memory Populations,” J Immunol. 205(4):901-6 (2020); Ataide et al., “BATF3 programs CD8(+) T cell memory,” Nat Immunol. 21(l l):1397-407 (2020); Murphy et al., “Specificity through cooperation: BATF-IRF interactions control immune-regulatory networks,” Nature Reviews Immunology 13(7):499-509 (2013). The specification further provides lymphocytes expressing BATF3.

[0005] Genetically engineered effector lymphocytes (GEELs) can efficiently eliminate cancer cells in humans leading to durable complete remission in patients. Clinical responses are correlated with the expansion and persistence of GEELs; thus, improving these parameters is critical to increasing the efficacy of GEEL-based therapies. Our group and others have shown that interleukin- 15 (IL15) enhances GEEL antitumor activity. See Batra et al., “Glypican-3- Specific CAR T Cells Coexpressing IL15 and IL21 Have Superior Expansion and Antitumor Activity against Hepatocellular Carcinoma,” Cancer Immunol Res. 8(3):309-20 (2020); Xu et al., “NKT Cells Coexpressing a GD2-Specific Chimeric Antigen Receptor and IL15 Show Enhanced In Vivo Persistence and Antitumor Activity against Neuroblastoma,” Clinical Cancer Research 25(23):7126-38 (2019); Hoyos et al., “Engineering CD19-specific T lymphocytes with interleukin- 15 and a suicide gene to enhance their anti-lymphoma/leukemia effects and safety,” Leukemia 24(6): 1160-70 (2010); Park et al., “Long-Term Follow-up of CD19 CAR Therapy in Acute Lymphoblastic Leukemia,” N Engl J Med. 378(5):449-59 (2018): and Markley and Sadelain, “IL-7 and IL-21 are superior to IL-2 and IL- 15 in promoting human T cell-mediated rejection of systemic lymphoma in immunodeficient mice,” Blood 115(17):3508-19 (2010). Expression of IL15 in natural killer T cells (NKTs), a subset of invariant lymphocytes redirected with a chimeric antigen receptor (CAR) specific for tumor antigen glypican-3 (GPC3). NKTs expressing the 15.GPC3-CAR construct controlled tumors in a murine model of HCC significantly better than NKTs expressing the GPC3-CAR without IL15 (Fig 1). (See Metelitsa “Anti -tumor potential of type-I NKT cells against CD Id-positive and CD Id-negative tumors in humans,” Clin Immunol. 140(2): 119-29 (2011)),

[0006] As provided herein, BATF3 overexpression enhances proliferation of lymphocytes such as Natural Killer T cells (NKTs), a subset of innate lymphocytes, without inducing autonomous growth and boosting antitumor activity.

SUMMARY

[0007] The present disclosure comprises, in one form thereof, engineered Natural Killer T- cell (NKT cell), or a plurality thereof, comprising an expression construct that encodes a basic leucine zipper ATF-like transcription factor (BATF) polypeptide. BATF polypeptides known in the art are included in the methods and compositions disclosed herein.

[0008] More particularly, the present disclosure includes a composition comprising a plurality of engineered Natural Killer T-cells (NKT cells) comprising an expression construct that encodes basic leucine zipper ATF-like transcription factor (BATF), basic leucine zipper ATF-like transcription factor 2 (BATF2), or basic leucine zipper ATF-like transcription factor 3 (BATF3) and a chimeric antigen receptor.

[0009] In another form, the present disclosure includes a recombinant nucleic acid comprising nucleic acid sequences encoding a polypeptide for basic leucine zipper ATF-like transcription factor (BATF), basic leucine zipper ATF-like transcription factor 2 (BATF2), or basic leucine zipper ATF-like transcription factor 3 (BATF3), and encoding a polypeptide for a chimeric antigen receptor (CAR) having an antigen recognition domain directed to at least one tumor- associated antigen.

[0010] An aspect, the present specification provides a method for treating a cancer in a subject in need thereof comprising administering a therapeutically effective amount of a plurality of engineered Natural Killer T-cells (NKT cells) engineered to exogenously express basic leucine zipper ATF-like transcription factor (BATF), basic leucine zipper ATF-like transcription factor 2 (BATF2), or basic leucine zipper ATF-like transcription factor 3 (BATF3) and a chimeric antigen receptor.

[0011] A further aspect of the present specification is a method of producing Natural Killer T- cell (NKT cell) cells for immunotherapy comprising isolating human NKT cells from human peripheral blood mononuclear cells (PBMCs); culturing the human NKT cells to prepare a culture having a majority of CD62L-positive Type I NKT cells by culturing in the presence of at least (1) one or more cytokines selected from the group consisting of IL-21, IL-7, IL-15, IL-12, TNF-alpha, and a combination thereof; and (2) irradiated NKT-depleted PBMCs or irradiated artificial antigen-presenting cells (aAPCs) loaded with alpha-Galactosylceramide (aGalCer); genetically modifying the CD62L-positive Type I NKT cells to exogenously express a basic leucine zipper ATF-like transcription factor (BATF) polypeptide.

[0012] The present specification provides for, and includes, a method of treating an individual for a medical condition using immunotherapy, comprising the steps of: isolating human NKT cells from human peripheral blood mononuclear cells (PBMCs); culturing the human NKT cells in the presence of at least: one or more cytokines selected from the group consisting of IL-21, IL- 7, IL-15, IL-12, TNF-alpha, and a combination thereof; and irradiated NKT-depleted PBMCs or irradiated artificial antigen-presenting cells (aAPCs) loaded with alpha-Galactosylceramide (aGalCer); to prepare a culture having a majority of CD62L-positive Type I NKT cells; genetically modifying the CD62L-positive Type I NKT cells to exogenously express a basic leucine zipper ATF-like transcription factor (BATF) polypeptide and a chimeric antigen receptor (CAR) having an antigen recognition domain directed to at least one tumor-associated antigen and providing a therapeutically effective amount of the co-stimulated CD62L+ NKT cells to the individual.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The present disclosure is disclosed with reference to the accompanying drawings, wherein:

[0014] Figure 1 presents a diagram of a chimeric antigen receptor (CAR) according to embodiments of the present specification. Figure 1 A identifies the essential features of a CAR. Figure IB presents an exemplary, non-limiting embodiment of a CAR having a CH8H hinge region, a CD8 transmembrane region from CD8a, a CD28 co-stimulatory domain and a CD3^ stimulatory domain.

[0015] Figure 2 presents diagrams of representative expression constructs of the present specification.

[0016] Figure 3 presents diagrams of expression constructs of representative embodiments according to the present specification.

[0017] Figure 4 presents a presents certain embodiments of the expression constructs showing exemplary regulatory sequences.

[0018] Figure 5 presents additional exemplary embodiments presenting combinations of expressed sequences and regulatory sequences.

[0019] Figure 6 presents additional exemplary embodiments presenting combinations of expressed sequences and regulatory sequences.

[0020] Figure 7 presents representative results of an exemplary embodiment of NKT cells engineered to express a CAR having an antigen recognition domain directed to the tumor antigen glypican-3 (GPC) with or without expression of the growth factor IL-15. As shown, GPC3-CAR s induce robust in vivo antitumor activity. Figure 7 presents results from NSG mice (n= 5-11 per group) injected with 2xl0⁶ GPC3-positive Huh-7.Ffluc tumor cells IP and injected intravenously after 7 days, with 8xl0⁶ GPC3-CARNKT cells or control cells (NT: non-transduced parental NKT cells, IL-15 only, or CD19-CAR expressing NKTs). Figure 7A presents representative weekly tumor bioluminescence imaging of mice at indicated time points. Figure 7B presents tumor bioluminescence over time. Figure 7C presents Kaplan-Meier survival curves of tumorbearing mice after treatment with GPC3-CAR NKT cells. GPC3-CAR vs 15.GPC3-CAR groups p < 0.001 by Mantel-Cox analysis of a representative experiment.

[0021] Figure 8 presents gene expression results of an exemplary embodiment showing that BATF3 expression is elevated in 15.GPC3-CAR NKTs after tumor cell engagement. 15.GPC3- CAR and GPC3-CARNKTs are co-cultured with HCC tumor cells and gene expression is assessed 48 hrs post-tumor cell engagement by RNAseq. Log2 fold change in gene expression versus -log 10 transformed p values are shown.

[0022] Figure 9 presents results of BATF3 overexpression in genetically engineered NKT cells and shows enhanced in vitro proliferation of NKTs. Wild-type and engineered NKTs expressing indicated transgenes are plated with fresh a- galactosylceramide loaded CDld+ Jurkat cells every 3-4 days (cycles). NKT numbers are quantified by flow cytometry at each timepoint. Figure 9A presents absolute number of NKTs in indicated groups over time. Figure 9B presents Area under the curve (AUC) of NKT groups up to cycle 7. Figure 9C presents the fold expansion of NKTs up to cycle 7. Comparison via one-way ANOVA and post-test Tukey. * p<0.05; ** p<0.01 and *** p<0.001 is indicated.

[0023] Figure 10 presents results of an exemplary experiment showing that GPC3-CAR NKTs kill HCC cell lines and co-expression of IL15 or BATF3 does not impact cytotoxic capacity. Indicated effector and control cells are plated at a 1:1 ratio with tumor cells and monitored with the xCelligence system for 100 hrs.

[0024] Figure 11 presents results of an exemplary experiment showing that BATF3.GPC3- CAR engineered NKTs induce robust antitumor activity in vivo. NSG mice are injected with 2x10⁶ GPC3-positive Huh-7. Ffluc tumor cells IP. After 7 days, 8x10⁶ GPC3-CARNKT cells or controls (NT: non-transduced parental NKT cells and GFP-, IL15-, or BATF3 -transduced NKTs) are injected IV. Figure 11 A presents weekly tumor bioluminescence imaging of mice at indicated time points. Figure 1 IB presents tumor bioluminescence graphed over time. Figure 11C presents Kaplan-Meier survival curve of tumor-bearing mice after treatment with GPC3- CAR NKT cells. 15.GPC3-CAR vs BATF3.GPC3-CAR groups p<0.001 by Mantel-Cox analysis.

[0025] Figure 12 presents results of an exemplary experiment showing that autonomous growth of NKTs does not result from expression of BATF3 either alone or in combination with a CAR in the engineered NKT cells of the present specification. NKT cells are transduced with indicated constructs and quantified at indicated timepoints while maintained under standard culture conditions without the use of cytokines or other stimulants.

[0026] Corresponding reference characters indicate corresponding parts throughout the several views. The examples set out herein illustrate several embodiments of the present disclosure but should not be construed as limiting the scope of the present disclosure in any manner.

DETAILED DESCRIPTION

[0027] The present application is directed to genetically modified natural killer T cells (NKT cells). NKT cells are a distinct cell type that share some features of both T and NK cells but are distinct from both conventional T cells and also NK cells. NKT cells have divergent development from conventional T cells and NK cells and different functions driven by a unique set of transcriptional regulators. See Kronenberg M, Gapin L. The unconventional lifestyle of NKT cells. Nat. Rev. Immunol. 2(8): 557-568 (2002); Godfrey and Kronenberg, “Going both ways: immune regulation via CD Id-dependent NKT cells,” J. Clin. Invest. 114(10): 1379-88 (2004), Cohen NR, el al. “Shared and distinct transcriptional programs underlie the hybrid nature of iNKT cells,” Nat Immunol. 14(l):90-99. 2013). Godfrey etal., identify transcription factors, signal-transduction factors, cell surface molecules, cytokines, and other factors that selectively influence NKT cell development reflecting the unique programming associated with the NKT cell lineage. (Godfrey etal., “Raising the NKT cell family,” Nat. Immunol., 11(3): 197-206 (2010) (“Godfrey et al ”) hereby incorporated by reference in its entirety. See also Engel and Kronenberg, “Transcriptional control of the development and function of Va4i NKT cells,” Current Topics in Microbiology and Immunology, Volume 381, 2014. Many transcription factors and signaling molecules that affect NKT cells differentiation in the thymus do not affect other conventional T cell populations that develop there. As used throughout the present disclosure, the term “T cell” is limited to conventional T cells that are distinguishable from NKT cells. These differences result in different responses to stimuli and genetic changes such as engineered gains and losses of gene expression that make results in non-NKT cells unpredictable.

[0028] NKT cells are distinguishable based on whole genome transcription analysis and are equally distant from conventional and NK cell lineages. See Cohen et al. supra. Conventional T cells, also known as T lymphocytes, are an important cell type with the function of fighting pathogens and regulating the immune response. Two hall marks of these cells are expression of an antigen receptor encoded by segments of DNA that rearrange during cell differentiation to form a vast array of receptors. A number of cells fall within this generic definition of a T cell, for example: T helper cells (CD4+ cells) including the sub-types TH1, TH2, TH3, TH17, TFH; cytotoxic T cells (mostly CD8+ cells, also referred to a CTLs); memory T cells (including central memory T cells, effector memory T cells, and resident memory T cells); regulatory T cells, and mucosal associated invariant T cells. Cell surface markers of T cells include the T cell receptor and CD3. Generally T cells do not express CD56 (i.e. are CD56 negative).

[0029] NK cells and NKT cells are CD56+. In humans NK cells usually express the cell surface marker CD56, CD161, CDl lb, NKp46, NKp44, CD158 and IL-12R. NK cells express a limited repertoire of receptors with an entirely different structure, some of which are also found on NKT cells. Most NK receptors are not highly conserved comparing humans and rodents. NK cells express members of the family of killer-cell-immunoglobulin-like receptors (KIRs), which can be activating or inhibiting, as well as receptors that are members of the lectin (carbohydrate- binding) family of proteins such as NKG2D and CD94NKG2A/C. KIRs are not expressed on NKT cells. NK cells are activated by a number of cell surface receptors, such as KIRs in humans or Ly49 in mice, natural cytotoxic receptors (NCRs), NKG2D and CD94:NKG2 heterodimers. In addition cytokines and chemokines, such as IL-12, IL-15, IL-18, IL-2 and CCLS, play a significant role in NK cell activation.

[0030] NKT cells generally can be identified as CD3+CD56+ cells and express a T cell receptor. NKT cells express a T cell receptor and CD3 chains like T cells, but also have markers such CD56 and CD161, like NK cells. Having said that, it is now commonly accepted by experts that they are a distinct lineage of cells. That is they are very different from other T cells and their behavior and properties cannot be predicted from analysis of other T cells, nor are they NK cells. NKT cells are completely different cells to conventional T cells and to NK cells. Due to the unique properties of the NKT cell lineage, observations made with other populations of lymphocytes, such as T cells, NK cells, and B cells, may not predict functional consequences of NKT cell activation.

[0031] NKT cells can be identified from other cell types including CD4 T cells, CD8 T cells, regulatory T cells, y5 T cells, B cells, NK cells, monocytes and dendritic cells based on the expression of cell surface markers. See Park etal., “OMIP-069: Forty-Color Full Spectrum Flow Cytometry Panel for Deep Immunophenotyping of Major Cell Subsets in Human Peripheral Blood,” Cytometry Part A 97A: 1044-1051 (2020); Hertoghs etal., OMIP-064: A 27-Color Flow Cytometry Panel to Detect and Characterize Human NK Cells and Other Innate Lymphoid Cell Subsets, MAIT Cells, and y5 T Cells, Cytometry Part A 97A: 1019-1023 (2020); Sahir et al., Development of a 43 color panel for the characterization of conventional and unconventional T- cell subsets, B cells, NK cells, monocytes, dendritic cells, and innate lymphoid cells using spectral flow cytometry, Cytometry 2020:1-7.

[0032] NKT cells are divided into two main types, Type I and Type II. The most significant form of NKT cells, known as type I NKT cells or invariant NKT cells (“iNKT”), have an invariant T cell receptor alpha chain (Va4i mouse or Va24i human). Type I NKT (iNKT) cells can be readily detected by the binding of CD Id- based tetramers loaded with aGalCer analogs. The form of the antigen receptor is a limited repertoire due to an invariant alpha chain paired with one of a relatively small number of beta chains, inhibition, or therapeutic use. The antigens recognized by this invariant receptor are glycolipids, for example those found in bacterial cells. The invariant receptor recognizes alpha-galatosylceramide (a-GalCer) a glycolipid originally derived from marine sponges. This compound is similar to microbial glycolipids, and it is now generally assumed to be derived from a microbial symbiont associated with the sponge. NKT cells require antigen presented on a molecule CD Id.

[0033] Type II NKT cells also require antigen presentation from CD1 d but have a more diverse but still limited TCR repertoire. Type II NKT cells express low levels of the transcription factor PLZF. While Type I NKT cells only recognize a-GalCer, Type II NKT cells recognize sulfatide, lyso-sulfatide, Lyso-PC and Lyso-GLl. Type II NKT cells are more prevalent in humans, but less prevalent in mice. See Dhodpkar and Kumar, “Type II NKT Cells and Their Emerging Role in Health and Disease,” J Immunol. 198(3): 1015-1021 (2017). [0034] Two pathways are known for NKT cell activation. NKT cells respond stimulation through their T cell receptor via antigen presented on CD Id molecules. This does not depend upon the involvement of a CD4 or CD8 co-receptor to generate a TCR signal, and the response of these cells is somewhat less dependent on a co-stimulatory signal. In addition, a mechanism for activation of NKT cells exists in the absence of antigen engaging the T cell receptor, via innate inflammatory stimuli, such as IL- 12 and IL- 18. Once activated T cells are found in the peripheral blood. Similarly NK cells are found in the peripheral blood. In contrast the majority of NKT cells are found in tissues and they migrate away from peripheral blood to the site of tumors, for example as mediated via a two-step process involving CCR2 and CCR6. The mechanisms involved in this migration are specific to NKT cells and not general mechanisms that apply to other lymphocytes.

[0035] iNKT cells are readily distinguishable from other T-cell types. See Table 1. Only a small fraction of expanded T cells (a subset of CD4 T cells) can produce tumor-protective Th2 cytokines (IL-4, IL-5, IL-13, IL-10) upon activation either via the T cell receptor (TCR). The majority of T cells (including all CD8+ T cells) and all NK cells produce only anti-tumor Thl cytokines (i.e. IFN-gamma, GM-CSF, TNF-alpha). In contrast, NKT cells simultaneously produce Thl and Th2 cytokines." Depending on the balance of Thl and Th2 cytokines produced after T cell receptor (TCR) activation, NKT cells can either activate or suppress the immune response. Thus NKT cells have an intriguing paradoxical dual function of immune activation and immune suppression. In contrast other immune cells usually have one primary function, for example fighting pathogens, whilst other subsets of cells are dedicated to regulating the immune response.

Table 1: Distinguishing features of iNKT cells

[0036] NKT cells also develop in the thymus, however, the positive selection of Type I NKT cells is mediated by CD Id positive thymocytes. NKT cells are also subject to negative selection by dendritic cells. See Godfrey et al., at Figure 2 summarizing the development and maturation of T cells and NKT cells in the thymus.

[0037] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invent ion belongs. The following references provide one of skill with a general definition of many of the terms used in this disclosure: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger etal. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.

[0038] The terms “comprises”, “comprising”, “includes”, “including”, “having” and their conjugates mean “including but not limited to.” The term “consisting of’ means “including and limited to.” The term “consisting essentially of’ means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

[0039] As used herein, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” or “at least one cell” may include a plurality of cells, including mixtures thereof.

[0040] As used herein the term “about” refers to plus/minus 10 %.

[0041] By “increase” is meant to alter positively by at least 5%. An alteration may be by 5%, 10%, 25%, 30%, 50%, 75%, or even by 100%.

[0042] By “decrease” or “reduce” is meant to alter negatively by at least 5%. An alteration may be by 5%, 10%, 25%, 30%, 50%, 75%, or even by 100%.

[0043] By “modulate” is meant positively or negatively alter. Exemplary modulations include a 1%, 2%, 5%, 10%, 25%, 50%, 75%, or 100% change.

[0044] Throughout this application, various embodiments of this disclosure may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0045] As used herein the term “method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

[0046] As used herein, “treatment” refers to clinical intervention in an attempt to alter the disease course of the individual or cell being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Therapeutic effects of treatment include, without limitation, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastases, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. By preventing progression of a disease or disorder, a treatment can prevent deterioration due to a disorder in an affected or diagnosed subject or a subject suspected of having the disorder, but also a treatment may prevent the onset of the disorder or a symptom of the disorder in a subject at risk for the disorder or suspected of having the disorder.

[0047] Nucleic acid molecules encoding polypeptides useful in the methods of the disclosure include any nucleic acid molecule that encodes the polypeptide of the disclosure or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will encode a polypeptide that is identical to the recited sequences. Exogenous nucleic acids of the present application provide for and include modifications such as optimized codons, fusions, and other non-function changing changes.

[0048] The terms “subject,” “individual,” and “patient,” are used interchangeably herein and refer to any vertebrate subject, including, without limitation, mammals, preferably a humans and other primates, and including laboratory animals including rodents such as mice, rats and guinea pigs. The term does not denote a particular age. Thus, both adult and newborn individuals are intended to be covered.

[0049] By “effective amount” is meant an amount sufficient to have a therapeutic effect. In one embodiment, an “effective amount” is an amount sufficient to arrest, ameliorate, or inhibit the continued proliferation, growth, or metastasis (e.g., invasion, or migration) of a neoplasia. [0050] The terms “isolated,” “purified,” or “biologically pure” refer to material that is free to varying degrees from components which normally accompany it as found in its native state. “Isolate” denotes a degree of separation from original source or surroundings. “Purify” denotes a degree of separation that is higher than isolation. A “purified” or “biologically pure” protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of this disclosure is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high performance liquid chromatography. The term “purified” can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified. [0051] The term, “operably linked”, as used herein, is meant the linking of two or more biomolecules so that the biological functions, activities, and/or structures associated with the biomolecules are at least retained. In reference to polypeptides, the term means that the linking of two or more polypeptides results in a fusion polypeptide that retains at least some of the respective individual activities of each polypeptide component. The two or more polypeptides may be linked directly or via a linker. In reference to nucleic acids, the term means that a first polynucleotide and a second polynucleotide are part of a single continuous nucleic acid molecule. In aspects, multiple polypeptides and regulatory sequences can be readily combined into a single operably linked nucleic acid. In aspects, operably linked nucleic acids are configured as a single linked expression vector. Combined vectors can reduce the number of steps to engineer the claims cells and can reduce cell to cell variability. Operably linked nucleic acids can include polypeptide encoding sequences and non-coding sequences including, but not limited to, regulatory sequences (promoters, transcriptional enhancer sequences, IRES sequences, autonomous intra-ribosomal self-processing peptide sequences separating polypeptides expressed as polyproteins). Operably linked nucleic acids can include sequences for suppressing the expression of an endogenous, for example using DNA sequences encoding a small hairpin RNA (shRNA) sequence, antisense, small inhibiting RNA (siRNA) and others known in the art. The operably linked sequences can be directly joined to each other, or separated by non-regulatory sequences such as linkers, cloning sites, and spacers that are known to persons of skill in the art. Unless indicated otherwise, the order of operably linked sequences can vary. [0052] As used herein, “populations of cells” refer to pluralities of cells and may further comprise mixtures of different cell types as well as homogenous populations.

[0053] The term, “recognize” is meant selectively binds a target. An immune cell that recognizes a cell typically expresses a receptor that binds an antigen expressed by the cell. Immune cells in aspects according to the present disclosure express a CAR that binds to a tumor associated antigen, for example tumor associated antigens and recognition domains presented in Table . As will be understood by those skilled in the art, the specification provides for and includes CARS having alternative recognition domains that bind to tumor associated antigens. [0054] As used herein, an “autonomous intra-ribosomal self-processing peptide” is a small peptide of 18 amino acids that avoids the need of proteinases to process a polyprotein into separate proteins. First discovered in foot-and-mouth disease virus, when introduced as a linker between two proteins, these peptides provides for the autonomous intra-ribosomal selfprocessing of polyproteins. Similar sequences have been identified in other members of the pircornaviradae. See de Felipe, “Skipping the co-expression problem: the new 2A ‘CHYSEL’ technology,” Genetic Vaccines and Therapy 2: 13 (2004).

[0055] As used herein, the term “engineering” refers to the genetic modification of a cell to introduce one or more exogenous nucleic acid sequences. Generally, the specification provides for engineering introduced exogenous nucleic acid sequences that are transcribed and translated to express a protein (or a polyprotein). Introducing exogenous nucleic acid sequences can be performed using methods known in the art including transformation, transfection and transduction.

[0056] As used herein, a “genetically engineered natural killer T (NKT) cell” or “engineered NKT cell” is an NKT cell that comprises at least one recombinant nucleic acid encoding exogenous protein or an endogenous protein downstream of a non-native promoter. In aspects, genetically engineered NKT cells comprise a recombinant nucleic acid encoding a basic leucine zipper ATF-like transcription factor (BATF) polypeptide. In other aspects, genetically engineered NKT cells comprising a recombinant nucleic acid encoding a BATF polypeptide and a chimeric antigen receptor. In additional aspects, the genetically engineered NKT cells can include additional proteins and regulatory nucleic acids. As used throughout, preparation of the engineered cells can be performed using combined expression vectors or using separate vectors. Combined vectors are illustrated in Figures 1 to 3, however each polypeptide or regulatory sequence can be introduced in separate steps. Combined vectors have advantages such as ease of introduction, ability to integrate in a single locus, transmissibility and heritability.

[0057] By “endogenous” is meant a nucleic acid molecule or polypeptide that is normally expressed in a cell or tissue.

[0058] By “exogenous” is meant a nucleic acid molecule or polypeptide that is not endogenously present in the cell, or not present at a level sufficient to achieve the functional effects obtained when over-expressed. The term “exogenous” would therefore encompass any recombinant nucleic acid molecule or polypeptide expressed in a cell, such as foreign, heterologous, and over-expressed nucleic acid molecules and polypeptides.

[0059] As used herein, the term “artificial microRNAs (amiRNAs)” are molecules that have been developed to promote gene silencing in a similar manner to naturally occurring miRNAs. amiRNAs are generally constructed by replacing the mature miRNA sequence in the pre-miRNA stem-loop with a sequence targeting a gene of interest. These molecules offer a great alternative to silencing approaches that are based on shRNAs and siRNAs because they present the same efficiency as these options and are less cytotoxic. As used herein, the term “embedded” in an artificial microRNA scaffold” refers to the process of replacing a mature miRNA sequence in the pre-miRNA stem-loop with a sequence targeting a gene of interest. In some aspects, the amiR used in the instant disclosure is amiR155. Lagos-Quintana etal., “Identification of tissue-specific microRNAs from mouse.” CurrBiol. 2002 Apr 30;12(9):735-9. In another aspect, the amiR used in the instant disclosure is amiR30. Fellmann etal., “An optimized microRNA backbone for effective single-copy RNAi.” Cell Rep. 2013 Dec 26;5(6):1704-13. In further aspects, the amiR used in the instant disclosure is an artificial microRNA scaffold known in the art, e.g., in WO 2022/226353, hereby incorporated by reference in its entirety.

[0060] A “short hairpin RNA,” “small hairpin RNA” or “shRNA” is an artificial RNA molecule with a tight hairpin turn that can be used to silence target gene expression via RNA interference (RNAi). They typically consist of a stem of 19-29 base pairs (bp), a loop of at least 4 nucleotides (nt), and a dinucleotide overhang at the 3' end. In some aspects, the term “shRNA” in the instant disclosure may refer to the sense strand or the antisense strand of the “stem” part of a small hairpin RNA. In other aspects, the term “shRNA” may include the sense strand, the antisense strand, and the loop in between. [0061] As used herein, a small hairpin RNA (shRNA) “targeting” a gene of interest refers to an shRNA comprising a sequence of at least 19 contiguous nucleotides that is essentially identical to, or is essentially complementary to, a gene of interest. Aspects of shRNAs functional in this disclosure have sequence complementarity that need not be 100% but is at least sufficient to permit hybridization to RNA transcribed from the target gene to form a duplex under physiological conditions in a cell to permit cleavage by a gene silencing mechanism. Thus, in aspects the segment is designed to be essentially identical to, or essentially complementary to, a sequence of 19 or more contiguous nucleotides in either the target gene or messenger RNA transcribed from the target gene. By “essentially identical” is meant having 100% sequence identity or at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity when compared to the sequence of 19 or more contiguous nucleotides in either the target gene or RNA transcribed from the target gene; by “essentially complementary” is meant having 100% sequence complementarity or at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence complementarity when compared to the sequence of 19 or more contiguous nucleotides in either the target gene or RNA transcribed from the target gene. In some aspects of this disclosure shRNAs are designed to comprise a sequence having 100% sequence identity with or complementarity to one allele of a given target gene; in other aspects the shRNAs are designed to comprise a sequence having 100% sequence identity with or complementarity to multiple alleles of a given target gene.

[0062] Sequence identity is typically measured using sequence analysis software widely available in the art. Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e'³ and e'¹⁰⁰ indicating a closely related sequence.

[0063] As used herein, the term “transcriptional activator in the Wnt signaling pathway,” identified also as “Wnt activator,” generally refers to proteins, that when exogenously expressed in a cell, activate genes downstream of Wnt/ 0-catenin signaling pathway. A transcriptional activator in the Wnt signaling pathway includes the expression of positive regulators of Wnt signaling such as LEF1 and inhibition of negative regulators, such as GSK30. Also included in transcriptional activators of Wnt signaling are small molecule activators including, but not limited to those described in Blagodatski et al., “Small Molecule Wnt Pathway Modulators from Natural Sources: History, State of the Art and Perspectives,” Cells 9:589 (2020) and Verkaar et al., “Discovery of Novel Small Molecule Activators of P-catenin Signaling,” PLoS ONE 6(4): el9185 (2011) and include inhibitors of negative regulators of Wnt signaling, such as TWS119 (See Ding etal., Synthetic small molecules that control stem cell fate,” PNAS 100(13):7632-7 (2003)).

[0064] As used herein, the phrase “expresses a growth factor” refers to the exogenous expression of one or more growth factors, generally under the control of a heterologous promoter and more usually as part of a polyprotein downstream of a CHYSEL sequence.

[0065] As used herein, the phrase “expresses a growth factor” refers to the exogenous expression of one or more growth factors, generally under the control of a heterologous promoter and more usually as part of a polyprotein downstream of a CHYSEL sequence.

[0066] The present specification provides for, and includes, genetically engineered NKT cells modified to express a basic leucine zipper ATF-like transcription factor (BATF) polypeptide. As provided herein, in aspects, a genetically engineered NKT cell is a Type I NKT cell. In an aspect the Type I NKT cell is a CD62L positive (CD62L+) NKT cell. Generally, the NKT cells of the present disclosure are isolated from human peripheral blood and have undergone less than 20 days of culture prior to introducing a gene construct to produce a genetically engineered NKT cell.

[0067] In aspects, the genetically engineered NKT cells of the present disclosure are further characterized by the expression of the cell markers CD4, CD28, 4- IBB, CD45RO (Gene ID5788), 0X40, CCR7, and combinations thereof. The expression of these markers is closely associated with trafficking of the NKT cells to the tumor site where they can mediate anti-tumor responses. In further aspects, the genetically engineered NKT cells express markers of NKT cell survival and memory such as, but not limited to, S1PR1, IL-7Ra, IL21R. In aspects, the genetically engineered NKT cells of the present disclosure express low levels of the exhaustion markers TIM-3, LAG3, and PD-1.

[0068] In aspects, the BATF is selected from the group consisting of basic leucine zipper ATF-like transcription factor (BATF), basic leucine zipper ATF-like transcription factor 2 (BATF2), or basic leucine zipper ATF-like transcription factor 3 (BATF3). The BATF sequences (e.g., SEQ ID NO: 1) are known in the art. In an aspect, the genetically engineered NKT cells further comprise an expression construct with a sequence encoding chimeric antigen receptor (CAR) having an antigen recognition domain directed to at least one tumor-associated antigen. Exemplary tumor-associated antigens and antigen recognition domain directed thereto are provided at Table . In aspects, the genetically engineered NKT cell comprises two expression constructs a first encoding expression of a CAR and a second encoding for expression of a BATF protein. Also provided for are expression constructs that express a protein encoding for a BATF protein and up to three additional protein sequences. In aspects, the expression construct encodes a polyprotein comprising a CAR and a BATF. The expression construct encoding a polyprotein comprising a CAR and a BATF may include a transcriptional activator in the Wnt signaling pathway, and may further include additional coding sequences (e.g. cistrons). In aspects, the expression construct encoding a polyprotein comprising CAR and a BATF. Exemplary combinations are presented in Figures 2 to 5. Engineered NKT cells of the present specification expressing BATF polypeptides exhibit enhanced proliferation. The claimed cells benefit from the cell-autonomous activity of the BATF polypeptides as non-specific effects on endogenous cells are reduced or eliminated. Importantly, expression of BATF does not impact or reduce cytotoxic activity and CAR and BATF expressing cells exhibit robust activity against tumor cells in vivo.

[0069] The present specification provides for and includes, genetically engineered NKT cells expressing a BATF polypeptide and a CAR as illustrated in Figure 2. In general, an ectodomain of the CAR encompasses a signal peptide, antigen recognition domain, and a spacer that links the antigen recognition domain to the transmembrane domain. The antigen recognition domain generally will comprise a single chain variable fragment (scFv) specific for a particular cancer antigen. However, in cases wherein there are two or more CARs in the same cell, the second CAR may comprise an scFv specific for another particular antigen. In aspects, the NKT cells are engineered using a single linked expression construct, though the introduction of the claimed sequences can be achieved in one or more steps using one or more vectors by methods known in the art. The genetically engineered NKT cells expressing a BATF polypeptide are not limited to any specific CAR, though CARs targeting certain tumor associated antigens are specifically envisioned. Included, and provided by the present disclosure are cancer antigens such as Melanoma-associated antigen (MAGE), Preferentially expressed antigen of melanoma (PRAME), CD19, CD20, CD22, K-light chain, CD30, CD33, CD123, CD38, CD138, R0R1, ErbB2, ErbB3/4, EGFr vIII, carcinoembryonic antigen, EGP2, mesothelin, TAG72, PSMA, NKG2D ligands, B7-H6, IL-13 receptor a2, MUC1, MUC16, CA9, GD2, GD3, , CD171, Lewis Y, G250/CAIX, HLA-AI MAGE Al, HLA-A2 NY-ESO-1, PSC1, folate receptor-a, CD44v6, CD44v7/8, 8H9, NCAM, VEGF receptors, 5T4, Fetal AchR, or CD44v6. In an aspect, the cancer antigen is selected from the group consisting of CD 19, GD2, and GPC3. In another aspect, the cancer antigen is CD 19. In an aspect, the cancer antigen is GD2. In yet another aspect, the cancer antigen is GPC3. Non-limiting examples of suitable tumor antigen sequences and CARs having an antigen recognition domain directed to tumor-associated antigens are provided in Table .

Table 2: Tumor associated antigens and antigen recognition domains thereto

[0070] Also included and provided for by the present disclosure are genetically engineered NKT cells comprising a BATF polypeptide and two or more CAR molecules that recognize cancer antigens selected from the group consisting of MAGE, PRAME, CD 19, CD20, CD22, K- light chain, CD30, CD33, CD 123, CD38, CD 138, ROR1, ErbB2, ErbB3/4, EGFr vIII, carcinoembryonic antigen, EGP2, mesothelin, TAG72, PSMA, NKG2D ligands, B7-H6, IL- 13 receptor a2, MUC1, MUC16, CA9, GD2, GD3, , CD171, Lewis Y, G250/CAIX, HLA-AI MAGE Al, HLA-A2 NY-ESO-1, PSC1, folate receptor-a, CD44v6, CD44v7/8, 8H9, NCAM, VEGF receptors, 5T4, Fetal AchR, and CD44v6. Also included and provided for by the present disclosure are genetically engineered NKT cells comprising a BATF polypeptide and two or more CAR molecules that recognize cancer antigens selected from the group of tumor associated antigens listed in Table . Expression constructs can be prepared separately, or more conveniently as one or more polyprotein expression constructs with CHYSEL sequences.

[0071] The present specification provides for, and includes, genetically engineered NKT cells expressing a BATF polypeptide and a sequence encoding a polypeptide for a transcriptional activator in the Wnt signaling pathway as illustrated in Figure 2, constructs 3 and 4. In some aspects, the BATF polypeptide and Wnt activator are provides as a polyprotein separated by a CHYSEL sequence. In some aspects, the BATF polypeptide comprises the amino terminal polypeptide of a polyprotein with a Wnt activator. In other aspects the BATF polypeptide comprises the carboxy terminal polypeptide of a polyprotein with a Wnt activator. Also included, and provided for by the present application are sequences encoding a BATF polypeptide and a Wnt activator wherein the polypeptides are expressed from different promoters. Such dual promoter constructs and the genetically engineered NKT cells prepared using them provide for differential expression of the two polypeptides. In an aspect, the BATF polypeptide may be expressed from regulatory sequence that leads to high expression while the Wnt signaling polypeptide is expressed using a promoter that provides lower levels of activity. Such differential expression cannot be attained when the two polypeptide sequences are expressed as a polyprotein. Thus, in aspects, the relative levels of expression of BATF polypeptides and Wnt activator can be controlled through selection of appropriate promoters In some aspects, the NKT cells can be engineered using a single vector, for example as shown in Figure 2, constructs 3 to 5. Also included and provided are genetically engineered NKT cells prepared using separate vectors.

[0072] In aspects, the genetically engineered NKT cells modified to express a basic leucine zipper ATF-like transcription factor (BATF) polypeptide and a transcriptional activator in the Wnt signaling pathway are Type I NKT cells. In an aspect the Type I NKT cells engineered to express BATF and Wnt activator is a CD62L positive (CD62L+) NKT cell. Generally, the NKT cells of the present disclosure are isolated from human peripheral blood and have undergone less than 20 days of culture prior to introducing a gene construct to produce a genetically engineered NKT cell.

[0073] In aspects of the present specification, the genetically engineered NKT cells modified to express a basic leucine zipper ATF-like transcription factor (BATF) polypeptide and a transcriptional activator in the Wnt signaling pathway express BATF3. In another aspect, the BATF polypeptide is BATF2. In a further aspect, the BATF polypeptide is BATF. Exemplary, non-limiting vectors for modifying NKT cells are presented in Figure 1, constructs 1 to 5.

[0074] The genetically engineered NKT cells can be modified to express a CAR, a BATF polypeptides, and a Wnt activator. Exemplary constructs for the preparation of such cells are provided in Figure 2, construct 5 and 6. It is to be understood that the polypeptide order can be changed without changing the overall function of the genes in the engineered NKT cell. In aspects, the CAR is selected from the CARS Table , combined with BATF3, and Wnt activator LEF1. In aspects, the CAR is selected from the CARS Table , combined with BATF2, and Wnt activator LEF1. In aspects, the CAR is selected from the CARS Table , combined with BATF, and Wnt activator LEF1.

[0075] Major histocompatibility complex (MHC) class I and class II proteins play a pivotal role in the adaptive branch of the immune system. Both classes of proteins share the task of presenting peptides on the cell surface for recognition by T cells.

[0076]

[0077] The human leukocyte antigen (HLA) system or complex is a group of related proteins that are encoded by the MHC gene complex in humans. These cell-surface proteins are responsible for the regulation of the immune system.

[0078] The present disclosure provides for, and includes, genetically engineered NKT cells expressing a BATF polypeptide and suppressing the expression of an endogenous major histocompatibility complex (MHC) gene comprising a DNA sequence encoding a small hairpin RNA (shRNA) sequence targeting an MHC class I or MHC class II gene, where the shRNA sequence is embedded in an artificial microRNA (amiR) scaffold. An example of suitable constructs to prepare engineered cells are presented in Figure 4, constructs 1 to 4. In some aspects, the amiR used in the instant disclosure is amiR155. In another aspect, the amiR used in the instant disclosure is amiR30. In further aspects, the amiR used in the instant disclosure is an artificial microRNA scaffold known in the art.

[0079] In some aspect, the MHC class I and class II genes are human leukocyte antigen (HLA) class I and class II genes.

[0080] In some aspects, the MHC class I gene encodes a P2-microglobulin (B2M).

[0081] In some aspects, the MHC class II gene encodes an invariant chain (li) or a class II transactivator (CIITA).

[0082] In some aspects, the recombinant constructs as disclosed herein comprise a first shRNA sequence embedded in a first amiR scaffold and a second shRNA sequence embedded in a second amiR scaffold. Non-limiting examples of BATF expressing constructs having a first and second shRNA sequence are illustrated in Figure 4, constructs 5 and 6. In some aspects, the first shRNA sequence targets a MHC class I gene and the second shRNA sequence targets a MHC class II gene. In other aspects the order of the shRNA sequences can be reversed. As shown in Figure 4, the regulatory sequences are a suitable promoter. In one aspect, the first amiR scaffold and the second amiR scaffold are from the same amiR sequence. In other aspects, the first amiR scaffold and the second amiR scaffold are from different amiR sequences.

[0083] The present disclosure also provides for, and includes, a method for limiting rejection of an engineered natural killer T (NKT) cell by the immune system of an allogeneic host, comprising transducing an NKT cell with the recombinant constructs disclosed herein, where the expression of the endogenous MHC gene in the NKT cell is suppressed by the shRNA.

[0084] Also included, and provide for, are genetically engineered NKT cells expressing a BATF polypeptide, a CAR, and sequences for suppressing the expression of an endogenous major histocompatibility complex (MHC) gene comprising a DNA sequence encoding a small hairpin RNA (shRNA) sequence targeting an MHC class I or MHC class II gene (“MHC gene suppressor sequence”). Non-limiting examples of constructs to prepare the engineered NKT cells are presented in Figure 5, constructs 1 and 2. In other aspects, the engineered NKT cells can further comprise a Wnt activator. Constructs suitable to prepare such cells are presented in Figure 4, constructs 3 and 4.

[0085] In aspects, the genetically engineered NKT cells modified to express a BATF and polypeptide and sequences for suppressing the expression of an endogenous major histocompatibility complex (MHC) gene are Type I NKT cells. In an aspect the Type I NKT cells engineered to express BATF and MHC gene suppressor activator is a CD62L positive (CD62L+) NKT cell. The specification further provides for Type I NKT cells expressing a CAR, BATF polypeptide, a Wnt activator, and MHC gene suppressor sequences. In a further aspect, the Type I NKT cells expressing a CAR, a BATF polypeptide, a Wnt activator, and MHC gene suppressor sequence are CD62L positive NKT cell. In aspects, the NKT cells comprise a plurality of cells.

[0086] The genetically engineered NKT cells of the present specification may further harbor a polynucleotide that encodes the CAR, and the polynucleotide may further comprise a suicide gene. The construct described throughout can have the suicide gene sequences embedded in the constructs used to prepare them either as part of a polyprotein or driven from a separate promoter. Also included are cells engineered using a second construct to introduce the suicide gene sequences.

[0087] In one aspect, the expression constructs or chimeric antigen receptor expression constructs according to the present disclosure, and the cells prepared therefrom, further comprises an inducible suicide gene. Non-limiting examples of inducible suicide genes include an inducible caspase-9 suicide gene and a thymidine kinase (sr39 TK). In one aspect, the inducible caspase-9 suicide gene in the expression construct is activated by AP20187, API 903, or a mixture thereof. In another aspect, the In one aspect, the thymidine kinase in the expression construct is activated by ganciclovir. In one aspect, methods according to the present disclosure may comprise administering AP20187, API 903, or a mixture thereof to the subject to activate the inducible caspase-9 suicide gene. In another aspect, methods according to the present disclosure may comprise administering ganciclovir to the subject to activate the thymidine kinase.

[0088] In one aspect, the expression constructs or chimeric antigen receptor expression constructs according to the present disclosure further comprises a protein coding sequence for a CD34 tag.

[0089] The present disclosure provides for, and includes, recombinant nucleic acids useful for preparing genetically engineered cells. As used herein, it will be understood that when referring to polypeptides in recombinant nucleic acids, the nucleic acids provide sequences encoding said polypeptide. In aspects, the nucleic acids can be codon-optimized for improved expression of the polypeptides. For polyproteins, additional sequences encoding CHYSEL sequences are included. In other aspects, polypeptides in polycistronic expression cassettes can each be independently driven by one or more promoters. It should be understood that it is the expression of the polypeptides (or the suppression sequences for MHC genes) in the genetically engineered cell that provide the improvements and alternative approaches leading to the expression of the desired cistrons can be accomplished through multiple vectors and approaches.

[0090] In an aspect, the recombinant nucleic acids are prepared as polycistronic expression cassettes, generally illustrated in Figures 2 to 5. In aspects, the recombinant nucleic acids comprise a transcriptional regulatory element operably linked to a polycistronic polynucleotide that comprises nucleic acid sequences encoding a basic leucine zipper ATF-like transcription factor (BATF), basic leucine zipper ATF-like transcription factor 2 (BATF2), or basic leucine zipper ATF-like transcription factor 3 (BATF3), and a polypeptide for a chimeric antigen receptor (CAR) having an antigen recognition domain directed to at least one tumor-associated antigen. Suitable CAR sequences and tumor-associated antigens are presented in Table . [0091] The general construction of CARs are presented in Figure 1 and comprise an ectodomain, a transmembrane domain, and an endodomain. The ectodomain comprises the antigen recognition domain, generally as a scfv but other alternatives are known in the art. The transmembrane domain can be obtained from CD8a, but the CAR constructs should not be considered to be so limited. Alternative transmembrane regions are generally known in the art. The endodomain comprises a stimulatory domain, often but not limited to CD3g, and can further include a co-stimulatory domain obtained for example from CD28.

[0092] Exemplary recombinant constructs are presented in Figure 2, construct 1 and 2. In an aspect, the polycistronic expression cassette comprises a CAR polypeptide encoding sequence, regulatory sequence, and a BATF polypeptide sequence. In aspects the regulatory sequence is a CHYSEL sequence, and the polycistronic expression cassettes expresses the CAR polypeptide, the CHYSEL peptide sequence, and BATF polypeptide as a polyprotein. The expressed polyprotein undergoes autonomous intra-ribosomal self-processing of the polyproteins to produce a CAR polypeptide fused at the carboxy terminal end to a partial CHYSEL peptide and a BATF polypeptide. In other aspects, the polycistronic expression cassette regulatory sequence is an IRES sequence. IRES sequences allow for ribosomal binding and expression of the downstream protein encoding sequences as a separate polypeptide. In some aspects, the regulatory sequence is a promoter. The selection of appropriate regulatory sequences is known in the art. Generally polypeptide encoding sequences can be placed downstream of a promoter, a CHYSEL, or an IRES sequence, and combinations thereof. Non-coding sequences, for example the MHC shRNA targeting sequences, can be expressed from a promoter driving expression of the polycistronic cassette, but as illustrated in Figure 4, can have a promoter as a regulatory sequence. Incorporation of a separate promoter provides for a greater level of control of expression. In aspects, strong promoters are used as a regulatory sequence to ensure high levels of the inhibitory shRNA, antisense RNA, or other sequences.

[0093] The specification further provides for recombinant nucleic acids and vectors useful for preparing genetically engineered cells expressing a BATF polypeptide, a polypeptide for a chimeric antigen receptor (CAR) having an antigen recognition domain directed to at least one tumor-associated antigen, and further including a polypeptide encoding a Wnt activator from a polycistronic expression cassette. Exemplary expression cassettes of this type are illustrated in Figure 2, constructs 5 and 6. The present specification provides for, and includes, constructs incorporating the BATF, CAR, and Wnt Activator in different arrangements, for example as illustrated in the exemplary constructs of Figure 3. The selection of appropriate regulatory sequences (e.g, CHYSEL, IRES, promoter) is within the skill of the artisan, for examples based on desired expression levels, activities, and convenience. In aspects, polycistronic sequences encode the CAR polypeptides presented in Table , a 2 A CHYSEL sequence, a BATF polypeptide, a second CHYSEL 2A sequence, and the Wnt activator LEF1 (see Figure 3, construct 3). In the alternative, the order of the BATF polypeptide and Wnt activator can be switched as illustrated in Figure 3, constructs 4 and 6, and the regulatory sequence replaced, for example with a U6 promoter as illustrated in Figure 3, constructs 5 and 6. Additional variations will be understood by a skilled artisan in view of the present specification.

[0094] In aspects, the polycistronic expression cassette comprises a CAR polypeptide of Table 2, BATF3, and LEF1 and further include one or more regulatory sequences. In another aspect, the polycistronic expression cassette comprises a CAR polypeptide of Table , BATF2, and LEF1 and further include one or more regulatory sequences. In another aspect, the polycistronic expression cassette comprises a CAR polypeptide of Table , BATF, and LEF1 and further include one or more regulatory sequences. In aspects, the CAR polypeptide recognizes the GD2 antigen, the BATF polypeptide is BATF3, and the Wnt activator is LEF1. In aspects, the CAR polypeptide recognizes the CSPG4 antigen, the BATF polypeptide is BATF3, and the Wnt activator is LEF1. In aspects, the CAR polypeptide recognizes the GPC3 antigen, the BATF polypeptide is BATF3, and the Wnt activator is LEF1. In aspects, the CAR polypeptide recognizes the CD 19 antigen, the BATF polypeptide is BATF3, and the Wnt activator is LEF1. In aspects, the CAR polypeptide recognizes the GD2 antigen, the BATF polypeptide is BATF2, and the Wnt activator is LEF1. In aspects, the CAR polypeptide recognizes the CSPG4 antigen, the BATF polypeptide is BATF2, and the Wnt activator is LEF1. In aspects, the CAR polypeptide recognizes the GPC3 antigen, the BATF polypeptide is BATF2, and the Wnt activator is LEF1. In aspects, the CAR polypeptide recognizes the CD 19 antigen, the BATF polypeptide is BATF2, and the Wnt activator is LEF1. In aspects, the CAR polypeptide recognizes the GD2 antigen, the BATF polypeptide is BATF2, and the Wnt activator is LEF1. In aspects, the CAR polypeptide recognizes the CSPG4 antigen, the BATF polypeptide is BATF, and the Wnt activator is LEF1. In aspects, the CAR polypeptide recognizes the GPC3 antigen, the BATF polypeptide is BATF, and the Wnt activator is LEF1. In aspects, the CAR polypeptide recognizes the CD 19 antigen, the BATF polypeptide is BATF, and the Wnt activator is LEFl.

[0095] Also included and provide for by the present specification are polycistronic expression cassettes comprising a BATF polypeptide and a suppressor sequence for an MHC class I gene, and MHC class II gene, or both an MHC class I and MHC class II gene. Exemplary constructs are presented in Figure 4, constructs 1 to 6. In aspects, the cistrons can be separated by a regulatory sequence as shown in Figure 4, or the BATF sequences and MHC suppressor sequences can be driven from a single regulatory sequence (e.g., a promoter). As with other constructs, the order of the cistrons is not critical to the effectiveness of the constructs in preparing the engineered NKT cells of the present specification.

[0096] In aspects, the polycistronic expression cassettes comprising a BATF polypeptide and a suppressor sequence for an MHC gene comprises a polypeptide encoding BATF3 and a suppressor sequence for an MHC class I gene, and MHC class II gene, or both an MHC class I and MHC class II gene. In another aspect, the polycistronic expression cassettes comprising a BATF polypeptide and a suppressor sequence for an MHC gene comprises a polypeptide encoding BATF2 and a suppressor sequence for an MHC class I gene, and MHC class II gene, or both an MHC class I and MHC class II gene. In a further aspect, the polycistronic expression cassettes comprising a BATF polypeptide and a suppressor sequence for an MHC gene comprises a polypeptide encoding BATF and a suppressor sequence for an MHC class I gene, and MHC class II gene, or both an MHC class I and MHC class II gene.

[0097] The specification further provides, and includes, recombinant nucleic acid constructs for the expression of a BATF polypeptide, a suppressor sequence for an MHC gene, and a CAR. Exemplary constructs are presented in Figure 5, constructs 1 and 2. In an aspect, the recombinant nucleic acid comprises a polycistronic expression cassette comprising a CAR, a BATF polypeptide, and a suppressor sequence for an MHC gene. In an aspect, the recombinant nucleic acid comprises a polycistronic expression cassette comprising a CAR, a BATF polypeptide, and a suppressor sequence for an MHC class I gene and an MHC class II gene. In aspects, the polycistronic expression cassette encodes a CAR specific for a tumor antigen as presented in Table 2, a BATF polypeptide, and a suppressor sequence for an MHC class I gene and an MHC class II gene. In an aspect, the polycistronic expression cassette encodes a CAR specific for GD2, a BATF3 polypeptide, and a suppressor sequence for an MHC class I gene and an MHC class II gene. In an aspect, the polycistronic expression cassette encodes a CAR specific for CSPG4, a BATF3 polypeptide, and a suppressor sequence for an MHC class I gene and an MHC class II gene. In an aspect, the polycistronic expression cassette encodes a CAR specific for GPC3, a BATF3 polypeptide, and a suppressor sequence for an MHC class I gene and an MHC class II gene. In another aspect, the polycistronic expression cassette encodes a CAR specific for CD 19, a BATF3 polypeptide, and a suppressor sequence for an MHC class I gene and an MHC class II gene. In a further aspect, the polycistronic expression cassette encodes a CAR specific for GD2, a BATF2 polypeptide, and a suppressor sequence for an MHC class I gene and an MHC class II gene. In an aspect, the polycistronic expression cassette encodes a CAR specific for CSPG4, a BATF2 polypeptide, and a suppressor sequence for an MHC class I gene and an MHC class II gene. In another aspect, the polycistronic expression cassette encodes a CAR specific for GPC3, a BATF2 polypeptide, and a suppressor sequence for an MHC class I gene and an MHC class II gene. In another aspect, the polycistronic expression cassette encodes a CAR specific for CD 19, a BATF2 polypeptide, and a suppressor sequence for an MHC class I gene and an MHC class II gene. In an additional aspect, the polycistronic expression cassette encodes a CAR specific for GD2, a BATF polypeptide, and a suppressor sequence for an MHC class I gene and an MHC class II gene. In a further aspect, the polycistronic expression cassette encodes a CAR specific for CSPG4, a BATF polypeptide, and a suppressor sequence for an MHC class I gene and an MHC class II gene. In an aspect, the polycistronic expression cassette encodes a CAR specific for GPC3, a BATF polypeptide, and a suppressor sequence for an MHC class I gene and an MHC class II gene. In yet another aspect, the polycistronic expression cassette encodes a CAR specific for CD 19, a BATF polypeptide, and a suppressor sequence for an MHC class I gene and an MHC class II gene.

[0098] The specification further provides, and includes, recombinant nucleic acid constructs for the expression of a BATF polypeptide, a Wnt activator, and a suppressor sequence for an MHC gene, and a CAR. Exemplary constructs are presented in Figure 5, constructs 3 and 4. Additional specific aspects are presented in Figure 6, constructs 1 to 6. In an aspect, the recombinant nucleic acid comprises a polycistronic expression cassette comprising a CAR, a BATF polypeptide, a Wnt activator, and a suppressor sequence for an MHC gene. In an aspect, the recombinant nucleic acid comprises a polycistronic expression cassette comprising a CAR, a BATF polypeptide, a Wnt activator, and a suppressor sequence for an MHC class I gene and an MHC class II gene. In aspects, the polycistronic expression cassette encodes a CAR specific for a tumor antigen as presented in Table 2, a BATF polypeptide, a Wnt activator, and a suppressor sequence for an MHC class I gene and an MHC class II gene. In an aspect, the the polycistronic expression cassette encodes a CAR specific for GD2, a BATF3 polypeptide, the Wnt activator LEF1, and a suppressor sequence for an MHC class I gene and an MHC class II gene. In an aspect, the polycistronic expression cassette encodes a CAR specific for CSPG4, a BATF3 polypeptide, the Wnt activator LEF1, and a suppressor sequence for an MHC class I gene and an MHC class II gene. In an aspect, the polycistronic expression cassette encodes a CAR specific for GPC3, a BATF3 polypeptide, the Wnt activator LEF1, and a suppressor sequence for an MHC class I gene and an MHC class II gene. In another aspect, the polycistronic expression cassette encodes a CAR specific for CD 19, a BATF3 polypeptide, the Wnt activator LEF1, and a suppressor sequence for an MHC class I gene and an MHC class II gene. In a further aspect, the polycistronic expression cassette encodes a CAR specific for GD2, a BATF2 polypeptide, the Wnt activator LEF1, and a suppressor sequence for an MHC class I gene and an MHC class II gene. In an aspect, the polycistronic expression cassette encodes a CAR specific for CSPG4, a BATF2 polypeptide, the Wnt activator LEF1, and a suppressor sequence for an MHC class I gene and an MHC class II gene. In another aspect, the polycistronic expression cassette encodes a CAR specific for GPC3, a BATF2 polypeptide, the Wnt activator LEF1, and a suppressor sequence for an MHC class I gene and an MHC class II gene. In another aspect, the polycistronic expression cassette encodes a CAR specific for CD 19, a BATF2 polypeptide, the Wnt activator LEF1, and a suppressor sequence for an MHC class I gene and an MHC class II gene. In an additional aspect, the polycistronic expression cassette encodes a CAR specific for GD2, a BATF polypeptide, the Wnt activator LEF1, and a suppressor sequence for an MHC class I gene and an MHC class II gene. In a further aspect, the polycistronic expression cassette encodes a CAR specific for CSPG4, a BATF polypeptide, the Wnt activator LEF1, and a suppressor sequence for an MHC class I gene and an MHC class II gene. In an aspect, the polycistronic expression cassette encodes a CAR specific for GPC3, a BATF polypeptide, the Wnt activator LEF1, and a suppressor sequence for an MHC class I gene and an MHC class II gene. In yet another aspect, the polycistronic expression cassette encodes a CAR specific for CD 19, a BATF polypeptide, the Wnt activator LEF1, and a suppressor sequence for an MHC class I gene and an MHC class II gene. [0099] The present specification further provides for, and includes, recombinant nucleic acids that further include a nucleic acid sequence encoding an inducible suicide gene. The addition of a cistron encoding an inducible suicide gene can be under the regulation of its own regulatory sequence such as a promoter, or can be transcribed from a common promoter and having either a CHYSEL sequence or an IRES sequence.

[00100] The present application provides for, and includes, methods of treatment comprising administering a therapeutically effective amount of a plurality of engineered Natural Killer T- cells (NKT cells). In aspects, the engineered NKT cells are engineered to express a BATF polypeptide. In an aspect, the BATF polypeptide is BATF3. In another aspect the BATF polypeptide is BATF2. In a further aspect, the BATF polypeptide is BATF. Included and provided for are engineered NKT cells for use in a method of treatment comprise a CAR in combination with a BATF polypeptide. In a further aspect, the engineered NKT cells for use in a method of treatment comprise a CAR in combination with a BATF polypeptide and a Wnt activator. Also included, are engineered NKT cells for use in a method of treatment comprising a CAR in combination with a BATF polypeptide, a Wnt activator and a suppressor of a MHC class I gene, an MHC class II gene, or both.

[00101] The present application provides for, and includes, methods of treatment comprising administering a therapeutically effective amount of a plurality of engineered Natural Killer T- cells (NKT cells). In aspects, the engineered NKT cells are engineered to express a BATF polypeptide and further comprise an exogenously expressed inducible suicide gene. In an aspect, the BATF polypeptide is BATF3. In another aspect the BATF polypeptide is BATF2. In a further aspect, the BATF polypeptide is BATF. Included and provided for are engineered NKT cells for use in a method of treatment comprise a CAR in combination with a BATF polypeptide. In a further aspect, the engineered NKT cells for use in a method of treatment comprise a CAR in combination with a BATF polypeptide and a Wnt activator. Also included, are engineered NKT cells for use in a method of treatment comprising a CAR in combination with a BATF polypeptide, a Wnt activator and a suppressor of a MHC class I gene, an MHC class II gene, or both. The methods of the present specification provide for providing a therapeutically effective amount of a plurality of engineered NKT cells, allowing for a treatment period in a patient, and further comprising elimination of the administered engineered NKT cells in the patient by activating said inducible suicide gene. In aspects, the inducible suicide gene is inducible caspase-9 suicide gene. In another aspect, the method further comprises administering AP20187, API 903, or a mixture thereof to the subject to activate said inducible caspase-9 suicide gene. [00102] The present application provides for, and includes, methods of treatment comprising administering a therapeutically effective amount of a plurality of engineered Natural Killer T- cells (NKT cells) further comprising an exogenously expressed thymidine kinase (sr39 TK) as an inducible suicide gene. In an aspect, the BATF polypeptide is BATF3. In another aspect the BATF polypeptide is BATF2. In a further aspect, the BATF polypeptide is BATF. Included and provided for are engineered NKT cells for use in a method of treatment comprise a CAR in combination with a BATF polypeptide, and further comprising an exogenously expressed thymidine kinase (sr39 TK) as an inducible suicide gene. In a further aspect, the engineered NKT cells for use in a method of treatment comprise a CAR in combination with a BATF polypeptide, a Wnt activator, and an exogenously expressed thymidine kinase (sr39 TK) as an inducible suicide gene. Also included are engineered NKT cells for use in a method of treatment comprising a CAR in combination with a BATF polypeptide, a Wnt activator and a suppressor of a MHC class I gene, an MHC class II gene, or both, and an exogenously expressed thymidine kinase (sr39 TK) as an inducible suicide gene. The methods of the present specification provide for providing a therapeutically effective amount of a plurality of engineered NKT cells, allowing for a treatment period in a patient, and further comprising elimination of the administered engineered NKT cells in the patient by activating said inducible suicide gene. In another aspect, the method further comprises administering ganciclovir to the subject to activate they thymidine kinase.

[00103] The specification provides for, and includes, methods of treatment of cancer comprising administering a therapeutically effective amount of a plurality of engineered Natural Killer T-cells (NKT cells) as described. In aspects, the cancer is a tumor. In aspects, the cancer is selected from the group consisting of breast cancer, cervical cancer, ovary cancer, endometrial cancer, melanoma, bladder cancer, lung cancer, pancreatic cancer, colon cancer, prostate cancer, hematopoietic tumors of lymphoid lineage, leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, B-cell lymphoma, Burkitt's lymphoma, multiple myeloma, Hodgkin's lymphoma, Non-Hodgkin's lymphoma, myeloid leukemia, acute myelogenous leukemia (AML), chronic myelogenous leukemia, thyroid cancer, thyroid follicular cancer, myelodysplastic syndrome (MDS), tumors of mesenchymal origin, fibrosarcoma, rhabdomyosarcoma, melanoma, uveal melanoma, teratocarcinoma, neuroblastoma, glioma, glioblastoma, benign tumor of the skin, renal cancer, anaplastic large-cell lymphoma, esophageal squamous cell carcinoma, hepatocellular carcinoma, follicular dendritic cell carcinoma, intestinal cancer, muscle-invasive cancer, seminal vesicle tumor, epidermal carcinoma, spleen cancer, bladder cancer, head and neck cancer, stomach cancer, liver cancer, bone cancer, brain cancer, cancer of the retina, biliary cancer, small bowel cancer, salivary gland cancer, cancer of uterus, cancer of testicles, cancer of connective tissue, prostatic hypertrophy, myelodysplasia, Waldenstrom's macroglobinaemia, nasopharyngeal, neuroendocrine cancer myelodysplastic syndrome, mesothelioma, angiosarcoma, Kaposi's sarcoma, carcinoid, oesophagogastric, fallopian tube cancer, peritoneal cancer, papillary serous mullerian cancer, malignant ascites, gastrointestinal stromal tumor (GIST), and a hereditary cancer syndrome selected from Li-Fraumeni syndrome and Von Hippel-Lindau syndrome (VHL). In an aspect, the cancer is neuroblastoma.

[00104] In aspects of the methods for treating cancer, the engineered NKT cells of the present specification are autologous cells derived from the cancer patient in need of cancer treatment. The engineered NKT cells can be prepared as described in the Examples and transformed with the recombinant nucleic acids described herein.

[00105] The method for treating cancer include providing a therapeutically effective amount of the engineered NKT cells of the present specification systemically. In another aspect the therapeutically effective amount of the engineered NKT cells can be provide parenterally. In yet another aspect, the therapeutically effective amount of the engineered NKT cells can be administered locally to the tumor.

[00106] The present specification provides for combination treatments of comprising a therapeutically effective amount of the engineered NKT cells of the present specification with one or more additional cancer therapies to the subject. In some aspects, one or more therapies in addition to the immunotherapy of the disclosure may be provided to the subject, such as surgery, radiation, hormone therapy, another, nonidentical immunotherapy, chemotherapy, or a combination thereof.

[00107] The present specification further includes, and provides for methods of producing engineered NKT cells for use in immunotherapy comprising isolating human NKT cells from human peripheral blood mononuclear cells (PBMCs), culturing the human NKT cells in the presence of (1) IL-21 and at least one or more cytokines selected from the group consisting of IL-7, IL-15, IL-12, TNF-alpha, and a combination thereof and (2) irradiated NKT-depleted PBMCs or irradiated artificial antigen-presenting cells (aAPCs) loaded with alpha- Galactosylceramide (aGalCer) to prepare a culture having a majority of CD62L-positive Type I NKT cells, and genetically modifying the CD62L-positive Type I NKT cells to exogenously express a basic leucine zipper ATF-like transcription factor (BATF) polypeptide.

In an aspect, the method further includes genetically modifying the CD62L-positive Type I NKT cells to express one or more sequences selected from the group consisting of: a sequence encoding chimeric antigen receptor (CAR) having an antigen recognition domain directed to at least one tumor-associated antigen; a sequence encoding a polypeptide for a transcriptional activator in the Wnt signaling pathway a recombinant sequence for suppressing the expression of an endogenous major histocompatibility complex (MHC) gene; one or more regulatory sequences comprising a promoter, an internal ribosomal entry site (IRES), a cis-acting hydrolase element (CHYSEL); and a combination thereof. Suitable vectors for genetically modifying the cells are described above.

[00108] In an aspects of the present specification, the CD62L-positive Type I NKT cells are modified to exogenously express a CAR polypeptide recognizing the tumor-associated antigens of Table 2, and BATF3 polypeptide. In another aspect, the CD62L-positive Type I NKT cells are modified to exogenously express a CAR polypeptide recognizing the tumor-associated antigens of Table 2, a BATF3 polypeptide, and a Wnt Activator. In an aspect, the Wnt activator is LEF1. Also included in aspect, is genetically modifying the CD62L-positive Type I NKT cells to exogenously express a CAR polypeptide recognizing the tumor-associated antigens of Table 2, a BATF3 polypeptide, and a Wnt Activator, and a one or more recombinant sequences for suppressing the expression of endogenous major histocompatibility complex (MHC) genes. In further aspects, the genetically engineered CD62L-positive Type I NKT cells are expanded by culturing in the presence of of (1) IL-21 and at least one or more cytokines selected from the group consisting of IL-7, IL- 15, IL- 12, TNF-alpha, and a combination thereof and (2) irradiated NKT-depleted PBMCs or irradiated artificial antigen-presenting cells (aAPCs) loaded with alpha-Galactosylceramide (aGalCer) to prepare a therapeutic amount of genetically engineered CD62L-positive Type I NKT cells comprising an exogenously expressed CAR and a BATF polypeptide. In an aspect, the BATF polypeptide is BATF3. The methods further include providing a therapeutically effective amount of the expanded genetically engineered CD62L- positive Type I NKT cells to an individual.

[00109] While the present disclosure has been described with reference to certain embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof to adapt to particular situations without departing from the scope of the present disclosure. Therefore, it is intended that the present disclosure not be limited to the particular embodiments disclosed as the best mode contemplated for carrying out the present disclosure, but that the present disclosure will include all embodiments falling within the scope and spirit of the appended claims.

[00110] Any references cited herein are incorporated by reference in their entireties.

[00111] While the present disclosure has been described with reference to preferred embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof to adapt to particular situations without departing from the scope of the present disclosure. Therefore, it is intended that the present disclosure not be limited to the particular embodiments disclosed as the best mode contemplated for carrying out the present disclosure, but that the present disclosure will include all embodiments falling within the scope and spirit of the appended claims.

[00112] Some examples of embodiments include, but are not limited to, the following.

1. An engineered Natural Killer T-cell (NKT cell), or a plurality thereof, comprising an expression construct that encodes a basic leucine zipper ATF-like transcription factor (BATF) polypeptide.

2. The engineered Natural Killer T-cell (NKT cell), or a plurality thereof, of embodiment 1, further comprising one or more sequences selected from the group consisting of: a sequence encoding chimeric antigen receptor (CAR) having an antigen recognition domain directed to at least one tumor-associated antigen; a sequence encoding a polypeptide for a transcriptional activator in the Wnt signaling pathway a recombinant sequence for suppressing the expression of an endogenous major histocompatibility complex (MHC) gene; one or more regulatory sequences comprising a promoter, an internal ribosomal entry site (IRES), a cis-acting hydrolase element (CHYSEL); and a combination thereof.

3. The engineered Natural Killer T-cell (NKT cell), or a plurality thereof, of embodiment 1, wherein said BATF polypeptide is basic leucine zipper ATF-like transcription factor (BATF), basic leucine zipper ATF-like transcription factor 2 (BATF2), or basic leucine zipper ATF-like transcription factor 3 (BATF3).

4. The engineered Natural Killer T-cell (NKT cell), or a plurality thereof, of embodiment 1, wherein said engineered T-cell is a Type I Natural Killer T-cell (NKT cell).

5. The engineered Natural Killer T-cell (NKT cell), or a plurality thereof, of embodiment 1, wherein said engineered NKT cell is a CD62L-positive Type I NKT cell.

6. The engineered Natural Killer T-cell (NKT cell), or a plurality thereof, of embodiment 1, wherein said NKT cell is a human cell.

7. The engineered Natural Killer T-cell (NKT cell), or a plurality thereof, of embodiment 1, wherein the expression construct comprises a vector.

8. The engineered Natural Killer T-cell (NKT cell), or a plurality thereof, of embodiment 7, wherein the vector is a retroviral vector, lentiviral vector, adenoviral vector, adeno-associated viral vector, or plasmid.

9. The engineered Natural Killer T-cell (NKT cell), or a plurality thereof, of embodiment 1, wherein said plurality is a therapeutically effective amount.

10. The engineered Natural Killer T-cell (NKT cell), or a plurality thereof, of embodiment 1, further comprising a CAR expression construct encoding an antigen recognition domain directed to at least one tumor-associated antigen.

11. The engineered Natural Killer T-cell (NKT cell), or a plurality thereof, of embodiment 1, further comprising engineered nucleic acid sequences encoding a protein sequence for a transcriptional activator in the Wnt signaling pathway.

12. The engineered Natural Killer T-cell (NKT cell), or a plurality thereof, of embodiment 9, wherein said engineered nucleic acid sequences encoding a protein sequence for a transcriptional activator in the Wnt signaling pathway and BATF polypeptide are provided as single linked expression construct. The engineered Natural Killer T-cell (NKT cell), or a plurality thereof, of embodiment 11, wherein said transcriptional activator of the Wnt-signaling pathway is selected from the group consisting of lymphoid enhancer binding factor 1 (LEF1, Gene ID 51176), beta- catemn ((CTNNB1, Gene ID 1499)), Smad3 (Gene ID 4088), HNF1 homeobox A (HNF1 A, Gene ID: 6927 (alt. TCF1), transcription factor 7 (TCF7, Gene ID:6932 (alt. TCF1) and TLE family member 1, transcriptional corepressor (TLE 1, Gene ID 7088). The engineered Natural Killer T-cell (NKT cell), or a plurality thereof, of embodiment 13, wherein said transcriptional activator of the Wnt-signaling pathway is LEF1. The engineered Natural Killer T-cell (NKT cell), or a plurality thereof, of embodiment 1, further comprising a recombinant sequences for suppressing the expression of an endogenous major histocompatibility complex (MHC) gene. The engineered Natural Killer T-cell (NKT cell), or a plurality thereof, of embodiment 15, wherein said recombinant sequences for suppressing the expression of an endogenous major histocompatibility complex (MHC) gene comprise a DNA sequence encoding a small hairpin RNA (shRNA) sequence targeting an MHC class I, a MHC class II gene, or both. The engineered Natural Killer T-cell (NKT cell), or a plurality thereof, of embodiment 13, wherein the shRNA sequence is embedded in an artificial microRNA (amiR) scaffold. The engineered Natural Killer T-cell (NKT cell), or a plurality thereof, of embodiment 15, wherein said recombinant sequences for suppressing the expression of an endogenous major histocompatibility complex (MHC) gene and BATF polypeptide are provided as single linked expression construct. The engineered Natural Killer T-cell (NKT cell), or a plurality thereof, of embodiment 18, wherein said single linked expression construct further comprises sequences encoding a protein sequence for a transcriptional activator in the Wnt signaling pathway. The engineered Natural Killer T-cell (NKT cell), or a plurality thereof, of embodiment 1, wherein said construct that encodes a basic leucine zipper ATF-like transcription factor (BATF) polypeptide comprises a single linked expression construct that further comprises a sequence encoding chimeric antigen receptor (CAR) having an antigen recognition domain directed to at least one tumor-associated antigen; a sequence encoding a polypeptide for a transcriptional activator in the Wnt signaling pathway a recombinant sequence for suppressing the expression of an endogenous major histocompatibility complex (MHC) gene; and one or more regulatory sequences comprising a promoter, an internal ribosomal entry site (IRES), a cis-acting hydrolase element (CHYSEL), or a combination thereof.

21. The engineered Natural Killer T-cell (NKT cell), or a plurality thereof, of embodiment 19, wherein said NKT cells are CD62L-positive Type I NKT cells.

22. The engineered Natural Killer T-cell (NKT cell), or a plurality thereof, of embodiment 15, wherein said cell has reduced or no detectable expression of: a. endogenous beta-2-microglobulin (B2M); b. endogenous MHC class Il-associated invariant chain (II); or c. both.

23. The engineered Natural Killer T-cell (NKT cell), or a plurality thereof, of embodiment 10, wherein said tumor-associated antigen is selected from the group consisting of GD2, chondroitin sulfate proteoglycan 4 (CSPG4), GPC3, melanoma-associated antigen (MAGE), expressed antigen of melanoma (PRAME), CD19, CD20, CD22, K-light chain, CD30, CD33, CD123, CD38, CD138, R0R1, ErbB2, ErbB3/4, EGFr vIII, carcinoembryonic antigen, EGP2, mesothelin, TAG72, PSMA, NKG2D ligands, B7-H6, IL- 13 receptor a2, MUCI, MUC16, CA9, GD3, CD171, Lewis Y, G250/CAIX, HLA-AI MAGE Al, HLA-A2 NY- ESO-1, PSCI, folate receptor-a, CD44v6, CD44v7/8, 8H9, NCAM, VEGF receptors, 5T4, Fetal AchR, NKG2D ligands, CEA, or combinations thereof.

24. The engineered Natural Killer T-cell (NKT cell), or a plurality thereof, of embodiment 7, wherein the CAR comprises co-stimulatory endodomains selected from the group consisting of CD28 (Gene ID: 940), TNF receptor superfamily member 9 (Gene ID 3604, e.g., 4- IBB or CD137), CD247 (Gene ID 919, CD3-Q, 2B4 (Gene ID:51744, CD244), Interleukin 21 (IL- 21, Gene ID 59067), hematopoietic cell signal transducer (HCST, Gene ID 10870 e.g., DAP10), and transmembrane immune signaling adaptor (TYROBP, Gene ID 7305; DAP12), TNF receptor superfamily member 4 (Gene Id 7293, TNFRSF4, 0X40), and CD40 (Gene ID 958).

25. The engineered Natural Killer T-cell (NKT cell), or a plurality thereof, of embodiment 7, wherein the CAR comprises a transmembrane domain selected from the group consisting of CD28 (Gene ID:940, 12487), CD3- (Gene ID:919;12503 CD247 ), CD4 (Gene ID:920, 12504), CD8 (Gene ID:924, 12525 ), CD16 (Gene ID:2214; 14131; Fcgr3 ), NKp44 (Gene ID: 9436, NCR2), NKp46 (Gene ID: 9437, 17086, NCR1 ), and NKG2d (Gene ID:22914;27007 KLRK1).

26. The engineered Natural Killer T-cell (NKT cell), or a plurality thereof, of embodiment 7, wherein the expression construct that encodes BATF3 and the expression construct that encodes a CAR are provided as an operably linked expression construct.

27. The engineered Natural Killer T-cell (NKT cell), or a plurality thereof, of embodiment 26, wherein said BATF polypeptide and said CAR are expressed as a self-processing polyprotein.

28. The engineered Natural Killer T-cell (NKT cell), or a plurality thereof, of embodiment 25, wherein expression of BATF3 and expression of the CAR are regulated by the same regulatory sequences.

29. The engineered Natural Killer T-cell (NKT cell), or a plurality thereof, of embodiment 1, wherein said cell further comprises an expression construct that encodes an inducible suicide gene.

30. The engineered Natural Killer T-cell (NKT cell), or a plurality thereof, of embodiment 29, wherein the inducible suicide gene is inducible caspase-9 suicide gene.

31. The engineered Natural Killer T-cell (NKT cell), or a plurality thereof, of embodiment 29, wherein the inducible suicide gene is thymidine kinase (sr39 IK).

32. The engineered Natural Killer T-cell (NKT cell), or a plurality thereof, of embodiment 1, further comprising a CD34 tag.

33. A composition comprising a plurality of engineered Natural Killer T-cells (NKT cells) comprising an expression construct that encodes basic leucine zipper ATF-like transcription factor (BATF), basic leucine zipper ATF-like transcription factor 2 (BATF2), or basic leucine zipper ATF-like transcription factor 3 (BATF3) and a chimeric antigen receptor.

34. A recombinant nucleic acid comprising nucleic acid sequences encoding a polypeptide for basic leucine zipper ATF-like transcription factor (BATF), basic leucine zipper ATF-like transcription factor 2 (BATF2), or basic leucine zipper ATF-like transcription factor 3 (BATF3), and encoding a polypeptide for a chimeric antigen receptor (CAR) having an antigen recognition domain directed to at least one tumor-associated antigen. 35. The recombinant nucleic acid of embodiment 34, further comprising one or more regulatory sequences between said polypeptide coding sequences that comprise an internal ribosomal entry sites (IRESs), an in-frame autonomous intra-ribosomal self-processing of peptide, or a promoter.

36. The recombinant nucleic acid of embodiment 34, further comprising a nucleic acid sequence encoding an inducible suicide gene; or a protein sequence for a transcriptional activator in the Wnt signaling pathway.

37. The recombinant nucleic acid of embodiment 34, wherein said nucleic acid sequence further comprises one or more synthetic nucleic acids that target and reduce the expression of a gene selected from the MHC class I gene, the MHC class II gene, the B2M gene, the li gene, or a combination thereof.

38. The recombinant nucleic acid of embodiment 37, wherein said synthetic nucleic acids that target and reduce the expression of a gene are small hairpin RNA (shRNA) sequences.

39. A method for treating a cancer in a subject in need thereof comprising administering a therapeutically effective amount of a plurality of engineered Natural Killer T-cells (NKT cells), said cells engineered to exogenously express basic leucine zipper ATF-like transcription factor (BATF), basic leucine zipper ATF-like transcription factor 2 (BATF2), or basic leucine zipper ATF-like transcription factor 3 (BATF3) and a chimeric antigen receptor.

40. The method for treating a cancer of embodiment 39, wherein said plurality of engineered NKT cells further comprise an exogenously expressed inducible suicide gene.

41. The method for treating a cancer of embodiment 40, said method further comprising inducing the elimination of the administered engineered NKT cells in said patient in need by activating said inducible suicide gene.

42. The method for treating a cancer of embodiment 40, wherein said inducible suicide gene is inducible caspase-9 suicide gene.

43. The method for treating a cancer of embodiment 42, further comprising administering AP20187, API 903, or a mixture thereof to said subject to activate said inducible caspase-9 suicide gene. 44. The method for treating a cancer of embodiment 40, wherein said inducible suicide gene is thymidine kinase (sr39 TK).

45. The method for treating a cancer of embodiment 44, further comprising administering ganciclovir to said subject to activate said thymidine kinase.

46. The method for treating a cancer of embodiment 39, wherein the natural killer T-cell cell further comprises an exogenously expressed CD34 tag.

47. The method for treating a cancer of embodiment 39, wherein the cancer is a tumor.

48. The method for treating a cancer of embodiment 39, wherein the cancer is selected from the group consisting of breast cancer, cervical cancer, ovary cancer, endometrial cancer, melanoma, bladder cancer, lung cancer, pancreatic cancer, colon cancer, prostate cancer, hematopoietic tumors of lymphoid lineage, leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, B-cell lymphoma, Burkitt's lymphoma, multiple myeloma, Hodgkin's lymphoma, Non-Hodgkin's lymphoma, myeloid leukemia, acute myelogenous leukemia (AML), chronic myelogenous leukemia, thyroid cancer, thyroid follicular cancer, myelodysplastic syndrome (MDS), tumors of mesenchymal origin, fibrosarcoma, rhabdomyosarcoma, melanoma, uveal melanoma, teratocarcinoma, neuroblastoma, glioma, glioblastoma, benign tumor of the skin, renal cancer, anaplastic large-cell lymphoma, esophageal squamous cell carcinoma, hepatocellular carcinoma, follicular dendritic cell carcinoma, intestinal cancer, muscle-invasive cancer, seminal vesicle tumor, epidermal carcinoma, spleen cancer, bladder cancer, head and neck cancer, stomach cancer, liver cancer, bone cancer, brain cancer, cancer of the retina, biliary cancer, small bowel cancer, salivary gland cancer, cancer of uterus, cancer of testicles, cancer of connective tissue, prostatic hypertrophy, myelodysplasia, Waldenstrom's macroglobinaemia, nasopharyngeal, neuroendocrine cancer myelodysplastic syndrome, mesothelioma, angiosarcoma, Kaposi's sarcoma, carcinoid, oesophagogastric, fallopian tube cancer, peritoneal cancer, papillary serous mullerian cancer, malignant ascites, gastrointestinal stromal tumor (GIST), and a hereditary cancer syndrome selected from Li-Fraumeni syndrome and Von Hippel-Lindau syndrome (VHL).

49. The method for treating a cancer of embodiment 39, wherein the cancer is a neuroblastoma.

50. The method for treating a cancer of embodiment 39, wherein said NKT cells exogenously express human BATF3. 51. The method for treating a cancer of embodiment 39, wherein said engineered NKT cells are autologous cells derived from said subject in need of cancer treatment.

52. The method for treating a cancer of embodiment 39, wherein the administration is systemic.

53. The method for treating a cancer of embodiment 39, wherein the administration is parenteral.

54. The method for treating a cancer of embodiment 39, wherein the natural killer T-cell is administered locally to the tumor.

55. The method for treating a cancer of embodiment 39, further comprising administering one or more additional cancer therapies to the subject.

56. A method of producing Natural Killer T-cell (NKT cell) cells for immunotherapy comprising isolating human NKT cells from human peripheral blood mononuclear cells (PBMCs); culturing said human NKT cells to prepare a culture having a majority of CD62L-positive

Type I NKT cells by culturing in the presence of at least:

(1) one or more cytokines selected from the group consisting of IL-21, IL-7, IL-15, IL- 12, TNF-alpha, and a combination thereof; and

(2) irradiated NKT-depleted PBMCs or irradiated artificial antigen-presenting cells (aAPCs) loaded with alpha-Galactosylceramide (aGalCer); and genetically modifying said CD62L-positive Type I NKT cells to exogenously express a basic leucine zipper ATF-like transcription factor (BATF) polypeptide.

57. The method of embodiment 56, further comprising modifying said CD62L-positive Type I NKT cells to exogenously express one or more sequences selected from the group consisting of: a sequence encoding chimeric antigen receptor (CAR) having an antigen recognition domain directed to at least one tumor-associated antigen; a sequence encoding a polypeptide for a transcriptional activator in the Wnt signaling pathway a recombinant sequence for suppressing the expression of an endogenous major histocompatibility complex (MHC) gene; one or more regulatory sequences comprising a promoter, an internal ribosomal entry site (IRES), a cis-acting hydrolase element (CHYSEL); and a combination thereof. 58. The method of embodiment 56, wherein said genetically modifying comprises transforming said CD62L-positive Type I NKT cells with a nucleic acid sequence encoding BATF polypeptide and a chimeric antigen receptor (CAR) having an antigen recognition domain directed to at least one tumor-associated antigen as a single linked expression construct.

59. The method of embodiment 57, further comprising expanding said genetically modified CD62L-positive Type I NKT cells to prepare a therapeutic amount of cells.

60. A method of treating an individual for a medical condition using immunotherapy, comprising the steps of: a. isolating human NKT cells from human peripheral blood mononuclear cells (PBMCs); b. culturing said human NKT cells in the presence of at least: i. one or more cytokines selected from the group consisting of IL-21, IL-7, IL- 15, IL- 12, TNF-alpha, and a combination thereof; and ii. irradiated NKT-depleted PBMCs or irradiated artificial antigen-presenting cells (aAPCs) loaded with alpha-Galactosylceramide (aGalCer); to prepare a culture having a majority of CD62L-positive Type I NKT cells; c. genetically modifying said CD62L-positive Type I NKT cells to exogenously express a basic leucine zipper ATF-like transcription factor (BATF) polypeptide and a chimeric antigen receptor (CAR) having an antigen recognition domain directed to at least one tumor- associated antigen; and d. providing a therapeutically effective amount of the co-stimulated CD62L+ NKT cells to the individual.

61. The method of embodiment 60, wherein said genetically modifying said CD62L-positive Type I NKT cells to exogenously express one or more sequences selected from the group consisting of: a sequence encoding a polypeptide for a transcriptional activator in the Wnt signaling pathway a recombinant sequence for suppressing the expression of an endogenous major histocompatibility complex (MHC) gene; one or more regulatory sequences comprising a promoter, an internal ribosomal entry site (IRES), a cis-acting hydrolase element (CHYSEL); and a combination thereof. 62. The method of embodiment 61, wherein said genetically modifying said CD62L-positive Type I NKT cells comprises introducing said one or more sequences into said CD62L- positive Type I NKT cells as a single construct.

63. The use of engineered Natural Killer T-cells (NKT cells) for the preparation of a medicament for immunotherapy wherein said NKT cells are genetically modified to exogenously express a basic leucine zipper ATF-like transcription factor (BATF) polypeptide.

EXAMPLES

EXAMPLE 1: NKT-cell isolation, expansion and in vivo injection

[00113] Isolate PBMCs via apheresis. Buffy Coats (Gulf Coast Regional Blood Center) are obtained. Samples are diluted with equal volume of PBS. 15ml Ficoll-Paque is placed in 50ml centrifugation tube, and is carefully over lay ed with 35ml of the peripheral blood/ PBS onto Ficoll-Paque without disturbance of the interface. The tubes are centrifuged at 800xg for 30 min at RT with no brake. The upper PBS layer is carefully aspirated, leaving about 10 mis of PBS. The PBMCs are carefully harvested at the PBS/Ficoll-Paque using a serological pipette. The harvested PBMCs are washed 3 times with 50ml PBS by centrifugation at 800xg for 5 mins at RT. PBMCs are resuspended in 50 ml MACS buffer and count using trypan blue. Proceed to iNKT isolation.

[00114] Isolate NKT cells with Miltenyi microbeads. Cell number is first determined from the previous step. Cell suspension is centrifuged at 300 g for 10 minutes. The supernatant is aspirated completely. Cell pellet is resuspended in 400 pL of MACS buffer per 10 total cells. 100 pL of Anti-iNKT MicroBeads (Miltenyi Biotec) is added per 10 total cells. The cells and the MicroBeads are mixed well and incubated for 15 minutes in the refrigerator (2-8°C). The cells are washed by adding 1-2 mL of MACS buffer per 10 cells and centrifuged at 300 g for 10 minutes. The supernatant is aspirated completely. Up to 10 cells are resuspended in 500 pL of MACS buffer. The column is placed in the magnetic field of a suitable MACS Separator. The column is prepared by rinsing with the appropriate amount of MACS buffer: LS: 3 mL. Cell suspension is applied onto the column. Flow-through containing unlabeled cells is collected. The column is washed with the appropriate amount of MACS buffer. Unlabeled cells that pass through: LS: 3x3 mL are collected. The column is then removed from the separator and placed on a suitable collection tube. The appropriate amount of MACS buffer is pipetted onto the column. The magnetically labeled cells are immediately flushed out by firmly pushing the plunger into the column. LS: 5 mL.

[00115] NKT primary stimulation including transduction. NKT cells are centrifuged at 400g for 5 mins at RT and resuspended in 1 ml complete RPMI media and plated in 1 well of 24- well plate. Cells are counted and small aliquot is taken for purity staining at this step. PBMCs are counted. An appropriate amount of PBMCs is irradiated with 2.5 Gy by setting irradiator to Level 5, and irradiated for 10 minutes, 40 seconds. After irradiation, PBMCs are washed and resuspended at 5x10⁶ cells/mL. 1ml of PBMCs (5 million cells) are added to NKT cells in 24- well plate. lOOng/ml (2pL) aGalCer (stock: lOOpg/mL), 200IU/mL (2pL) IL-2 (Stock: 200 lU/pL), and lOng/mL IL-21 are added. Cells are incubated at 37°C, 5% CO2 for 10 days, and are fed with 200IU/ml IL-2 and 10 ng/mL IL-21 every other day. Media is changed and/or wells are split as necessary. On day 8 of primary expansion, NKT cell transduction is performed as follows. After the transduction, cells are transferred to a 6-well G-Rex plate once NKT number exceeds 10xl0⁶ cells and continue to expand for 10-12 days total. At the end of primary expansion, NKT cells can either be frozen or proceed to secondary stimulation.

[00116] NKT cell transduction. Retronectin-coated plate is prepared: i). Determine the number of wells needed for transduction; ii). Make a suspension of Retronectin at 7 ug/ml in PBS for each well and add 1 ml of Retronectin suspension to each well of a non-tissue culture coated plate; iii) Seal the edges of the plate with Parafilm and incubate overnight at 4°C. Alternatively, for same-day use, incubate Retronectin-coated plate for 4 hours at 37°C. The Retronectin-coated plate is then removed from 4°C and warmed in hood for about 10 min. At the same time, retroviral supernatant(s) are thawed. Retronectin suspension is aspirated and discarded. 1ml of retroviral supernatant is added to each well. The plate is centrifuged at 4600G for Ihr, 30°C. NKT cells are collected and prepared at a concentration of 0.25xl0⁶ cells/ml. IL-2 200IU/ml and IL-21 lOng/ml are added to NKT suspension. Retroviral supernatant is aspirated. NKT suspension is plated into each well for a final concentration of 0.5xl0⁶ NKTs per well. The plate is spun at 400g for 10 minutes. The plate is then incubated at 37°C, 5% CO2 for 48 hours. On day 9 of primary expansion, transfer NKT cells into a 24-well tissue culture plate with fresh media. Wells are generally pooled together in order to maintain approximately 1x10⁶ cells/ml concentration. [00117] NKT secondary expansion. Following end of primary stimulation/transduction, or working with primary- expanded frozen NKT cells, NKT cells are resuspended at 2x10⁶ cells/ml. If using PBMCs for secondary stimulation, frozen aliquot is thawed and irradiated at Level 5 for 10 minutes and 40 seconds. If using artificial APC (B-8-2), cells are resuspended at 1x10⁶ cells/ml and irradiated at Level 5 for 27 minutes. Irradiated cells are washed and co-cultured with NKT cells at a 1:5 NKTPBMC or a 2: 1 NKT: aAPC ratio in a 24 well plate. lOO ng/ml (2pL) aGalCer (stock: lOOpg/mL), 200 lU/mL (2pL) IL-2 (Stock: 200 lU/pL), and 10 ng/mL IL-21 are added. Cells are incubated at 37°C, 5% CO2 for 10 days, and are fed with 200 lU/ml IL-2 and 10 ng/mL IL-21 every other day. Media is changed and/or wells are split as necessary. Cells are transferred to G-Rex 10 once NKT number exceeds 10xl0⁶ cells and continue to expand for 10- 12 days total.

[00118] Cell lines. Cells are kept in culture for less than 6 consecutive months, after which aliquots from the original expanded vial are used. All tumor cell lines are routinely tested to exclude contamination with Mycoplasma and assessed for the expression of transgenes and tumor markers by flow cytometry to confirm identity.

[00119] Day 0: Establish lymphoma xenografts using firefly luciferase/GFP+ CD 19+ Daudi cells. NOD/SCID/EL2ynull (NSG) mice are maintained at the Small Animal Core Facility of Texas Children’s Hospital and are treated according to the protocols approved by Baylor College of Medicine’s Institutional Biosafety Committee and Institutional Animal Care and Use Committee (IACUC) — refer to animal research protocol number AN-5194. On Day 0, NSG mice are injected via tail vein with 2x10⁵ firefly luciferase/GFP+ Daudi cells to establish disease.

Cells are washed with PBS. 300ul PBS is added and the samples are run on LSRII or iQue. First, gate on live lymphocytes in the FSC vs SSC plot. Gate directly on CAR/CD19+ positive cells, using non-transduced NKT cells to set up the CAR+ gate.

[00120] Day 3: Inject transduced NKTs. Three days after injecting Daudi xenografts, NSG mice carrying Daudi tumors are injected via tail vein with 5x10⁶ transduced NKT cells (15.GPC3-CAR, GPC3-CAR, CD19 CAR, etc.) as indicated followed by intraperitoneal injection of IL-2 (2000 U/mouse) every other day for two weeks. Tumor size/distribution is monitored every week using bioluminescence imaging as follows. Just prior to imaging, each mouse is injected with 100 pL luciferin at 30 mg/mL via intraperitoneal injection. After 5 min, the mice are imaged using an IVIS® Lumina II Quantitative Fluorescent and Bioluminescent imaging system under a bioluminescent channel at Texas Children’s Hospital, Small Animal Imaging Facility. Bioluminescence counts are then analyzed using Living Image® software. [00121] In vitro cytotoxicity assay. Cultures of luciferase positive Daudi or Raji cells are established in RPMI-1640/GlutaMAX/10% (v/v) FBS. Luciferase expression is confirmed prior to beginning experiment and the number of target cells is determined to use in cytotoxicity assay (A standard curve is set up with 200,000 cells at the highest concentration, then 1:2 serial dilutions are performed and evaluated for luciferase expression. Ensure that the number of target cells used in assay falls within linear range of standard curve.). A suspension of Daudi cells is prepared at 0.2x10⁶ cells/mL (or number of cells calculated based on standard curve) in RPMI/20% (v/v) FBS medium. 100 pL (20,000 cells) is plated in appropriate wells of black clear bottom 96-well plates. At least three wells are set up with target cells only and three wells are set up for media only controls. The wells are placed in 37°C in a 5% CCh-in-air, fully humidified atmosphere while effector cells are processed. Effector cells are harvested and counted. The cells are diluted to appropriate concentration for 10:1, 5: 1, 2.5: 1, and 1.25:1 effectortarget ratios, ensuring that transduction rate is normalized across all CAR-transduced NKT cells. Effector cells are added to target for each concentration in triplicate. Cells are cultured for 6 hours at 37°C in a 5% CCh-in-air, fully humidified atmosphere. Tecan Spark 10M plate reader is set up to warm to 37°C, bioluminescence signal is read, and an acquisition template is set up. 100 pL of medium is carefully removed from all wells of each plate while avoiding contact with base of wells. Immediately prior to use, required amount of 1.5 mg/ml working stock of luciferin is prepared. 100 ul of luciferin is added to all wells of each plate. The plates are incubated for 5 minutes at 37°C in a 5% CCh-in-air, fully humidified atmosphere. Plates are removed from incubator, the lid are then removed, and bioluminescence is read using Tecan Spark 10M plate reader. For data analysis: acquire data and calculate percentage killing/lysis as the difference of total luciferase and the read bioluminescence signal divided by the different between total luciferase and spontaneous luceriferase and the result converted to a percentage.

[00122] Retroviral constructs and retrovirus production. CAR. CD 19, CAR.GD2, and CAR.GPC3 constructs are made as previously described (Heczey et al., 2014; Pule etal., 2005) and contained a scFv from the CD19-specific antibody FMC-63 or the GD2-specific antibody 14G2a connected via a short spacer derived from the IgGl hinge region to the transmembrane domain derived from CD8a, followed by signaling endodomain sequences of 4- IBB fused with z chain.

[00123] DESCRIPTION OF NEW CONSTRUCTS

[00124] Flow cytometry. Immunophenotyping is performed using the following mAbs to: HLA-C EMR8-5, CD Id CDld42, CD86 2331, 4-1BBL C65-485, OX40L ik-1, CD3 OKT, Va24-Jal8 6B11, CD4 SK3, CD62L DREG-56, CD134 ACT35, CD137 4B4-1, PD-1 EH12.1, GATA3 L50-823 (BD Biosciences), LAG- 3 Polyclonal, TEVI-3 344823 (R&D System), and rabbit anti-LEFl EP2030Y mAb (ABC AM). BD or R&D-suggested fluorochrome and isotypematching Abs is used as negative controls. The expression of CAR.CD19 on NKTs is determined using anti-Id (clone 136.20.1) CD19-CAR specific mAb (Torikai H, etal. Toward eliminating HLA class I expression to generate universal cells from allogeneic donors. fi/oo<i.2013;122(8):1341-1349) and goat anti-mouse IgG (BD Biosciences).

[00125] NKT-cell phenotypic analysis. NKT-cell phenotype is assessed using monoclonal antibodies (mAbs) for CD3 (UCHT1), Va24-Jal 8 (6B11), CD4 (RPA-T4), granzyme B (GB11), CD62L (DREG-56; BD Biosciences, San Jose, CA), vpi l (C21; Beckman Coulter, Brea, CA), and IL-21R (17A12; BioLegend, San Diego, CA and BD Biosciences). CD19-CAR expression by transduced NKTs is detected using anti-Id mAb (clone 136.20.1) (25), a gift from Dr. B. Jena (MD Anderson Cancer Center, Houston, TX). Intracellular staining is performed using a fixation/permeabilization solution kit (BD Biosciences) with mAbs for Bcl2 (N46-467; BD Biosciences) and BIM (Y36; Abeam, Cambridge, MA) followed by staining with a secondary goat anti-rabbit IgG-AF488 mAb (Abeam). Phosflow staining is performed using Cytofix buffer (BD Biosciences) and Perm buffer III (BD Biosciences) with mAb for Stat3 (pY705; Clone 4; BD Biosciences). Detection of Stat3 phosphorylation is performed after 15 minutes of treatment with IL-21. Fluorochrome- and isotype-matching antibodies suggested by BD Biosciences or R&D Systems is used as negative controls.

[00126] Analysis is performed on an LSR-II 5-laser flow cytometer (BD Biosciences) using BD FACSDiva software version 6.0 and FlowJo 10.1 (Tree Star, Ashland, OR).

[00127] Gene expression analysis. Total RNA is collected using the Direct-zol™ RNA MiniPrep Kit (Zymo Research, Irvine, CA). Gene expression analysis is performed using the Immunology Panel version 2 (NanoString, Seattle, WA) with the nCounter Analysis System by the BCM Genomic and RNA Profiling Core. Data is analyzed using nSolver 3.0 software (NanoString). Differences in gene expression levels between CD62L+ and CD62L- subsets in the two culture conditions are evaluated using the paired moderated t-statistic of the Linear Models for Microarray Data (Limma) analysis package (26).

[00128] In vivo experiments. NSG mice are obtained from the Jackson Laboratory and maintained at the BCM animal care facility. Mice are injected intravenously (IV) with 2 x 10⁵ luciferase-transduced Daudi lymphoma cells to initiate tumor growth. On day 3, mice are injected IV with 4 x 10 x 10⁶ of the indicated engineered NKTs followed by intraperitoneal (IP) injection of IL-2 (1,000 U/ mouse) only or a combination of IL-2 (1,000 U/mouse) and IL-21 (50 ng/mouse) every other day for two weeks. Tumor growth is assessed once per week by bioluminescent imaging (Small Animal Imaging core facility, Texas Children’s Hospital).

[00129] Statistics. The Shapiro-Wilk test is used to assess normality of continuous variables. Normality is rejected when the P value is less than 0.05. For non-normally distributed data, the Mann- Whitney U test is used to evaluate differences in continuous variables between two groups. To evaluate differences in continuous variables, a two-sided paired Student’s t-test is used to compare two groups, one-way ANOVA with post-test Bonferroni correction is used to compare more than two groups, and two-way ANOVA with Sidak’s post-hoc test is used to compare in a two-by-two setting. Survival is analyzed using the Kaplan-Meier method with the log-rank (Mantel-Cox) test to compare two groups. Statistics are computed using GraphPad Prism 7 (GraphPad Software, San Diego, CA). Differences are considered significant when the P value was less than 0.05.

[00130] Immunophenotyping. T cells are stained with antibodies (Ab) against CD3 (APC- H7, clone SK7), CD45Ra (PE, clone HI100), CCR7 (FITC, clone 150503), CTLA4 (BV421, clone BNI3), PD-1 (PE- Cy7, clone EH12.1), LAG3 (PE, clone T47-530), UM3 (BV711, clone 7D3) an CD45 (APC, clone 2D1) from BD Biosciences. Anti-CD45 (PerCP, clone REA747) and anti CD69 (APC, clone REA824) from REAffinity by Miltenyi Biotec. Tumor cells are stained with Abs against CD276 (BV421, clone 7-517) from BD Biosciences and with the 763.74 mAb (anti-CSPG4) followed by the staining with a secondary Rat anti -Mouse IgGi (PE, clone X56) from BD Biosciences. The expression of the 763.74(A) and (B) CAR is assessed using an anti-idyotipic antibody, the expression of CTR CAR (anti-CD19 CAR) is assessed using an anti-idiotypic antibody (obtained from Dr Ferrone), followed by the staining with a secondary Rat anti-Mouse IgGi (PE, clone X56) from BD Biosciences. The expression of the h763.74 CAR followed by the staining with Streptavidin Protein RPE conjugate from Invitrogen. Data acquisition is performed on BD LSRFortessa or Canto II flow cytometer using the BD FACS- Diva software or on a MACSQuant (Miltenyi Biotec). Data analyses are performed with the FlowJo software (Version 9 or 10) or FlowLogic software (Version 7.2, Miltenyi Biotec).

[00131] Magnetic Resonance Imaging (MRI). MRI is performed using a horizontal-bore preclinical scanner (BioSpec 70/20 USR, Bruker, Ettlingen, Germany). The system has a magnetic field strength of 7 T (1H frequency 300 MHz) and a 20 cm bore diameter. The scanner is equipped with an actively shielded gradient system with integrated shims set up to 2nd order. The maximum gradient amplitude is 440 mT/m. All acquisitions are carried out using a cross coil configuration: a 72 mm linear birdcage coil is used for radiofrequency excitation and a mouse brain surface coil received signal. Mice are anaesthetized with 1.5 - 2% isoflurane (60:40 N2O:O2 (vol: vol), flow rate 0.8 L/min). To detect the depth of anesthesia and the animal health condition during the study, the respiratory rate is monitored by a pneumatic sensor. Mice are positioned on an animal bed equipped with a nose cone for gas anesthesia and a three pointfixation system (tooth-bar and ear-plugs). Mice injected with GBM-NS and treated with CAR-T cells undergo high resolution MRI investigation at different time points with the following protocol: a T2- weighted Rapid Acquisition with Reduced Echoes (RARE) sequence (TR = 3360 ms, IE = 35 ms, in plane resolution = 100 x 100 um², slice thickness = 400 um, 4 averages, total acquisition time of 5 min 36 sec) and two T1 -weighted RARE sequences (TR = 510 ms, TE = 8 ms, in plane resolution = 78 x 78 um², slice thickness = 400 um, 6 averages, total acquisition time of 9 min 47 sec) acquired before and after intraperitoneal administration of Gadoliunium- based contrast medium. All sequences are acquired along the same coronal geometry (400 um thick continuous slices), with slice package posterior to olfactory bulb and anterior to cerebellum. Contrast agent induced T1 signal enhancement is interpreted as being due to a Blood Brain Barrier lesion.

EXAMPLE 2: IL-15 enhances the in vivo antitumor activity of GPC3-CAR NKTs

[00132] Genetically engineered effector lymphocytes (GEELs) can efficiently eliminate cancer cells in humans leading to durable complete remission in patients. Clinical responses are correlated with the expansion and persistence of GEELs; thus, improving these parameters is critical to increasing the efficacy of GEEL-based therapies. Interleukin- 15 (IL15) enhances GEEL antitumor activity. Expression of IL15 in natural killer T cells (NKTs), a subset of invariant lymphocytes, redirected with a chimeric antigen receptor (CAR) specific for tumor antigen glypican-3 (GPC3) show enhanced in vivo persistence and antitumor activity against neuroblastoma. (Xu et al. 2019). NKTs expressing the 15.GPC3-CAR construct control tumors in a murine model of Hepatocellular Carcinoma (HCC) significantly better than NKTs expressing the GPC3-CAR without IL15 (Fig. 7).

EXAMPLE 3: Basic Leucine Zipper ATE-like Transcription Factor 3 (BATF3) is overexpressed in 15.GPC3-CAR NKTs

[00133] To identify drivers of 15.GPC3-CARNKT antitumor activity, the gene expression profile of these cells is evaluated post-tumor cell encounter and compared with the profile of NKTs expressing the GPC3-CAR without IL15. This analysis yields 21 differentially expressed genes including the transcription factor BATF3 which is upregulated in 15.GPC3-CARNKTs. This gene belongs to the AP-1 family of transcription factors, which form heterodimers with other AP-1 members and interact with several other transcription factors including nuclear factor of activated T cells (NF AT) and play key roles in lymphocyte survival and function. (Fig. 8).

EXAMPLE 4: BATF3 co-expression does not increase the in vitro cytolytic activity of GPC3-CAR NKTs

[00134] Following the observations that 15.GPC3-CAR NKTs demonstrate enhanced antitumor activity and that BATF3 is upregulated in these cells, transgenic expression of is examined for its effect on the antitumor activity of GPC3-CAR NKTs. To determine how BATF3 impacts the ability of NKTs to proliferate following multiple rounds of tumor cell exposure, NKTs are repeatedly plated with fresh tumor cells every 3-4 days in a serial tumor challenge assay. NKTs expressing IL15 versus BATF3 are compared. Non-transduced or GFP- transduced NKTs are controls. To avoid interference from CAR mediated non-physiologic signaling, a-galactosylceramide-loaded CDld+ Jurkat cells are used as tumor cells to activate NKTs via the invariant TCR. As expected, co-expression of IL15 significantly improves the ability of NKTs to proliferate when exposed to tumor cells versus wild-type or GFP control NKTs. Surprisingly, overexpression of BATF3 boosted NKT proliferation beyond the levels in the IL 15 -expressing group (Fig. 9). [00135] BATF3 transgenic expression is examined for impact on NKT in vitro cytotoxic activity. NKTs expressing the GPC3-CAR alone or co-expressing IL15 (15.GPC3-CAR) or BATF3 (BATF3.GPC3-CAR) are evaluated in an xCelligence assay (quantification platform to quantify tumor cell survival by impedance-based measurements in a 96 well system) with Huh7 and HepG2 HCC cell lines. Control NKTs (non-transduced and IL15- or BATF3 -transduced) do not show cytotoxic activity while all three GPC3-CARNKT groups demonstrate a robust ability to kill both HCC lines. Co-expression of IL15 or BATF3 does not significantly impact the cytotoxicity of GPC3-CAR NKTs.

EXAMPLE 5: BATF3 co-expression enhances the in vivo antitumor activity of GPC3-CAR NKTs more than IL15

[00136] The impact of BATF3 co-expression on the in vivo antitumor activity of GPC3-CAR NKTs is examined in mice with established Huh7 cell line derived HCC xenografts. BATF3.GPC3-CAR NKTs is injected and the tumor burden (measured by bioluminescence of Ffluc+ Huh7 tumor cells). Animal survival is compared to other treatment groups including 15.GPC3-CAR. NKTs and controls (non-transduced and GFP-, BATF3-, or IL15-transduced). Compared to controls, 15.GPC3-CARNKTs reduce tumor burden and significantly improve the survival of mice. Importantly, BATF3.GPC3-CAR NKTs mediated better tumor control than 15.GPC3-CAR NKTs as assessed by the above parameters and completely eliminates established tumors in some animals (Fig. 10).

EXAMPLE 6: BATF3 overexpression does not induce uncontrolled growth of NKTs

[00137] Mutation and overexpression of AP-1 family members has been shown to occur in malignant cells. Overexpression of BATF3 does not induce leukemic transformation in NKTs resulting in uncontrolled proliferation. Wild-type non-transduced NKTs and cells transduced with BATF3, GPC3-CAR, or BATF3.GPC3-CAR are maintained in optimal tissue culture conditions without adding homeostatic cytokines, TCR-mediated stimulation, or other stimuli. NKTs expressing BATF3 with and without GPC3-CAR do not demonstrate autonomous growth in vitro, showing similar duration of survival to control groups (Fig. 11).

[00138] Fig. 12 presents results of an exemplary experiment showing that autonomous growth of NKTs does not result from expression of BATF3 either alone or in combination with a CAR in the engineered NKT cells of the present specification. NKT cells are transduced with indicated constructs and quantified at indicated timepoints while maintained under standard culture conditions without the use of cytokines or other stimulants.

Claims

CLAIMS:

2. The engineered Natural Killer T-cell (NKT cell), or a plurality thereof, of claim 1, further comprising one or more sequences selected from the group consisting of: a sequence encoding chimeric antigen receptor (CAR) having an antigen recognition domain directed to at least one tumor-associated antigen; a sequence encoding a polypeptide for a transcriptional activator in the Wnt signaling pathway a recombinant sequence for suppressing the expression of an endogenous major histocompatibility complex (MHC) gene; one or more regulatory sequences comprising a promoter, an internal ribosomal entry site (IRES), a cis-acting hydrolase element (CHYSEL); and a combination thereof.

3. The engineered Natural Killer T-cell (NKT cell), or a plurality thereof, of claim 1, wherein said BATF polypeptide is basic leucine zipper ATF-like transcription factor (BATF), basic leucine zipper ATF-like transcription factor 2 (BATF2), or basic leucine zipper ATF-like transcription factor 3 (BATF3).

4. The engineered Natural Killer T-cell (NKT cell), or a plurality thereof, of claim 1, wherein said engineered T-cell is a Type I Natural Killer T-cell (NKT cell).

5. The engineered Natural Killer T-cell (NKT cell), or a plurality thereof, of claim 1, wherein said engineered NKT cell is a CD62L-positive Type I NKT cell. The engineered Natural Killer T-cell (NKT cell), or a plurality thereof, of claim 1, wherein said NKT cell is a human cell. The engineered Natural Killer T-cell (NKT cell), or a plurality thereof, of claim 1, wherein the expression construct comprises a vector. The engineered Natural Killer T-cell (NKT cell), or a plurality thereof, of claim 7, wherein the vector is a retroviral vector, lentiviral vector, adenoviral vector, adeno-associated viral vector, or plasmid. The engineered Natural Killer T-cell (NKT cell), or a plurality thereof, of claim 1, wherein said plurality is a therapeutically effective amount. The engineered Natural Killer T-cell (NKT cell), or a plurality thereof, of claim 1, further comprising a CAR expression construct encoding an antigen recognition domain directed to at least one tumor-associated antigen. The engineered Natural Killer T-cell (NKT cell), or a plurality thereof, of claim 1, further comprising engineered nucleic acid sequences encoding a protein sequence for a transcriptional activator in the Wnt signaling pathway. The engineered Natural Killer T-cell (NKT cell), or a plurality thereof, of claim 9, wherein said engineered nucleic acid sequences encoding a protein sequence for a transcriptional activator in the Wnt signaling pathway and BATF polypeptide are provided as single linked expression construct. The engineered Natural Killer T-cell (NKT cell), or a plurality thereof, of claim 11, wherein said transcriptional activator of the Wnt-signaling pathway is selected from the group consisting of lymphoid enhancer binding factor 1 (LEF1, Gene ID 51176), beta-catenin

((CTNNB1, Gene ID 1499)), Smad3 (Gene ID 4088), HNF1 homeobox A (HNF1A, Gene ID: 6927 (alt. TCF1), transcription factor 7 (TCF7, Gene ID:6932 (alt. TCF1) and TLE family member 1, transcriptional corepressor (TLE 1, Gene ID 7088). The engineered Natural Killer T-cell (NKT cell), or a plurality thereof, of claim 13, wherein said transcriptional activator of the Wnt-signaling pathway is LEF1. The engineered Natural Killer T-cell (NKT cell), or a plurality thereof, of claim 1, further comprising a recombinant sequences for suppressing the expression of an endogenous major histocompatibility complex (MHC) gene. The engineered Natural Killer T-cell (NKT cell), or a plurality thereof, of claim 15, wherein said recombinant sequences for suppressing the expression of an endogenous major histocompatibility complex (MHC) gene comprise a DNA sequence encoding a small hairpin RNA (shRNA) sequence targeting an MHC class I, a MHC class II gene, or both. The engineered Natural Killer T-cell (NKT cell), or a plurality thereof, of claim 13, wherein the shRNA sequence is embedded in an artificial microRNA (amiR) scaffold. The engineered Natural Killer T-cell (NKT cell), or a plurality thereof, of claim 15, wherein said recombinant sequences for suppressing the expression of an endogenous major histocompatibility complex (MHC) gene and BATF polypeptide are provided as single linked expression construct. The engineered Natural Killer T-cell (NKT cell), or a plurality thereof, of claim 18, wherein said single linked expression construct further comprises sequences encoding a protein sequence for a transcriptional activator in the Wnt signaling pathway. The engineered Natural Killer T-cell (NKT cell), or a plurality thereof, of claim 1, wherein said construct that encodes a basic leucine zipper ATF-like transcription factor (BATF) polypeptide comprises a single linked expression construct that further comprises a sequence encoding chimeric antigen receptor (CAR) having an antigen recognition domain directed to at least one tumor-associated antigen; a sequence encoding a polypeptide for a transcriptional activator in the Wnt signaling pathway a recombinant sequence for suppressing the expression of an endogenous major histocompatibility complex (MHC) gene; and one or more regulatory sequences comprising a promoter, an internal ribosomal entry site (IRES), a cis-acting hydrolase element (CHYSEL), or a combination thereof. The engineered Natural Killer T-cell (NKT cell), or a plurality thereof, of claim 19, wherein said NKT cells are CD62L-positive Type I NKT cells. The engineered Natural Killer T-cell (NKT cell), or a plurality thereof, of claim 15, wherein said cell has reduced or no detectable expression of: a. endogenous beta-2-microglobulin (B2M); b. endogenous MHC class Il-associated invariant chain (II); or c. both. The engineered Natural Killer T-cell (NKT cell), or a plurality thereof, of claim 10, wherein said tumor-associated antigen is selected from the group consisting of GD2, chondroitin sulfate proteoglycan 4 (CSPG4), GPC3, melanoma-associated antigen (MAGE), expressed antigen of melanoma (PRAME), CD19, CD20, CD22, K-light chain, CD30, CD33, CD123, CD38, CD 138, R0R1, ErbB2, ErbB3/4, EGFr vIII, carcinoembryonic antigen, EGP2, mesothelin, TAG72, PSMA, NKG2D ligands, B7-H6, IL-13 receptor a2, MUCI, MUC16, CA9, GD3, CD171, Lewis Y, G250/CAIX, HLA-AI MAGE Al, HLA-A2 NY-ESO-1, PSCI, folate receptor-a, CD44v6, CD44v7/8, 8H9, NCAM, VEGF receptors, 5T4, Fetal AchR, NKG2D ligands, CEA, or combinations thereof. The engineered Natural Killer T-cell (NKT cell), or a plurality thereof, of claim 7, wherein the CAR comprises co-stimulatory endodomains selected from the group consisting of CD28 (Gene ID: 940), TNF receptor superfamily member 9 (Gene ID 3604, e.g., 4- IBB or CD 137), CD247 (Gene ID 919, CD3-Q, 2B4 (Gene ID: 51744, CD244), Interleukin 21 (IL-21, Gene ID 59067), hematopoietic cell signal transducer (HCST, Gene ID 10870 e.g., DAP10), and transmembrane immune signaling adaptor (TYROBP, Gene ID 7305; DAP 12), TNF receptor superfamily member 4 (Gene Id 7293, TNFRSF4, 0X40), and CD40 (Gene ID 958). The engineered Natural Killer T-cell (NKT cell), or a plurality thereof, of claim 7, wherein the CAR comprises a transmembrane domain selected from the group consisting of CD28 (Gene ID:940, 12487), CD3- (Gene ID:919;12503 CD247 ), CD4 (Gene ID:920, 12504), CD8 (Gene ID:924, 12525 ), CD16 (Gene ID:2214; 14131; Fcgr3 ), NKp44 (Gene ID:9436, NCR2), NKp46 (Gene ID:9437, 17086, NCR1 ), and NKG2d (Gene ID:22914;27007 KLRK1). The engineered Natural Killer T-cell (NKT cell), or a plurality thereof, of claim 7, wherein the expression construct that encodes BATF3 and the expression construct that encodes a CAR are provided as an operably linked expression construct. The engineered Natural Killer T-cell (NKT cell), or a plurality thereof, of claim 26, wherein said BATF polypeptide and said CAR are expressed as a self-processing polyprotein. The engineered Natural Killer T-cell (NKT cell), or a plurality thereof, of claim 25, wherein expression of BATF3 and expression of the CAR are regulated by the same regulatory sequences. The engineered Natural Killer T-cell (NKT cell), or a plurality thereof, of claim 1, wherein said cell further comprises an expression construct that encodes an inducible suicide gene. The engineered Natural Killer T-cell (NKT cell), or a plurality thereof, of claim 29, wherein the inducible suicide gene is inducible caspase-9 suicide gene. The engineered Natural Killer T-cell (NKT cell), or a plurality thereof, of claim 29, wherein the inducible suicide gene is thymidine kinase (sr39 TK). The engineered Natural Killer T-cell (NKT cell), or a plurality thereof, of claim 1, further comprising a CD34 tag. A composition comprising a plurality of engineered Natural Killer T-cells (NKT cells) comprising an expression construct that encodes basic leucine zipper ATF-like transcription factor (BATF), basic leucine zipper ATF-like transcription factor 2 (BATF2), or basic leucine zipper ATF-like transcription factor 3 (BATF3) and a chimeric antigen receptor. A recombinant nucleic acid comprising nucleic acid sequences encoding a polypeptide for basic leucine zipper ATF-like transcription factor (BATF), basic leucine zipper ATF-like transcription factor 2 (BATF2), or basic leucine zipper ATF-like transcription factor 3 (BATF3), and encoding a polypeptide for a chimeric antigen receptor (CAR) having an antigen recognition domain directed to at least one tumor-associated antigen. The recombinant nucleic acid of claim 34, further comprising one or more regulatory sequences between said polypeptide coding sequences that comprise an internal ribosomal entry sites (IRESs), an in-frame autonomous intra-ribosomal self-processing of peptide, or a promoter. The recombinant nucleic acid of claim 34, further comprising a nucleic acid sequence encoding an inducible suicide gene; or a protein sequence for a transcriptional activator in the Wnt signaling pathway. The recombinant nucleic acid of claim 34, wherein said nucleic acid sequence further comprises one or more synthetic nucleic acids that target and reduce the expression of a gene selected from the MHC class I gene, the MHC class II gene, the B2M gene, the li gene, or a combination thereof. The recombinant nucleic acid of claim 37, wherein said synthetic nucleic acids that target and reduce the expression of a gene are small hairpin RNA (shRNA) sequences. A method for treating a cancer in a subject in need thereof comprising administering a therapeutically effective amount of a plurality of engineered Natural Killer T-cells (NKT cells), said cells engineered to exogenously express basic leucine zipper ATF-like transcription factor (BATF), basic leucine zipper ATF-like transcription factor 2 (BATF2), or basic leucine zipper ATF-like transcription factor 3 (BATF3) and a chimeric antigen receptor. The method for treating a cancer of claim 39, wherein said plurality of engineered NKT cells further comprise an exogenously expressed inducible suicide gene. The method for treating a cancer of claim 40, said method further comprising inducing the elimination of the administered engineered NKT cells in said patient in need by activating said inducible suicide gene. The method for treating a cancer of claim 40, wherein said inducible suicide gene is inducible caspase-9 suicide gene. The method for treating a cancer of claim 42, further comprising administering AP20187, API 903, or a mixture thereof to said subject to activate said inducible caspase-9 suicide gene. The method for treating a cancer of claim 40, wherein said inducible suicide gene is thymidine kinase (sr39 TK). The method for treating a cancer of claim 44, further comprising administering ganciclovir to said subject to activate said thymidine kinase. The method for treating a cancer of claim 39, wherein the natural killer T-cell cell further comprises an exogenously expressed CD34 tag. The method for treating a cancer of claim 39, wherein the cancer is a tumor. The method for treating a cancer of claim 39, wherein the cancer is selected from the group consisting of breast cancer, cervical cancer, ovary cancer, endometrial cancer, melanoma, bladder cancer, lung cancer, pancreatic cancer, colon cancer, prostate cancer, hematopoietic tumors of lymphoid lineage, leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, B-cell lymphoma, Burkitt's lymphoma, multiple myeloma, Hodgkin's lymphoma, Non-Hodgkin's lymphoma, myeloid leukemia, acute myelogenous leukemia (AML), chronic myelogenous leukemia, thyroid cancer, thyroid follicular cancer, myelodysplastic syndrome (MDS), tumors of mesenchymal origin, fibrosarcoma, rhabdomyosarcoma, melanoma, uveal melanoma, teratocarcinoma, neuroblastoma, glioma, glioblastoma, benign tumor of the skin, renal cancer, anaplastic large-cell lymphoma, esophageal squamous cell carcinoma, hepatocellular carcinoma, follicular dendritic cell carcinoma, intestinal cancer, muscle- invasive cancer, seminal vesicle tumor, epidermal carcinoma, spleen cancer, bladder cancer, head and neck cancer, stomach cancer, liver cancer, bone cancer, brain cancer, cancer of the retina, biliary cancer, small bowel cancer, salivary gland cancer, cancer of uterus, cancer of testicles, cancer of connective tissue, prostatic hypertrophy, myelodysplasia, Waldenstrom's macroglobinaemia, nasopharyngeal, neuroendocrine cancer myelodysplastic syndrome, mesothelioma, angiosarcoma, Kaposi's sarcoma, carcinoid, oesophagogastric, fallopian tube cancer, peritoneal cancer, papillary serous mullerian cancer, malignant ascites, gastrointestinal stromal tumor (GIST), and a hereditary cancer syndrome selected from Li- Fraumeni syndrome and Von Hippel-Lindau syndrome (VHL). The method for treating a cancer of claim 39, wherein the cancer is a neuroblastoma. The method for treating a cancer of claim 39, wherein said NKT cells exogenously express human BATF3. The method for treating a cancer of claim 39, wherein said engineered NKT cells are autologous cells derived from said subject in need of cancer treatment. The method for treating a cancer of claim 39, wherein the administration is systemic. The method for treating a cancer of claim 39, wherein the administration is parenteral. The method for treating a cancer of claim 39, wherein the natural killer T-cell is administered locally to the tumor. The method for treating a cancer of claim 39, further comprising administering one or more additional cancer therapies to the subject. A method of producing Natural Killer T-cell (NKT cell) cells for immunotherapy comprising isolating human NKT cells from human peripheral blood mononuclear cells (PBMCs); culturing said human NKT cells to prepare a culture having a majority of CD62L-positive

Type I NKT cells by culturing in the presence of at least: (1) one or more cytokines selected from the group consisting of IL-21, IL-7, IL-15, IL-

12, TNF-alpha, and a combination thereof; and

(2) irradiated NKT-depleted PBMCs or irradiated artificial antigen-presenting cells (aAPCs) loaded with alpha-Galactosylceramide (aGalCer); and genetically modifying said CD62L-positive Type I NKT cells to exogenously express a basic leucine zipper ATF-like transcription factor (BATF) polypeptide. The method of claim 56, further comprising modifying said CD62L-positive Type I NKT cells to exogenously express one or more sequences selected from the group consisting of: a sequence encoding chimeric antigen receptor (CAR) having an antigen recognition domain directed to at least one tumor-associated antigen; a sequence encoding a polypeptide for a transcriptional activator in the Wnt signaling pathway a recombinant sequence for suppressing the expression of an endogenous major histocompatibility complex (MHC) gene; one or more regulatory sequences comprising a promoter, an internal ribosomal entry site (IRES), a cis-acting hydrolase element (CHYSEL); and a combination thereof. The method of claim 56, wherein said genetically modifying comprises transforming said CD62L-positive Type I NKT cells with a nucleic acid sequence encoding BATF polypeptide and a chimeric antigen receptor (CAR) having an antigen recognition domain directed to at least one tumor-associated antigen as a single linked expression construct. The method of claim 57, further comprising expanding said genetically modified CD62L- positive Type I NKT cells to prepare a therapeutic amount of cells. A method of treating an individual for a medical condition using immunotherapy, comprising the steps of: a. isolating human NKT cells from human peripheral blood mononuclear cells (PBMCs); b. culturing said human NKT cells in the presence of at least: i. one or more cytokines selected from the group consisting of IL-21, IL-7, IL-

15, IL-12, TNF-alpha, and a combination thereof; and ii. irradiated NKT-depleted PBMCs or irradiated artificial antigen-presenting cells (aAPCs) loaded with alpha-Galactosylceramide (aGalCer); to prepare a culture having a majority of CD62L-positive Type I NKT cells; c. genetically modifying said CD62L-positive Type I NKT cells to exogenously express a basic leucine zipper ATF-like transcription factor (BATF) polypeptide and a chimeric antigen receptor (CAR) having an antigen recognition domain directed to at least one tumor-associated antigen; and d. providing a therapeutically effective amount of the co-stimulated CD62L+ NKT cells to the individual. The method of claim 60, wherein said genetically modifying said CD62L-positive Type I NKT cells to exogenously express one or more sequences selected from the group consisting of: a sequence encoding a polypeptide for a transcriptional activator in the Wnt signaling pathway a recombinant sequence for suppressing the expression of an endogenous major histocompatibility complex (MHC) gene; one or more regulatory sequences comprising a promoter, an internal ribosomal entry site

(IRES), a cis-acting hydrolase element (CHYSEL); and a combination thereof. The method of claim 61, wherein said genetically modifying said CD62L-positive Type I NKT cells comprises introducing said one or more sequences into said CD62L-positive Type I NKT cells as a single construct. The use of engineered Natural Killer T-cells (NKT cells) for the preparation of a medicament for immunotherapy wherein said NKT cells are genetically modified to exogenously express a basic leucine zipper ATF-like transcription factor (BATF) polypeptide.