NKT cell subset for in vivo persistence and therapeutic activity and propagation of same

Abstract

To provide methods and composition for producing immunotherapeutically effective NKT cells, and methods and compositions for providing an effective amount of NKT cells to an individual in need of immunotherapy.SOLUTION: In specific embodiments, the NKT cells are CD62L positive, and maintaining the CD62L expression with exposure to one or more co-stimulatory agents. Optionally, the NKT cells may be engineered to incorporate a chimeric antigen receptor.SELECTED DRAWING: Figure 3A

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 151,690, filed April 23, 2015, and U.S. Provisional Patent Application No. 62 / 309,525, filed March 17, 2016, both of which are incorporated herein by reference in their entireties.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under RO1CA116548 and P50CA126752 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] FIELD OF THE INVENTION Embodiments of the present disclosure encompass at least the fields of cell biology, molecular biology, immunology, and medicine, including at least cancer medicine. [Background technology]

[0004] Type I NKT cells (NKTs) are an evolutionarily conserved subset of innate lymphoid cells that express the invariant TCR α-chain Vα24-Jα18 and respond to self- or microbe-derived glycolipids presented by the monomorphic HLA class I-like molecule CD1d (Porcelli et al., 1993; Lantz and Bendelac, 1994; Bendelac et al., 1995; Kim et al., 2015). The potential importance of NKTs for tumor immunity and immunotherapy has been demonstrated in multiple mouse models of cancer and in early clinical trials in cancer patients (McEwen-Smith et al., 2015; Dhodapkar, 2009; Exley and Nakayama, 2011; Motohashi et al., 2011; Yamasaki, 2011; Taniguchi et al., 2015). In contrast to T cells, NKT cells can effectively mobilize to tumor sites and mediate antitumor responses through direct killing of CD1d+ tumor cells, inhibition of tumor-supporting macrophages, or transactivation of NK cells (Metelitsa, 2011). Several studies have demonstrated a strong and clear association between the number of tumor-infiltrating or circulating NKT cells and improved disease outcomes in patients with a variety of tumor types (Dhodapkar, 2009; Metelitsa et al., 2004; Tachibana et al., 2005; Molling et al., 2007; Cariani et al., 2012; Cariani et al., 2012). Conversely, tumor progression is often accompanied by a decrease in the number or functional activity of NKT cells (16) or downregulation of CD1d expression on malignant tumor cells (Dhodapkar et al., 2003). To counter these tumor escape mechanisms, methods have been developed to expand primary human NKT cells ex vivo to clinical scale and redirect their cytotoxicity against tumor cells via transgenic expression of chimeric antigen receptors (CARs) (Heczey et al., 2014). Similar to findings reported in CAR T cell clinical trials (Kalos and June, 2013; Dotti et al., 2014), there is a strong correlation between antitumor efficacy in xenogeneic tumor models and the in vivo persistence of CAR NKT cell products (Heczey et al., 2014).However, the mechanisms governing human NKT proliferation in vitro and subsequent persistence in vivo remain largely unknown, hindering the rational design of NKT cell-based cancer immunotherapies.

[0005] Recent global transcriptional profiling studies have demonstrated that NKT cells share characteristics with T cells and NK cells but are a distinct population of lymphocytes (Cohen et al., 2013). In mice, the developmental program and functional differentiation of NKT cells have been characterized fairly extensively over the last decade, as summarized in recent reviews (Kim et al., 2015; Contantinides and Bendelac, 2013). Several key features of mouse NKT cells have also been confirmed in their human counterparts. In both mice and humans, NKT cells diverge from T cells at the CD4+CD8+ (double-positive, DP) thymocyte stage. Unlike T cells, which are positively selected by thymic epithelial cells, NKT cells are selected by DP thymocytes expressing CD1d (Gapin et al., 2001). Immediately after positive selection, expression of promyelocytic leukemia zinc finger transcription factor (PLZF) allows NKT cells to proliferate within the thymus and differentiate into effector / memory-like cells (Savage et al., 2008). Peripheral NKT cells are long-lived lymphocytes, and their postthymic maintenance largely depends on slow, homeostatic proliferation mediated by IL-15 (Matsuda et al., 2002; Baev et al., 2004). In human peripheral blood, NKT cells are divided into two major functional subsets based on CD4 expression: CD4+ and CD4- (mostly CD8 / CD4 double negative, DN) (Lee et al., 2002). The CD4+ subset is highly enriched in neonatal NKT cells and undergoes fewer homeostatic divisions in adults compared with the CD4- subset (Baev et al., 2004), suggesting that CD4+ NKT cells may contribute to the long-term persistence of adoptively transferred therapeutic NKT cells under certain conditions. However, for example, ex vivo expansion of human NKT cells in response to antigen stimulation with α-galactosylceramide (αGalCer) yields similar numbers of CD4+ and CD4+ DN NKT cells (28). NKT cells also exhibit NK-like lineage differentiation by acquiring expression of CD161 and subsequently CD56. As in T cells, CD56 expression is associated with terminal differentiation and loss of proliferative capacity (Loza et al., 2002).

[0006] In contrast to peripheral T cells, which have a well-established developmental hierarchy from naive to central memory, then effector memory, and then terminal effector cells (Sallusto et al., 2004), NKT cells have been widely described as cells with an "activated / memory" phenotype without a naive state (D'Andrea et al., 2000; Kronenberg and Gapin, 2002). In umbilical cord blood, the majority of NKT cells are CD4+ and co-express CD45RO with CD62L and CCR7, lacking immediate effector function, thus resembling central memory CD4+ T cells (Baev et al., 2004; D'Andrea et al., 2000; Eger et al., 2006). In adult peripheral blood, NKT cells are evenly divided into CD4+ and CD4- subsets (although there is significant interindividual variability). Adult NKT cells lack a clear boundary between a "memory" and an "effector" state, as they express altered markers of memory and possess immediate effector functions such as cytokine production and cytotoxicity (Baev et al., 2004; Eger et al., 2006). Even in elderly individuals, the majority of adult NKT cells express CD28 (DelaRosa et al., 2002), distinguishing them from terminally differentiated T effector cells (Okada et al., 2008).

[0007] Recent reports have demonstrated that CD62L+ central memory T cells possess stem cell properties and superior therapeutic activity in cell therapy products (Graef et al., 2014; Wang et al., 2012; Sommermeyer et al., 2015). The functional significance of CD62L expression on NKT cells remains unknown. In this disclosure, the CD62L+ subset is required for NKT cell proliferation in vitro and persistence in vivo. Importantly, when engineered to express a CD19-specific CAR (CAR.CD19), CD62L+, but not CD62L-, CAR.CD19NKT cells produced sustained tumor regression in a B-cell lymphoma model in NSG mice. CD62L+ NKT cells can be maintained during ex vivo expansion when specific costimulatory ligands are provided. This knowledge allows for the engineering of costimulatory artificial antigen-presenting cells (aAPCs) that can be used, for example, to generate NKT and CAR-NKT cells with superior therapeutic activity in cancer patients. Summary of the Invention

[0008] The methods and compositions of the present disclosure relate to immunotherapy for individuals in need thereof. In some embodiments, the individual is in need of a therapy that targets specific antigen-bearing cells, such as cancer, for destruction. The present disclosure generally provides for the use of NKT cells for immunotherapy based on improved methods for generating clinically useful quantities and efficacy of NKT cells.

[0009] Embodiments of the present disclosure provide CD62L+ NKT cells with excellent in vivo persistence and anti-tumor activity. Embodiments of the present disclosure enable the effective expansion of NKT cells so that they can be used in therapeutic applications. NKTs of the present disclosure have improved survival and proliferation associated with expression of CD62L. CD62L expression is present on NKT cells and is maintained in the cells due to costimulation of the NKT cells. Embodiments of the present disclosure include costimulation of NKT cells by methods to maintain CD62L expression. NKT cells are exposed to costimulation using one or more methods, such as, for example, one or more cytokines (including at least IL-21), one or more agonist antibodies that bind to costimulatory receptors, and / or CD1d, and CD1d, such as by exposure to artificial antigen-presenting cells expressing ligands for one or more costimulatory receptors. Thus, in specific embodiments, artificial antigen-presenting cells can be used to generate CD62L-enriched NKTs for effective cancer immunotherapy.

[0010] In one embodiment, there is a method of preparing natural killer T (NKT) cells for use in immunotherapy, comprising enriching a population of NKT cells for CD62L-positive NKT cells. In a specific embodiment, the CD62L-positive NKT cells are activated by stimulation of a T cell receptor and co-stimulation with a costimulatory receptor and / or cytokine. Optionally, the method further comprises delivering a therapeutically effective amount of the cells to an individual in need of treatment. In certain aspects, the cells are engineered to express one or more chimeric antigen receptors, T cell receptors, one or more cytokines, one or more cytokine receptors, one or more chimeric cytokine receptors, or combinations thereof.

[0011] In certain embodiments, there is a method of treating an individual for a condition using immunotherapy, comprising: (a) enriching a population of NKT cells for CD62L-positive NKT cells or obtaining a population of NKT cells that is enriched for CD62L-positive NKT cells; and (b) providing a therapeutically effective amount of CD62L-positive NKT cells to the individual.

[0012] In embodiments, there are methods of treating an individual for a medical condition using immunotherapy, comprising: expanding CD62+ NKT cells from a population mixture of CD62+ NKT cells and CD62- NKT cells by exposing the population mixture to one or more costimulatory agents to enrich for costimulated CD62+ NKT cells and generating CD62+ NKT cells; and providing a therapeutically effective amount of costimulatory CD62+ NKT cells to the individual. In specific embodiments, the stimulatory and costimulatory agents include (a) one or more cytokines; (b) a substrate comprising an agonist antibody or ligand for a T cell receptor (e.g., OKT3mAb, 6B11mAb, or recombinant human CD1d with an agonist glycolipid such as conjugated alpha-galactosylceramide) and one or more agonist antibodies targeting the costimulatory receptor; or (c) antigen-presenting cells comprising expression of CD1d and expression of one or more ligands for one or more costimulatory receptors. In certain embodiments, the cytokine is selected from the group consisting of IL-21, IL-2, IL-7, IL-15, IL-12, TNF-alpha, and combinations thereof. The substrate may be a bead, a plate, or a gel. In specific embodiments, the antigen-presenting cells are transduced with one or more polynucleotides to express one or more ligands of one or more costimulatory receptors. The costimulatory receptor may be CD28, OX40, 4-1BB, ICOS, CD40, CD30, CD27, or a combination thereof. The ligand of the costimulatory receptor may be CD80, CD86, OX40L, 4-1BBL, ICOS ligand, CD154, CD30L, or a combination thereof.

[0013] In certain embodiments, the NKT cells encompassed by the present disclosure include genetic engineering. In specific aspects, the genetic engineering provides the cells with cancer cell targeting activity, such as targeting an antigen in cancer cells. The genetic engineering may include a T cell receptor and / or a chimeric antigen receptor. In some cases, the NKT cells are genetically engineered after exposing the population to one or more costimulatory agents. The NKT cells may be genetically engineered within 1, 2, 3, 4, 5, 6, or more days after exposing the population to one or more costimulatory agents.

[0014] In certain embodiments, there is a method of generating NKT cells for immunotherapy, comprising costimulating a population of NKT cells to maintain expression of CD62L on at least a portion of the NKT cells, optionally further comprising providing a therapeutically effective amount of the NKT cells to an individual in need thereof.

[0015] In one embodiment, there is a method of generating NKT cells for immunotherapy, comprising exposing a pre-selected population of CD62L+ NKT cells or a mixed population of CD62L+ NKT cells and CD62L- NKT cells to one or more costimulatory agents designed to intentionally enrich or preserve CD62L+ NKT cells. Optionally, the method further comprises obtaining the mixed population. In a specific embodiment, the mixed population is derived from an individual to whom the enriched population will be delivered. In certain aspects, the mixed population is derived from an individual different from the individual to whom the enriched population will be delivered. The mixed population may be derived from a deposit or may be commercially obtained.

[0016] In one embodiment, there is a composition of matter comprising non-naturally occurring cells that express CD1d and express one or more ligands for one or more costimulatory receptors. [Brief explanation of the drawings]

[0017] [Figure 1A](1A-1D) Figures showing accumulation of the CD62L+ subset in cultures following in vitro antigen stimulation of primary NKTs. (1A) CD62L expression was examined by FACS on primary NKTs derived from freshly isolated PBMCs (day 0) and on day 12 after stimulation with αGalCer and expansion in vitro in culture. (1B) Kinetics of CD62L expression on NKTs at the indicated intervals after primary stimulation (as in 1A) from individual donors (n=10). (1C) Expression of PD-1, TIM-3, and LAG-3 on the surface of NKT cells was measured by FACS on days 0 and 12. Representative figures from one of four donors (upper panel) or mean ± SD of MFI for all donors (lower panel). (1D) 12 days after primary stimulation, NKT cells were magnetically sorted into CD62L+ and CD62L- subsets, followed by RNA isolation and gene expression analysis using the Human Immunology Panel v2 and nCounter Analysis System. Heatmaps show the log2 fold change (CD62+ / CD62L-) of genes with p-values ​​less than 0.02 and mean fold change greater than 2. Data were generated from six NKT cell donors (12 paired samples). [Figure 1B] Same as above. [Figure 1C-1] Same as above. [Figure 1C-2] Same as above. [Figure 1C-3] Same as above. [Figure 1D] Same as above.

[0018] [Figure 2A](2A-2E) Functional characterization of CD62L+ and CD62L- NKTs. (2A) Luciferase-transduced CD1d+ DAOY cells were pulsed overnight with PBS (control) or αGalCer and then cocultured with magnetically sorted CD62L+ or CD62L- NKTs. Cytotoxicity was analyzed after 4 hours by measuring luciferase intensity using a plate reader. The left panel is representative of three donors with no difference in cytotoxicity between NKT subsets. The right panel is representative of three donors with significant difference in cytotoxicity between NKT subsets. (2B) IFNγ and IL-4 concentrations were measured in the 24-hour supernatants of CD62L+ or CD62L- NKTs stimulated with αGalCer by Luminex assay in three independent experiments with NKTs from three donors. (2C) CFSE-labeled NKTs were magnetically sorted into CD62L+ and CD62L- subsets (upper panel), as determined by post-sort FACS, and stimulated with αGalCer-pulsed irradiated APCs. Three days after stimulation, NKTs were analyzed by FACS for Annexin V and 7-AAD staining after gating on CFSE-positive events. Results are from a representative of five donors examined (middle panel). The corresponding bar graph (lower panel) shows the mean ± SD of the percentage of Annexin V+ NKTs at day 3 (N = 5). (2D) Cell proliferation was assessed at day 6 after stimulation, as measured by CFSE dilution. Results are from a representative of five donors examined (upper panel) and are the mean ± SD of the MFI of CFSE for all five donors (lower panel). (2E) After NKT cell stimulation, total cell counts were performed at the indicated time intervals. Shown is the mean ± SD of viable cells for a representative donor (top panel) or fold change for each of the five donors examined at day 6 post-stimulation. ***P<0.001, paired t-test. [Figure 2B] Same as above. [Figure 2C-1] Same as above. [Figure 2C-2] Same as above. [Figure 2D-1] Same as above. [Figure 2D-2] Same as above. [Figure 2E] Same as above.

[0019] [Figure 3A] (3A-3D) These figures show that CD62L+ NKT cells have superior in vivo persistence and antitumor activity. (3A) Luciferase-transduced NKT cells were sorted into CD62L+ and CD62L- subsets and injected into NSG mice. NKT cell in vivo persistence was monitored by bioluminescence imaging. (3B) Mean ± SD bioluminescence photon counts at the indicated days after injection of CD62L+ or CD62L- NKT cells (P = 0.008, paired t-test). (3C) Each mouse received an intravenous injection of 2 × 105 luciferase-transduced Daudi lymphoma cells (day 0), followed by an intravenous injection of 107 CAR.CD19-transduced NKT cells (day 4) with IL-2 (1000 U / mouse) or PBS as a control. Tumor growth was monitored weekly using bioluminescence imaging. (3D) Survival probability was analyzed by the Kaplan-Meier method (10 mice per group), and differences in survival were then compared using the log-rank test. [Figure 3B] Same as above. [Figure 3C] Same as above. [Figure 3D] Same as above.

[0020] [Figure 4A](4A-4D) Figures show that costimulation maintains CD62L+ NKTs and prevents their depletion. (4A) NKTs were sorted into CD62L+ and CD62L- subsets, stimulated with αGalCer, and examined for 4-1BB and OX40 expression by FACS before and 3 days after stimulation. Figures shown are from a representative of four donors. (4B) CD62L+ NKTs were stimulated on plates coated with the indicated agonist mAbs. Shown are the mean ± SD (N = 4) fold-change in absolute NKT cell numbers at day 7 post-stimulation compared to day 0. *P < 0.001, one-way ANOVA. (4C) CD62L+ NKTs were stimulated as in B and analyzed for CD62L expression (black) relative to isotype control (gray) at day 7. Shown are representative overlay histograms (top panel) and the mean ± SD percentage of CD62L+ cells, N = 4. (4D) CD62L+ NKTs were stimulated as in B and analyzed for PD-1 expression (black) relative to isotype control (gray) on day 12. Shown are representative overlay histograms (top panel) and mean ± SD of percentage of PD-1+ cells. ** or ***P < 0.01 or 0.001, one-way ANOVA. [Figure 4B] Same as above. [Figure 4C-1] Same as above. [Figure 4C-2] Same as above. [Figure 4C-3] Same as above. [Figure 4D-1] Same as above. [Figure 4D-2] Same as above. [Figure 4D-3] Same as above.

[0021] [Figure 5A](5A-5E) Phenotypic analysis of freshly isolated and in vitro expanded NKTs. (5A) CD4 and CD62L expression was examined by FACS in freshly isolated primary NKTs (gated on the CD3+Vα24-Jα18+ subset) in umbilical cord blood mononuclear cells (CBMCs). Figures are from a representative of five CBMC donors. (5B) CD4 and CD62L expression was examined in primary NKTs before (day 0) and after 12 days of stimulation with αGalCer and in vitro expansion, gating as in A. Figures are from a representative of ten PBMC donors. (5C) CCR7, CD27, and CD28 expression in relation to CD62L expression in primary NKTs before (day 0) and after 12 days of stimulation with αGalCer and in vitro expansion, gating as in A. Figures are from a representative of six PBMC donors. (5D) Expression of CD161, CD56, and IL7Rα in relation to CD62L expression 12 days after stimulation with αGalCer and in vitro expansion. Figure is from a representative of three PBMC donors. (5E) Expression of PLZF, LEF1, and GATA3, as well as co-expression of LEF1 and GATA3, in relation to CD62L expression was analyzed using intracellular flow cytometry 12 days after stimulation with αGalCer and in vitro expansion. Figure is from a representative of four PBMC donors. [Figure 5B] Same as above. [Figure 5C] Same as above. [Figure 5D] Same as above. [Figure 5E-1] Same as above. [Figure 5E-2] Same as above.

[0022] [Figure 6A-1](6A-6B) Comparison of the purity and absolute number of NKT cells after expansion with CD3 / CD28 agonist mAb compared with aGalCer-pulsed, irradiated PBMCs. NKT cells were isolated from PBMCs of four individuals. Half were stimulated with aGalCer-pulsed, irradiated autologous PBMCs, and the other half were stimulated on plates coated with CD3 / CD28 mAb. In both cases, cells were expanded in culture with IL-2 (200 U / ml) every other day. Cultures were analyzed on day 12. (6A) NKT cell purity was determined by flow cytometry as the percentage of cells expressing CD3 and iTCRα. (6B) Absolute cell counts of NKT cells were performed in triplicate using trypan blue exclusion assays. *P<0.05. Data were analyzed using paired t-tests after Log(2) transformation. [Figure 6A-2] Same as above. [Figure 6B] Same as above.

[0023] [Figure 7A] (7A-7B) NKT cells transduced with CAR.CD19. (7A) Schematic diagram of the CAR.CD19 construct. (7B) NKT cells were restimulated with autologous PBMCs (irradiated at 40 Gy). Three days after restimulation, 24-well non-tissue culture plates were coated with retronectin and, after washing, inoculated with 1 ml of retroviral supernatant containing CAR.CD19. The viral supernatant was then removed, and NKT cells were added to the wells in complete medium and 200 U / ml rhIL-2. NKT cells were then magnetically sorted into CD62L+ and CD62L- subsets, and surface expression of CAR.CD19 was analyzed by 2D3 mAb staining by FACS 12 days after transduction. Shown is a FACS image from a representative of three independent experiments. [Figure 7B] Same as above.

[0024] [Figure 8A](8A-8B) Expression of costimulatory receptors on resting and activated NKTs. (8A) FACS analysis of CD4-associated OX40 and 4-1BB expression on resting NKTs (12 days after primary stimulation) and NKTs 3 days after restimulation with αGalCer. Figures are from a representative of six PBMC donors. (8B) Magnetically sorted CD62L+ and CD62L- NKTs were analyzed for CD4-associated OX40 and 4-1BB expression 3 days after restimulation of NKT cells with αGalCer. Figures are from a representative of four PBMC donors. [Figure 8B] Same as above.

[0025] [Figure 9] Figure 1 shows a comparison of NKT cell proliferation using low and high concentrations of plate-bound OKT3 mAb. Resting NKT cells grown in vitro were stimulated with 20 ng / ml or 1 μg / ml of anti-CD3 OKT3 mAb alone or with 500 ng / ml of anti-CD28 CD28.2 mAb. Cells were expanded in culture with IL-2 (200 U / ml) added every other day. Absolute cell counts of NKT cells were performed in triplicate on day 12 using a trypan blue exclusion assay and divided by input count on day 0. Data are M ± SD, N = 4. **P = 0.01, paired t-test.

[0026] [Figure 10-1] FIG. 1 shows that IL-21 increases the frequency of CD62L+ NKT cells during primary expansion. [Figure 10-2] Same as above. [Figure 10-3] Same as above.

[0027] [Figure 11-1] FIG. 10 demonstrates that IL-21 increases the frequency of CD62L+ NKT cells during secondary expansion. [Figure 11-2] Same as above. [Figure 11-3] Same as above.

[0028] [Figure 12-1] FIG. 1 shows the expression of CD1d and costimulatory molecules on Ramos cells as an example. [Figure 12-2] Same as above.

[0029] [Figure 13-1] FIG. 1 shows that Ramos cells can expand primary NKT cells with high levels of CD62L expression. [Figure 13-2] Same as above. [Figure 13-3] Same as above.

[0030] [Figure 14] FIG. 1 shows that Ramos cells expand NKTs upon secondary stimulation with significant retention of CD62L expression. DETAILED DESCRIPTION OF THE INVENTION

[0031] This application incorporates by reference 62 / 151,690, filed April 23, 2015.

[0032] As used herein, "a" or "an" may mean one or more. As used herein in the claims, when used in conjunction with the word "comprising," the word "a" or "an" may mean one or more. As used herein, "another" may mean at least a second or more. In specific embodiments, aspects of the invention may, for example, "consist essentially of" or "consist of" one or more sequences of the invention. Some embodiments of the invention may consist of, or consist essentially of, one or more elements, method steps, and / or methods of the invention. It is contemplated that any method or composition described herein may be implemented in conjunction with any other method or composition described herein. The scope of the present application is not intended to be limited to the particular embodiments of the process, machine, manufacture, composition of matter, means, methods and steps described in the specification.

[0033] I. General Embodiments The present disclosure provides NKT cells suitable for use in immunotherapy because they can proliferate to and persist at sufficient levels in vivo to achieve a therapeutic effect. The NKT cells of the present disclosure are engineered to express and maintain expression of CD62L, which at least in part makes them highly therapeutically applicable. Such maintenance of CD62L expression on NKT cells occurs, at least in part, upon costimulation, including upon exposure to one or more costimulatory agents.

[0034] II. NKT Cells and Their Co-stimulation In certain embodiments, NKT cells are useful in therapeutic applications because they have improved in vitro proliferation and in vivo persistence following exposure to one or more costimulatory agents that enable the cells to maintain CD62L expression. The one or more costimulatory agents may be of any type, but in specific embodiments, they include one or more cytokines; (b) a substrate (e.g., beads, plates, etc.) comprising one or more agonist antibodies targeting costimulatory receptors; and / or (c) cells, such as antigen-presenting cells, that express CD1d and one or more ligands for one or more costimulatory receptors. When NKT cells are exposed to a cytokine, the cytokine may be of any suitable type, but in specific cases, the cytokine is IL-21, IL-2, IL-7, IL-15, IL-12, IL-18, TNF-alpha, or a combination thereof. In specific embodiments, CD62L+ NKT cells express IL-21 when cultured in the presence of IL-2, and IL-21 maintains CD62L expression.

[0035] In cases where NKT cells are exposed to one or more costimulatory agents that are agonistic antibodies (which, at least in some cases, are monoclonal) that immunologically recognize the costimulatory receptor, the receptor may be a costimulatory receptor. However, in specific embodiments, the receptor is, for example, CD28, 4-1BB, OX-40, ICOS, CD2, CD27, CD30, GITR, TIM1, LFA1, ICAM1, or HVEM. The antibody may be attached to a substrate that allows sufficient exposure of the cell population, e.g., a population of NKT cells, to the antibody. The antibody may be commercially available, obtained as a gift, or produced by means standard in the art.

[0036] When NKT cells are exposed to a therapeutically effective amount of cells with antigen-presenting cell activity, e.g., artificial antigen-presenting cells (e.g., non-naturally occurring cells with antigen-presenting cell activity), the cells may be transduced with one or more polynucleotides to express one or more ligands for one or more costimulatory receptors. The cells may be of any type so long as they express one or more ligands for one or more costimulatory receptors, although in at least some cases, the cells also express CD1d. In certain embodiments, the cells are antigen-presenting cells. In particular cases, cells that express CD1d and / or one or more ligands for one or more costimulatory receptors are naturally occurring and may be used in the methods encompassed herein. In other cases, cells that do not naturally express CD1d and / or do not naturally express one or more ligands for one or more costimulatory receptors are transduced to express the respective components and used in the methods encompassed herein. The ligand for the costimulatory receptor may be of any type; in specific embodiments, the ligand is CD80, CD86, 4-1BBL, OX40L, ICOSL, CD30L, GITRL, TIM4, LIGHT, or the like. When cells having antigen-presenting cell activity are transduced with one or more polynucleotides, the polynucleotides may be contained in a vector, including a viral vector or a non-viral vector (e.g., a plasmid). Viral vectors include retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, and the like. The vector will contain appropriate regulatory elements for expression in cells having antigen-presenting cell activity. In some cases, the polynucleotide transduced into cells having antigen-presenting cell activity encodes two or more coding regions, such as, for example, encoding two costimulatory receptor ligands. In such cases, the separate coding regions may or may not be regulated by the same regulatory element.

[0037] In embodiments in which CD62L expression on NKT cells is sustained due to costimulation of the NKT cells, there may or may not be a general order of steps to prepare the desired NKT cells. In certain embodiments, NKT cells are obtained from a suitable source (e.g., blood (including peripheral blood, umbilical cord blood, etc.)) by sorting (e.g., magnetic bead or FACS sorting) and are present in a mixed population of cells. Following this, the NKT cells are activated via TCR stimulation using a substrate containing an agonist antibody or ligand for the T cell receptor (e.g., OKT3 mAb, 6B11 mAb, or recombinant human CD1d with a bound agonist glycolipid such as alpha-galactosylceramide), or antigen-presenting cells expressing CD1d and a bound agonist glycolipid such as alpha-galactosylceramide. The TCR-activated NKT cells may be exposed to costimulation to generate higher levels of CD62L+ NKT cells from the population than occur in the absence of costimulation. In some embodiments, prior to delivery to an individual in need thereof and following exposure to one or more costimulatory agents, the NKT cells are engineered by recombinant means to incorporate one or more features, e.g., express one or more therapeutic agents or entities that render the NKT cells therapeutic. In certain embodiments, the cells are genetically engineered to provide the cells with the ability to target antigen-bearing cells. In specific embodiments, the engineering is by transducing the NKT cells to express one or more chimeric antigen receptors and / or T cell receptors, or those that target a particular antigen of interest. In specific embodiments, the antigen is a tumor antigen.

[0038] The NKT cells utilized to treat a condition in an individual may originate from the individual to whom they are administered, they may originate from another individual, or they may be obtained from a cell depository. The NKT cells, in a specific embodiment, are type 1 NKT cells.

[0039] NKT cells may or may not be selected prior to delivery to an individual. In a specific embodiment, CD62L-positive NKT cells are not selected from CD62L-negative NKT cells, although in alternative embodiments, they may be selected. For example, if cells are not selected based on whether they express CD2L, they can be enriched using costimulation of the cells, which results in the maintenance of CD62L expression, if the cells are not selected by physical separation.

[0040] In most cases, cells are not sorted based on a particular phenotype, although in some cases, cells may be sorted by methods that allow for enrichment of the desired cells, for example, by recovering the desired cells upon exposure to one or more substrates capable of specifically binding the cells. For example, separation of the desired cells using antibodies on a substrate (e.g., beads, particles, plates, gel matrices, etc.) may be utilized, where the antibodies may directly or indirectly bind the cells. In specific embodiments, magnetic separation may be employed.

[0041] III. Genetic manipulation of NKT cells In certain embodiments, the NKT cells are genetically engineered prior to delivery to an individual in need thereof. NKT cells are usually engineered after TCR stimulation and costimulation; in certain embodiments, genetic engineering occurs within 1, 2, 3, 4, 5, or more days after stimulation (and this may depend on the type of transduction used; for example, with retroviral vectors, it is within 2 days).

[0042] Genetic engineering of NKT cells can occur by human intervention, and in certain embodiments, the genetic engineering enables the cells to specifically target one or more cancer cells, e.g., cancer cells expressing a particular antigen. In specific embodiments, the engineering provides the NKT cells with a specific, non-native receptor for a particular cancer cell. The receptor can be of any type, but in specific embodiments, the receptor is, for example, a T cell receptor or a chimeric antigen receptor. In some cases, the chimeric antigen receptor is for CD19, CD22, CD30, GD2, GPC3, CSPG4, HER2, CEA, mesothelin, etc.

[0043] T cell receptors directed against MHC / peptide complexes derived from non-mutated tumor-associated antigens (e.g., survivin, MYCN, NY-ESO1, MAGE, PRAME, WT1, etc.) or patient-specific mutated tumor antigens revealed by sequencing of tumor DNA.

[0044] In some cases, NKT cells are engineered to express a CAR. Genetic engineering of NKT cells to express a tumor-directed chimeric antigen receptor (CAR) can generate anti-tumor effector cells that circumvent tumor immune escape mechanisms that are due to abnormalities in protein / antigen processing and presentation. Furthermore, transgenic receptors can be directed to tumor-associated antigens that are not protein-derived. In certain embodiments of the present disclosure, there are NKT cells that have been engineered to contain at least a CAR. In specific aspects, certain NKT cells contain expression of two or more CARs.

[0045] The present disclosure includes NKT cells expressing an artificial T cell receptor called a CAR (which may also be called a chimeric T cell receptor or chimeric immune receptor). In an embodiment of the present disclosure, it is specific for a cancer antigen. A CAR may generally include an extracellular domain, a transmembrane domain, and an intracellular domain. In specific embodiments, it may be first-generation, second-generation, or third-generation.

[0046] In certain cases, the NKT cells comprise a CAR that is chimeric, non-natural, and at least partially engineered by human hands, and directed against a particular cancer antigen of interest. In certain cases, the engineered CAR has one, two, three, four, or more components, and in some embodiments, one or more components direct the targeting or binding of the NKT cell to cancer cells that contain the cancer antigen. In specific embodiments, the CAR comprises an antibody against the cancer antigen, part or all of a cytoplasmic signaling domain, and / or one or more costimulatory molecules, e.g., part or all of the intracellular domain of a costimulatory molecule. In specific embodiments, the antibody is a single-chain variable fragment (scFv). In certain aspects, the antibody is directed against a cancer antigen, e.g., on the cell surface of a cancer cell expressing the antigen of interest. In certain embodiments, a cytoplasmic signaling domain, e.g., from the T cell receptor zeta chain, is employed as at least part of the chimeric receptor to generate a stimulatory signal for NKT cell proliferation and effector function following binding of the chimeric receptor to the target antigen. Examples would include, but are not limited to, intracellular domains derived from costimulatory molecules such as CD27, CD28, 4-1BB, and OX40, or signaling components of cytokine receptors such as IL7 and IL15. In certain embodiments, costimulatory molecules are employed to enhance the activation, proliferation, and cytotoxicity of NKT cells generated by the CAR after antigen binding. In a specific embodiment, the costimulatory molecules are CD28, OX40, and 4-1BB.

[0047] Generally, the extracellular domain of a CAR includes a signal peptide, an antigen-recognition domain, and a spacer linking the antigen-recognition domain to the transmembrane domain. The antigen-recognition domain will generally comprise a single-chain variable fragment (scFv) specific for a particular cancer antigen. However, when there are two or more CARs in the same cell, the second CAR may comprise an scFv specific for another particular antigen. Examples of cancer antigens include, for example, melanoma-associated antigens (MAGEs), preferentially expressed antigens in melanoma (PRAMEs), CD19, CD20, CD22, kappa-light chain, CD30, CD33, CD123, CD38, CD138, ROR1, ErbB2, ErbB3 / 4, EGFrvIII, carcinoembryonic antigen, EGP2, EGP40, HER2, mesothelin, TAG72, PSMA, NKG2D ligand, B7-H6, IL-13 receptor a2, MUC1, MUC16, CA9, GD2, GD3, HMW-MAA, CD171, Lewis Y, G250 / CAIX, HLA-AI MAGE A1, HLA-A2 The antibody may include any one of NY-ESO-1, PSC1, folate receptor-a, CD44v6, CD44v7 / 8, 8H9, NCAM, VEGF receptor, 5T4, fetal AchR, NKG2D ligand, or CD44v6.

[0048] Examples of hinge regions for extracellular domains include the CH2CH3 region of an immunoglobulin, a hinge region from IgG1, and a portion of CD3. In some cases, the transmembrane region is CD28, but it can be of any type.

[0049] Generally, the intracellular domain of the CAR of the present disclosure is used to signal the cell and receptor cluster after antigen recognition. The most commonly used intracellular domain component is CD3 zeta, which contains three ITAMs and transmits an activation signal to T cells after antigen binding. In some embodiments, additional costimulatory signaling is utilized, such as CD3 zeta in combination with CD28, 4-1BB, and / or OX40.

[0050] IV. Cells in general The cells of the present disclosure include both CD62L-positive NKT cells that have been costimulated by one or more costimulatory agents, as well as specific costimulatory agents that are themselves antigen-presenting cells (which may also be referred to as non-naturally occurring cells with antigen-presenting cell activity). In some embodiments, there are compositions of matter that are non-naturally occurring cells that express CD1d and are artificial antigen-presenting cells that express one or more ligands for one or more costimulatory receptors.

[0051] As used herein, the terms "cell," "cell line," and "cell culture" may be used interchangeably. All of these terms include their progeny, any and all subsequent generations. It is understood that not all progeny may be identical due to deliberate or inadvertent mutations. In the context of expressing a heterologous nucleic acid sequence, a "host cell" can refer to a prokaryotic or eukaryotic cell, including transformable organisms capable of replicating a vector and / or expressing a heterologous gene encoded by the vector. A host cell can be used and has been used as a recipient for a vector. A host cell may be "transfected" or "transformed," which refers to the process by which exogenous nucleic acid may be transfected or introduced into a host cell. Transformed cells include the primary subject cell and its progeny. As used herein, the terms "engineered" and "recombinant" cell or host cell are intended to refer to a cell into which an exogenous nucleic acid sequence, e.g., a vector, has been introduced. Thus, recombinant cells are distinguishable from naturally occurring cells that do not contain a recombinantly introduced nucleic acid.

[0052] In certain embodiments, it is contemplated that an RNA or proteinaceous sequence may be co-expressed with other selected RNAs or proteinaceous sequences in the same host cell. Co-expression may be achieved by co-transfecting a host cell with two or more different recombinant vectors. Alternatively, a single recombinant vector may be constructed to contain multiple different coding regions for RNAs, which may then be expressed in a host cell transfected with a single vector.

[0053] Some vectors may employ control sequences that allow them to be replicated and / or expressed in both prokaryotic and eukaryotic cells. One of skill in the art will further understand the conditions for incubating all of the above host cells to maintain them and allow for replication of the vector. Also understood and known are techniques and conditions that allow for the production of nucleic acids encoded by the vectors and their cognate polypeptides, proteins, or peptides, as well as large-scale production of the vectors.

[0054] Although prokaryotic cells may be employed for manipulation in recombinant engineering of vectors or DNA incorporated into vectors, the cells used in this disclosure are eukaryotic cells, including mammalian, particularly human, but also animal, particularly domestic animals such as horses, cows, murines, ovine, canines, felines, etc. for use in each animal.

[0055] Cells can be, for example, autologous, syngeneic, allogeneic, and even, in some cases, xenogeneic with respect to the individual receiving the cells. Cells may also be engineered by altering the properties of the major histocompatibility complex ("MHC"), by inactivating β2-microglobulin to prevent the formation of functional class I MHC molecules, by inactivating class II molecules, by providing expression of one or more MHC molecules, by enhancing or inactivating cytotoxic capacity by enhancing or inhibiting expression of genes associated with cytotoxic activity, etc.

[0056] An expression vector encoding a CAR can be introduced into cells as one or more DNA molecules or constructs, optionally with at least one marker that allows for selection of host cells containing the construct. Constructs can be prepared by conventional methods, whereby the gene and regulatory regions can be isolated, optionally ligated by restriction or sequencing or other means, cloned into a suitable cloning host, and analyzed. In particular, PCR can be used to isolate individual fragments containing all or part of the functional unit, optionally followed by the introduction of one or more mutations using "primer repair," ligation, or in vitro mutagenesis. Once completed and verified to have the appropriate sequence, the construct can then be introduced into CTLs by conventional means. For cell infection or transduction, the construct can be integrated into or packaged in a non-replicating, defective viral genome, such as adenovirus, adeno-associated virus (AAV), or herpes simplex virus (HSV), including retroviral vectors. The construct can also include viral sequences for transfection, if desired. Alternatively, the construct may be introduced by fusion, electroporation, gene gun, transfection, lipofection, etc. The host cells may be grown and expanded in culture prior to introduction of the construct, followed by appropriate treatment for introduction and integration of the construct. The cells are then expanded and screened for a marker present on the construct. Various markers that may be used successfully include hprt, neomycin resistance, thymidine kinase, hygromycin resistance, etc.

[0057] In many situations, one may wish to be able to kill engineered cells, where one wishes to terminate treatment and the cells become tumorous in studies where the absence of the cells after their presence is of interest or other events. To this end, one can provide for the expression of specific gene products that will enable one to kill the engineered cells under controlled conditions. A suicide gene product such as caspase 9 is an example of such a product.

[0058] By way of illustration, a patient with cancer or a patient susceptible to or suspected of having cancer may be treated as follows: engineered cells as described herein may be administered to the patient and maintained long-term; the individual may receive one or more administrations of the cells; the cells will be engineered and provided to the individual in need thereof.

[0059] V. Polynucleotides The present disclosure also encompasses compositions comprising a nucleic acid sequence encoding an antigen-specific CAR as defined herein, and cells harboring the nucleic acid sequence. The nucleic acid sequence is in certain embodiments a recombinant nucleic acid sequence, and may be synthetic. It may comprise DNA, RNA, as well as PNA (peptide nucleic acid), or it may be a hybrid thereof.

[0060] Furthermore, it is further envisaged for the purpose that the nucleic acid molecule may contain, for example, thioester bonds and / or nucleotide analogues. Manipulation may be useful for stabilizing the nucleic acid molecule against endonucleases and / or exonucleases in cells. The nucleic acid molecule may be transcribed by a suitable vector containing a chimeric gene that allows transcription of the nucleic acid molecule in cells. In this regard, it should also be understood that such polynucleotides can be used in "gene targeting" or "gene therapy" approaches. In another embodiment, the nucleic acid molecule is labeled. Methods for detecting nucleic acids are well known in the art, such as Southern and Northern blots, PCR, or primer extension. This embodiment may be useful in screening methods to verify successful introduction of the above-mentioned nucleic acid molecule during gene therapy approaches.

[0061] The nucleic acid molecule may be a recombinantly produced chimeric nucleic acid molecule comprising any of the foregoing nucleic acid molecules, alone or in combination. In a specific embodiment, the nucleic acid molecule is part of a vector.

[0062] Accordingly, the present disclosure also relates to compositions comprising vectors that include the nucleic acid molecules described in this disclosure.

[0063] Numerous suitable vectors are known to those skilled in the art of molecular biology, and their selection depends on the desired function. These include plasmids, cosmids, viruses, bacteriophages, and other vectors conventionally used in genetic engineering. Various plasmids and vectors can be constructed using methods well known to those skilled in the art. See, for example, the techniques described in Sambrook et al. (1989) and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, NY (1989), (1994). Alternatively, the polynucleotides and vectors of the present disclosure can be reconstituted into liposomes for delivery to target cells. Cloning vectors may be used to isolate individual sequences of DNA. Relevant sequences can be transferred into expression vectors where expression of a specific polypeptide is desired. Exemplary cloning vectors include pBluescript SK, pGEM, pUC9, pBR322, and pGBT9. Exemplary expression vectors include pTRE, pCAL-n-EK, pESP-1, and pOP13CAT.

[0064] In a specific embodiment, there is a vector comprising a nucleic acid sequence that is a regulatory sequence operably linked to a nucleic acid sequence encoding an antigen-specific CAR as defined herein. Such regulatory sequences (control elements) are known to those skilled in the art and may include promoters, splice cassettes, translation initiation codons, translation and insertion sites for introducing inserts into the vector. In a specific embodiment, the nucleic acid is operably linked to said expression control sequences that allow expression in eukaryotic or prokaryotic cells.

[0065] The vector is envisioned to be an expression vector comprising a nucleic acid molecule encoding an antigen-specific CAR as defined herein. In a specific embodiment, the vector is a viral vector, such as a lentiviral vector. Lentiviral vectors are commercially available, including, for example, from Clontech (Mountain View, CA) or GeneCopoeia (Rockville, MD).

[0066] The term "regulatory sequences" refers to DNA sequences necessary to achieve expression of coding sequences to which they are ligated. The nature of such control sequences varies depending on the host organism. In prokaryotes, control sequences generally include promoters, ribosomal binding sites, and terminators. In eukaryotes, control sequences generally include promoters, terminators, and, in some cases, enhancers, transactivators, or transcription factors. The term "control sequences" is intended to include, at a minimum, all components the presence of which are necessary for expression, and may also include additional advantageous components.

[0067] The term "operably linked" refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. A control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences. It will be apparent to those skilled in the art that where the control sequence is a promoter, double-stranded nucleic acid is preferably used.

[0068] Thus, the vectors referred to are, in certain embodiments, expression vectors. An "expression vector" is a construct that can be used to transform a selected host and provides for expression of a coding sequence in the selected host. Expression vectors can be, for example, cloning vectors, binary vectors or integrating vectors. Expression involves transcription of the nucleic acid molecule, preferably into translatable mRNA. Regulatory elements ensuring expression in prokaryotic and / or eukaryotic cells are well known to those skilled in the art. In the case of eukaryotic cells, they usually include a promoter ensuring initiation of transcription and, optionally, a polyA signal ensuring termination of transcription and stabilization of the transcript. Possible regulatory elements allowing expression in prokaryotic host cells include, for example, P L Examples of regulatory elements that allow expression in eukaryotic host cells, including the lac, trp or tac promoters, are the AOX1 or GAL1 promoters in yeast, or the CMV-, SV40-, RSV-promoter (Rous sarcoma virus), CMV-enhancer, SV40-enhancer, or globin intron in mammalian and other animal cells.

[0069] For example, elements involved in transcription initiation, such as regulatory elements, may also contain transcription termination signals downstream of the polynucleotide, such as, for example, an SV40 poly-A site or a tk poly-A site. Furthermore, depending on the expression system used, leader sequences capable of directing the polynucleotide to a cellular compartment or secreting it into the medium may be added to the coding sequence of the cited nucleic acid sequence, as are well known in the art. The leader sequence is produced in appropriate phase with the translation initiation and termination sequences, and preferably, the leader sequence is capable of directing the secretion of the translated protein or a portion thereof into the periplasmic space or the extracellular medium. Optionally, the heterologous sequence can encode a fusion protein containing an N-terminal specific peptide that confers a desired characteristic, such as stabilization or simplified purification of the expressed recombinant product; see above. In this context, suitable expression vectors are known in the art, such as, for example, the Okayama-Berg cDNA expression vectors pcDV1 (Pharmacia), pEF-Neo, pCDM8, pRc / CMV, pcDNA1, pcDNA3 (Invitrogen), pEF-DHFR and pEF-ADA (Raum et al. Cancer Immunol Immunother (2001), 50(3), 141-150) or pSPORT1 (GIBCO BRL).

[0070] In some embodiments, the expression control sequences are eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells, although control sequences for prokaryotic hosts may also be used. Once the vector is incorporated into a suitable host, the host is maintained under conditions suitable for high-level expression of the nucleotide sequences, followed by recovery and purification of the polypeptides of the present disclosure, as desired. In certain embodiments, one or more codable sequences are regulated by expression control sequences that are responsive to a low-oxygen environment.

[0071] Additional regulatory elements may include transcriptional as well as translational enhancers. Advantageously, the above-mentioned vectors of the present disclosure comprise selectable and / or scorable markers. Selectable marker genes useful for selecting transformed cells are well known to those skilled in the art and include, for example, antimetabolite resistance as the basis for selection for dhfr, which confers resistance to methotrexate (Reiss, Plant Physiol. (Life-Sci. Adv.) 13 (1994), 143-149), npt, which confers resistance to the aminoglycosides neomycin, kanamycin, and paromycin (Herrera-Estrella, EMBO J. 2 (1983), 987-995), and hygro, which confers resistance to hygromycin (Marsh, Gene, 32 (1984), 481-485). Additional selectable genes have been described: trpB, which allows cells to utilize indole instead of tryptophan; hisD, which allows cells to utilize histinol instead of histidine (Hartman, Proc. Natl. Acad. Sci. USA, 85 (1988), 8047); mannose-6-phosphate isomerase, which allows cells to utilize mannose (WO 94 / 20627), and ODC (ornithine decarboxylase), which confers resistance to the ornithine decarboxylase inhibitor 2-(difluoromethyl)-DL-ornithine, DFMO (McConlogue, 1987, In: Current Communications in Molecular Biology, Cold Spring Harbor Laboratory ed.), or deaminase from Aspergillus terreus, which confers resistance to blasticidin S (Tamura, Biosci. Biotechnol. Biochem. 59 (1995), 2336-2338).

[0072] Useful scorable markers are also known to those skilled in the art and are commercially available. Advantageously, the marker is a gene encoding luciferase (Giacomin, Pl. Sci. 116 (1996), 59-72; Scikantha, J. Bact. 178 (1996), 121), green fluorescent protein (Gerdes, FEBS Lett. 389 (1996), 44-47) or β-glucuronidase (Jefferson, EMBO J. 6 (1987), 3901-3907). This embodiment is particularly useful for simple and rapid screening of cells, tissues and organisms containing the cited vectors.

[0073] As described above, the cited nucleic acid molecules can be used alone in a cell or as part of a vector to express the encoded polypeptide in the cell. A nucleic acid molecule or vector containing a DNA sequence encoding any one of the specific CAR constructs is then introduced into a cell to produce the polypeptide of interest. The cited nucleic acid molecules and vectors may be designed for direct introduction into a cell or for introduction via a liposome or viral vector (e.g., adenovirus, retrovirus).

[0074] In accordance with the above, the present disclosure relates to methods for mobilizing vectors, particularly plasmids, cosmids, viruses, and bacteriophages, conventionally used in genetic engineering, containing nucleic acid molecules encoding the polypeptide sequences of antigen-specific CARs defined herein. Preferably, the vectors are expression vectors and / or gene transfer or targeting vectors. For example, expression vectors derived from viruses such as retroviruses, vaccinia viruses, adeno-associated viruses, herpes viruses, or bovine papillomaviruses may be used to deliver the referenced polynucleotides or vectors to targeted cell populations. Recombinant vectors can be constructed using methods well known to those skilled in the art; see, for example, the techniques described in Sambrook et al. (loc cit.), Ausubel (1989, loc cit.), or other standard textbooks. Alternatively, the referenced nucleic acid molecules and vectors can be reconstituted into liposomes for delivery to target cells. Vectors containing the nucleic acid molecules of the present disclosure can be transferred into host cells by well-known methods, which vary depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment or electroporation may be used for other cellular hosts; see Sambrook, supra.

[0075] XII. Pharmaceutical Compositions According to the present disclosure, the term "pharmaceutical composition" refers to a composition for administration to an individual. In certain aspects of the present disclosure, the pharmaceutical composition comprises a plurality of NKT cells. In preferred embodiments, the pharmaceutical composition comprises a composition for parenteral, transdermal, intracavitary, intraarterial, intrathecal, or intravenous administration, or for direct injection into a cancer. It is particularly contemplated that the pharmaceutical composition is administered to an individual via infusion or injection. Administration of suitable compositions may be achieved by various methods, for example, by intravenous, subcutaneous, intraperitoneal, intramuscular, topical, or intradermal administration.

[0076] The pharmaceutical compositions of the present disclosure further comprise a pharmaceutically acceptable carrier. Examples of suitable pharmaceutical carriers are well known in the art, and include phosphate-buffered saline solution, water, emulsions such as oil / water emulsions, various types of wetting agents, sterile solutions, etc. Compositions containing such carriers can be formulated by well-known conventional methods. These pharmaceutical compositions can be administered to subjects at a suitable dose.

[0077] The dosing regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, the dosage administered to a single patient will depend on many factors, including the patient's size, body surface area, age, the specific compound being administered, sex, time and route of administration, general condition, and other medications currently being administered. Progress can be monitored by periodic evaluation. CAR-engineered NKT may be administered via intravenous infusion. Doses of 1 x 10 7 / m 2 ~2×10 8 / m 2 In a specific embodiment, the range is 10 9 Up to 10 cells may be utilized.

[0078] The compositions of the present disclosure may be administered locally or systemically. Administration will generally be parenteral, e.g., intravenous; DNA may also be administered directly to the target site, e.g., by gene gun delivery to an internal or external target site or by a catheter to a site within an artery. In a preferred embodiment, the pharmaceutical composition is administered subcutaneously, and in an even more preferred embodiment, intravenously. Formulations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (e.g., those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, inert gases, and the like. In addition, the pharmaceutical compositions of the present disclosure may include a proteinaceous carrier or the like, such as serum albumin or an immunoglobulin, preferably of human origin. It is contemplated that the pharmaceutical compositions of the present disclosure may include, in addition to the CAR construct or the nucleic acid molecule or vector encoding it (as described in this disclosure), further biologically active agents depending on the intended use of the pharmaceutical composition.

[0079] XII. Therapeutic Uses of NKT Cells By way of illustration, a patient with cancer or a patient susceptible to cancer or suspected of having cancer may be treated as described herein. NKT cells engineered as described herein may be administered to the individual and maintained long-term. An individual may receive one or more administrations of the cells. In some embodiments, the engineered cells are encapsulated to inhibit immune recognition and are located at the site of the tumor.

[0080] In various embodiments, the expression constructs, vectors comprising same, host cells and / or pharmaceutical compositions are used in the prevention, treatment or amelioration of cancerous diseases, such as neoplastic diseases. In certain embodiments, the pharmaceutical compositions of the present disclosure may be particularly useful in preventing, ameliorating and / or treating cancers, including, for example, cancers with solid tumors.

[0081] As used herein, "treatment" or "treating" includes a beneficial or desired effect on the symptoms or condition of a disease or pathological condition, and may include even a minimal reduction in one or more measurable markers of the disease or condition being treated, e.g., cancer. Treatment can optionally involve reducing or ameliorating the symptoms of the disease or condition, or slowing the progression of the disease or condition. "Treatment" does not necessarily indicate a complete eradication or cure of the disease or condition, or its associated symptoms.

[0082] As used herein, "prevent" and similar words such as, for example, "prevented," "preventing," etc., refer to an approach that prevents, inhibits, or reduces the likelihood of the occurrence or recurrence of a disease or condition, such as cancer. It also refers to delaying the onset or recurrence of a disease or condition, or delaying the onset or recurrence of symptoms of a disease or condition. As used herein, "prevention" and similar words also include reducing the intensity, impact, symptoms, and / or burden of a disease or condition prior to the onset or recurrence of the disease or condition.

[0083] In certain embodiments, the present disclosure contemplates, in part, cells harboring expression constructs, nucleic acid molecules, and / or vectors that can be administered, alone or in combination with another therapy, and in at least some aspects, together with a pharmaceutically acceptable carrier or excipient. In certain embodiments, the nucleic acid molecule or vector may be stably integrated into the genome of the cell prior to administration of the cell. In specific embodiments, viral vectors may be used that are specific for and persist in particular cells or tissues. Suitable pharmaceutical carriers and excipients are well known in the art. Compositions prepared according to the present disclosure may be used to prevent, treat, or delay the diseases identified above.

[0084] Furthermore, the present disclosure relates to methods for preventing, treating, or ameliorating cancerous (including neoplastic) diseases, comprising administering to a subject in need thereof an effective amount of cells harboring a molecule of an antigen recognition moiety and a chemotherapy resistance molecule as contemplated herein and / or produced by a process as contemplated herein, a nucleic acid sequence encoding the same, or a vector encoding the same.

[0085] Exemplary possible indications for administration of compositions of engineered immune cells are cancerous diseases, including neoplastic diseases including breast, prostate, lung, and colon cancer or epithelial cancers / carcinomas, e.g., MM, breast cancer, colon cancer, prostate cancer, head and neck cancer, skin cancer, cancers of the urinary / genital tract, e.g., ovarian cancer, endometrial cancer, cervical cancer, and kidney cancer, lung cancer, stomach cancer, small intestine cancer, liver cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, esophageal cancer, salivary gland cancer, and thyroid cancer, neuroblastoma, medulloblastoma, glioblastoma, hematopoietic malignancies, etc. Exemplary indications for administration of compositions of cells are cancerous diseases, including, for example, malignancies expressing a particular antigen. In addition, this includes malignancies aberrantly expressing other tumor antigens, which may also be targeted. Administration of the compositions of the present disclosure is useful for all stages and types of cancer, including, for example, minimal residual disease, early stage cancer, advanced cancer, and / or metastatic and / or refractory cancer.

[0086] The present disclosure also encompasses co-administration protocols with other compounds that act via immune cells, such as bispecific antibody constructs, targeted toxins, or other compounds. Clinical dosing regimens for co-administration of the compounds of the present invention may include co-administration with, prior to, and / or after the administration of other components. Specific combination therapies include chemotherapy, radiation, surgery, hormone therapy, or other types of immunotherapy.

[0087] Embodiments relate to kits comprising one or more NKT cells as described herein, nucleic acid sequences as described herein, vectors as described herein, and / or hosts as described herein. Kits of the present disclosure are also contemplated to include pharmaceutical compositions as described herein above, alone or in combination with additional drugs to be administered to an individual in need of medical therapy or intervention.

[0088] NKT cells that have been engineered with the construct may then be grown in culture under selective conditions, and cells selected as carrying the construct may then be expanded and analyzed, for example, using the polymerase chain reaction to determine the presence of the construct in the host cells. Once engineered host cells have been identified, they may then be used as planned, for example, to expand in culture or to introduce into a host organism.

[0089] Depending on the nature of the cells, the cells may be introduced into a host organism, e.g., a mammal, in a variety of ways. In alternative embodiments, the cells develop or are engineered to develop cancer, while in specific embodiments, the cells may be introduced at the site of a tumor. The number of cells employed will depend on a number of circumstances, the purpose of the introduction, the lifespan of the cells, the protocol used, e.g., the number of administrations, the ability of the cells to proliferate, the stability of the recombinant construct, etc. The cells may be applied as a dispersion, generally injected at or near the site of interest. The cells may be in a physiologically acceptable medium.

[0090] The introduction of DNA does not necessarily result in integration in all cases.In some situations, the temporary maintenance of introduced DNA may be sufficient.In this way, one may have a short-term effect, in which cells can be introduced into a host, and then stimulated after a certain time, for example, after cells can home to a specific site.

[0091] The cells may be administered as desired. Depending on the desired response, the method of administration, the longevity of the cells, and the number of cells present, various protocols may be employed. The number of administrations will depend, at least in part, on the factors described above.

[0092] It should be appreciated that the system depends on many variables, such as the cellular response to the ligand, the efficiency of expression and, where appropriate, the level of secretion, the activity of the expression product, the particular needs of the patient which may vary with time and circumstances, the rate of loss of cellular activity as a result of cell loss, or the expression activity of individual cells, etc. Thus, even if there were a universal cell that could be administered to the population as a whole, it is expected that for each individual patient, each patient would be monitored for the appropriate dosage for that individual, and such practice of monitoring patients is routine in the art.

[0093] VI. Kits of the Disclosure Any of the compositions described herein may be included in the kit. In a non-limiting example, the kit may include cells or reagents for manipulating cells. In certain embodiments, the kit may include NKT cells or a population of cells containing NKT cells. Such kits may or may not include one or more reagents for manipulating cells. Such reagents include, for example, small molecules, proteins, nucleic acids, antibodies, buffers, primers, nucleotides, salts, and / or combinations thereof. Nucleotides encoding one or more cytokines or the cytokine itself may be included in the kit. Proteins such as cytokines or antibodies, including agonist monoclonal antibodies, may be included in the kit. The kit may include the antibody-containing or bare substrate itself, or in some embodiments, reagents for generating the antibody-bearing substrate. The substrate may be of any type, including beads or plates. Cells with antigen-presenting cell activity or reagents for generating them may be included in the kit. Nucleotides encoding chimeric antigen receptors or T cell receptors, including reagents for generating them, may be included in the kit.

[0094] In certain aspects, the kit includes a cell therapy of the present disclosure and another cancer therapy. In some cases, in addition to the cell therapy embodiment, the kit also includes a second cancer therapy, such as, for example, chemotherapy, hormone therapy, and immunotherapy. The kit may be customized for an individual for a particular cancer and may include each second cancer therapy for the individual.

[0095] A kit may include a suitably aliquoted composition of the present invention. The components of the kit may be packaged in aqueous media or in lyophilized form. The container means of the kit will generally include at least one vial, test tube, flask, bottle, syringe, or other container means into which the components may be placed, preferably into which the components may be suitably aliquoted. Where there is more than one component in the kit, the kit will also generally contain second, third, or other additional containers into which the additional components may be placed separately. However, various combinations of components may be included in a single vial. The kits of the present invention will also typically include a means for containing the composition under close custody for commercial sale and other reagent containers. Such containers may include injection- or blow-molded containers into which the desired vials are retained.

[0096] When the components of the kit are provided in one and / or more than one liquid solution, the liquid is an aqueous solution, with a sterile aqueous solution being particularly preferred. In that case, the container means may itself be a syringe, pipette, and / or other device, such as a syringe, pipette, and / or device from which the formulation may be applied to an infected area of ​​the body, injected into an animal, and / or applied to and / or mixed with the other components of the kit. However, the components of the kit may also be provided as a dry powder. When reagents and / or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is contemplated that the solvent may also be provided in a separate container means. [Example]

[0097] The following examples are included to demonstrate preferred embodiments of the present invention. It should be appreciated by those skilled in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventors to work well in the practice of the invention and can therefore be considered to constitute preferred modes for its practice. However, those skilled in the art should appreciate in light of the present invention that numerous changes can be made in the specific embodiments disclosed and still obtain the same or similar results without departing from the spirit and scope of the invention. Example 1 To identify the NKT cell subset responsible for in vivo persistence and define the conditions required for therapeutic activity and expansion of this subset in culture.

[0098] Adoptive transfer of invariant natural killer cells (NKT) is being developed as a promising therapeutic approach for the immunotherapy of cancer, autoimmune diseases, and other disorders. Because the frequency of NKT cells is low in human peripheral blood, such therapy requires extensive ex vivo expansion of primary NKT cells while preserving their longevity and function. However, the cellular and molecular mechanisms involved in maintaining NKT cells either in vivo or ex vivo remain largely unknown. Here, we demonstrate that in vitro antigen-induced expansion of primary human NKT cells is associated with the progressive accumulation of a CD62L-positive subset in all five individuals examined, regardless of the initial frequency of that subset. Following magnetic sorting of NKT cells into CD62L-positive and CD62L-negative subsets, only CD62L-positive cells survived and expanded in response to TCR stimulation, whereas approximately 90% of CD62L-negative cells underwent apoptosis within 3 days. Furthermore, after adoptive transfer into NSG mice, CD62L-positive NKT cells persisted five times longer than CD62L-negative ones. Importantly, CD62L-positive NKT cells had much higher therapeutic activity and significantly longer survival in xenogeneic lymphoma models. Expanding CD62L-positive cells downregulated or maintained CD62L expression when they were activated by TCR alone or with costimulatory receptors, respectively. In particular, specific combinations of agonistic mAbs against CD3, CD28, and / or 4-1BB enabled stable CD62L expression in in vitro stimulated NKT cells, which correlated with their maximal rate of proliferation and subsequent in vivo persistence. Thus, the results reveal a previously unanticipated functional hierarchy in human NKT cells that can be exploited for their effective ex vivo expansion for cell therapy applications.

[0099] Thus, identified herein is CD62L as a marker for human NKT cells with high proliferative potential and superior therapeutic activity. In vitro stimulatory conditions have been demonstrated that prevent downregulation of CD62L on NKT cells during ex vivo expansion, which is useful for generating NKT cell products with high therapeutic activity. Example 2 CD62L+NKT cells have excellent in vivo persistence and antitumor activity

[0100] CD62L+ cells accumulate in culture upon antigen stimulation of primary NKTs. Previous studies comparing the phenotype of human NKTs in adult peripheral blood with that in umbilical cord blood have found a much higher proportion of CD4+ and CD62L+ NKTs in newborns (Baev et al., 2004; D'Andrea et al., 2000; Eger et al., 2006) (Figure 5A). The predominance of CD4+CD62L+ NKTs in umbilical cord blood suggests that expression of CD4 and / or CD62L indicates a subset of NKTs with superior developmental potential and that may support ex vivo expansion of NKTs for therapeutic applications. To investigate this embodiment, immunophenotyping was performed on primary NKTs immediately after isolation from peripheral blood and at various time intervals in culture after stimulation with αGalCer, which consistently yielded higher frequencies and absolute numbers of NKTs compared to the use of T cell expansion protocols based on CD3 / CD28 stimulation (Figure 6). Despite significant interindividual variability in CD62L expression on freshly isolated NKTs, there was a significant accumulation of the CD62L+ fraction of NKTs, from 33.63% ± 27.62% in freshly isolated NKTs to 69.92% ± 10.57% at day 12 of culture (P < 0.001, Figures 1A and 1B). Although CD62L was more frequently expressed on CD4+ NKTs both before and after culture, the accumulation of CD62L+ cells could not be explained by preferential expansion of CD4+ NKTs. Indeed, at the end of 12 days of culture, the frequencies of CD62L+, CD4+, and CD62L+CD4+ NKTs increased 3.9 ± 1.8, 1.9 ± 0.7, and 3.8 ± 1.9-fold, respectively. Consistent with these results, there was an enrichment of the CD62L+CD4- subset at day 12 compared to day 0 (Figure 5B). Further multiparameter characterization of NKT cells showed that CD62L was frequently coexpressed with CCR7 before culture, but CCR7 expression was gradually lost during culture (Figure 5C). Before culture, nearly all NKT cells expressed both CD27 and CD28. Regardless of CD62L status, CD27 was downregulated in approximately half of NKT cells by day 12 of culture, whereas CD28 expression remained unchanged (Figure 5C).

[0101] CD62L- cells expressed high levels of CD161 and CD56 (indicating NK-like differentiation) but low levels of IL-7Rα (Figure 5D). Freshly isolated NKT cells rarely expressed exhaustion markers (PD-1, LAG-3, or TIM-3) in either the CD62L+ or CD62L- subsets, whereas by day 12 of NKT cell culture, the CD62L- subset preferentially expressed PD-1 and TIM3 (P = 0.0043, 0.0184, Figure 5C). Furthermore, immune-related gene expression analysis (nCounter® platform) of CD62L+ and CD62L- NKTs sorted on day 12 revealed upregulation of mRNA for genes associated with T cell survival / memory (e.g., LEF1, S1PR1, IL-7Rα, IL21R) in CD62L+ NKTs and genes associated with exhaustion / terminal differentiation (PD-1, LAG-3, TIM-3, CD244, CD161, CD56; Figure 1D) in CD62L- NKTs. The transcription factor lymphoid enhancer factor 1 (LEF1) was the top immune-related gene overexpressed in CD62L+ compared with CD62L- NKTs. Intracellular flow cytometry analysis revealed that CD62L+ NKTs uniformly expressed LEF1, whereas a major fraction of CD62L- cells was LEF1-negative. Because LEF1 has recently been shown to mediate the proliferation of mouse NKT cell precursors, in part through transcriptional activation of GATA3 gene expression (Carr et al., 2015), we analyzed GATA3 protein levels in human NKT cells in relation to LEF1 and CD62L levels. GATA3 expression strongly correlated with LEF1 and CD62L expression in human NKT cells (Figure 5E). Interestingly, CD62L+ and CD62L- NKT cells expressed similar levels of PLZF, a master transcriptional regulator of NKT cell functional differentiation (Cohen et al., 2013). Thus, the CD62L+ subset predominantly accumulates in culture upon antigen stimulation of primary NKT cells, and loss of CD62L expression is associated with NK-like terminal differentiation, exhaustion, and downregulation of proliferation-promoting transcriptional regulators.

[0102] CD62L+ NKT cells are Th-0-like cells capable of numerical expansion. Next, NKT cells were magnetically sorted from primary cultures into CD62L+ and CD62L- subsets and their functional characteristics were examined. Figure 2A shows that when target cells were pulsed with αGalCer, both subsets were equally cytotoxic against CD1d+ DAOY medulloblastoma cells (3 of 6 donors), or CD62L- NKT cells were more cytotoxic than CD62L+ NKT cells (3 of 6 donors). Analysis of cytokine production in αGalCer-stimulated NKT cells revealed significantly higher levels of both IFNγ and IL-4 production by the CD62L+ subset compared with the CD62L- subset (P<0.001, Figure 2B). Whereas CD62L+ cells exhibited a Th-0-like polarization (balanced production of IFNγ and IL-4, typical of the entire peripheral blood NKT population), the polarization profile of CD62L- cells could not be clearly determined due to the low absolute amounts of each cytokine. Despite a strong upregulation of IL-23R mRNA expression in the CD62L- subset as determined by nCounter analysis (Figure 1D, potential Th17 polarization), we did not detect IL-23R protein expression on the cell surface of NKT cells by FACS, nor did we detect IL-17 production upon TCR stimulation by ELISA.

[0103] To investigate whether the accumulation of CD62L+ NKT cells during in vitro expansion was due to their preferential survival or proliferation in response to antigen stimulation, we measured the rates of cell death and proliferation after stimulating sorted cells with αGalCer-pulsed antigen-presenting cells. At day 3 post-stimulation, 31% ± 21% and 74% ± 7.5% of CD62L+ and CD62L- cells, respectively, underwent apoptosis (Figure 2C). On day 6, there was a much greater rate of proliferation in the CD62L+ subset compared with the CD62L- subset, as measured by CFSE dilution (Figure 2D). Furthermore, the majority of surviving and proliferating cells in the CD62L- group expressed CD62L, suggesting that these cells were the descendants of a small subset of CD62L+ cells in the original CD62L- fraction. Consistent with these results, there was a striking difference in the number of NKTs generated after 6 days of culture of selected CD62L+ and CD62L- NKTs with IL-2 alone or with TCR stimulation. Indeed, Figure 2E (upper panel) shows that CD62L+ cells underwent a 2.5-fold and 8-fold numerical expansion with IL-2 alone or with TCR stimulation, respectively. In contrast, CD62L- NKTs failed to expand under either condition. Although the extent of NKT cell expansion in response to antigenic stimulation varied among donors, the CD62L- subset contributed little or nothing to NKT cell expansion in all five donors examined (Figure 2E, lower panel). Thus, CD62L+ NKTs survive and proliferate in response to antigenic stimulation and are responsible for the numerical expansion of NKT cells in culture.

[0104] The CD62L+ subset is involved in the in vivo persistence and therapeutic activity of NKT cells. To determine the role of the CD62L+ subset in the in vivo persistence of adoptively transferred NKT cells, we transduced NKT cells with firefly luciferase and magnetically sorted them into CD62L+ and CD62L- subsets. We then adoptively transferred the sorted cells into NSG mice. Longitudinal bioluminescence imaging demonstrated that signals from CD62L- cells were detectable up to 2 days, whereas CD62L+ cells remained detectable up to 10 days (P<0.001, Figure 3A, B). Next, we compared the in vivo therapeutic potential of CD62L+ and CD62L- NKT cell subsets in a model of CAR-redirected immunotherapy for lymphoma. NKT cells were transduced with a CD19-specific CAR (CAR.CD19, Figure 7) containing the 4-1BB costimulatory intracellular domain and then sorted into CD62L+ and CD62L- subsets. NOD / SCID / IL2Rγ(null) (NSG) mice were intravenously injected with luciferase-transduced CD19+ Daudi lymphoma cells. Four days later, mice were divided into two groups receiving either CD62L+ or CD62L- CAR.CD19NKT cells. Both CD62L+ and CD62L- CAR.CD19NKT cells prolonged the survival of treated animals compared with untreated controls (P<0.001). Importantly, only CD62L+ CAR.NKT cells induced sustained tumor regression in seven of the nine surviving treated animals, five of which remained tumor-free for at least three months. In contrast, all 10 mice treated with CD62L-CAR.NKT succumbed to tumor progression (P<0.001, Figures 3C and 3D). Thus, CD62L+NKT cells have prolonged biological persistence and superior therapeutic potential compared to CD62L-NKT cells.

[0105] Costimulation maintains CD62L+ NKTs and prevents their exhaustion. Growing evidence suggests that costimulation plays a role in NKT activation, survival, and proliferation (van den Heuvel et al., 2011). While resting NKTs express CD28 (Figure 5C), they express little or no late costimulatory receptors, such as 4-1BB and OX40 (Figure 8A). However, stimulation of NKTs with autologous PBMCs pulsed with αGalCer resulted in the rapid induction of 4-1BB in all NKTs and OX40 in CD4+ NKTs (Figure 8A). Because CD62L is transiently downregulated within the first 24–48 h of TCR stimulation (data not shown), we analyzed the kinetics of 4-1BB and OX40 expression in CD62L+ and CD62L- NKTs sorted prior to stimulation. We found that 71.13% ± 18.66% and 51.98% ± 18.83% of CD62L+ and CD62L- NKTs upregulated OX40 within 72 hours of stimulation (P = 0.0072, Figure 4A). Similarly, stimulated CD62L+ NKTs expressed higher levels of 4-1BB compared with CD62L- NKTs (P = 0.011, Figure 4A). OX40 was preferentially upregulated in the CD4+ subset of either CD62L+ or CD62L- NKTs, whereas 4-1BB was upregulated in all NKTs (Figure 8B).

[0106] Next, we investigated whether costimulation could counteract the depletion of NKT cells expanded in vitro. We stimulated CD62L+ selected NKT cells on plates coated with the anti-CD3 agonist mAb OKT3 alone or in combination with CD28, 4-1BB, or both. Because we did not have access to an anti-OX40 mAb with agonistic activity, OX40 was not tested in these settings. First, costimulation with anti-CD28, anti-4-1BB, or both increased the number of NKT cells generated within 7 days of culture compared with stimulation with 20 ng / ml anti-CD3 alone (P<0.001, Figure 4B). In the absence of costimulation, increasing the concentration of OKT3 from 20 ng / ml to 1 μg / ml did not affect the number of NKT cells generated, whereas costimulation was effective in increasing the number of NKT cells only when combined with a low concentration of OKT3 (Figure 9). Importantly, seven days after stimulation with OKT3 alone, fewer than half of the NKTs were positive for CD62L. Addition of anti-CD28, anti-4-1BB, or anti-CD28 with anti-4-1BB mAb resulted in the retention of CD62L expression in 58% ± 7.1% (P = 0.026), 73% ± 9.2% (P = 0.0036), and 73% ± 6.1% (P = 0.0002) of the NKTs, respectively (Figure 4C). Consistent with the retention of CD62L expression, NKTs receiving costimulatory signals expressed significantly less PD-1 (P < 0.05, Figure 4D). Thus, engagement of costimulatory receptors during antigen stimulation supports CD62L expression in proliferating NKTs and prevents their depletion.

[0107] Significance of Certain Embodiments of the Disclosure

[0108] Critical gaps in knowledge of human NKT cell biology have slowed the development of effective NKT cell-based cancer immunotherapy. Numerical expansion of NKT cells in vitro and their subsequent persistence in vivo, essential requirements for effective NKT cell-based cell and gene therapy applications, depend on the CD62L+ subset of peripheral blood NKT cells. Only CD62L+ NKT cells survive and proliferate in response to repeated TCR stimulation, while CD62L- cells undergo premature exhaustion and cell death. Continuous stimulation of NKT cells is associated with the loss of CD62L expression, but activation of costimulatory receptors during TCR stimulation can counteract this process. Specifically, we experimentally determined the unique combination of CD86, 4-1BBL, and OX40L molecules, as well as the level of their co-expression on the surface of aAPCs, that maximally preserves CD62L expression and enables highly efficient clinical-scale NKT cell expansion. In certain embodiments, CAR-NKTs generated using aAPCs as encompassed by the present disclosure exhibit long-term in vivo persistence and superior therapeutic activity against in vivo models of lymphoma and neuroblastoma (examples of cancer types).

[0109] The CD62L+ subset is responsible for the numerical expansion of NKT cells upon antigen stimulation in vitro. The following findings support this conclusion: (i) after stimulation of primary NKT cells, the fraction of CD62L+ cells dramatically increases; (ii) sorted CD62L+ cells proliferate in response to the same stimulation, whereas the majority of sorted CD62L- cells undergo apoptosis; (iii) CD62L-NKT cells show signs of terminal differentiation (expression of CD161 and CD56) in freshly isolated PBMCs and rapidly acquire an exhausted phenotype upon in vitro stimulation, as evidenced by a marked upregulation of PD-1 and TIM-3 expression and a reduced ability to produce cytokines. Similar characteristics are observed with therapeutic T cell products, which are associated with a subsequent lack of persistence or objective responses after adoptive transfer into cancer patients (Gattinoni et al., 2005; Klebanoff et al., 2005).

[0110] Proliferating NKTs eventually downregulate CD62L expression and acquire an exhaustion phenotype during in vitro culture. This finding likely reflects the ontogeny of human peripheral blood NKTs, as the frequency of CD62L+ NKTs is lower in adult peripheral blood compared to cord blood (Eger et al., 2006; Der Vliet et al., 2000). One report found that cord blood NKTs do not express CD62L (D'Andrea et al., 2000). The reason for the discrepancy between that report and others and the current results is, in certain embodiments, technical. M. Constantinides et al. identified a very rare, naive-like population of CD1d-restricted T cells in peripheral blood with high levels of CD62L (Constantinides et al., 2011). However, these cells do not express the invariant Vα24 chain and have low levels of PLZF expression compared to conventional NKTs. Both CD62L+ and CD62L- NKTs in this study expressed equally high levels of PLZF.

[0111] CD62L may represent a common marker for cells involved in the long-term maintenance of peripheral NKT cells and T cells. P. Graef et al. demonstrated that CD62L+ central memory T cells possess stem cell properties; they can proliferate while generating effector / memory and effector T cells (Graef et al., 2014). In a recently published study, D. Sommermeyer et al. demonstrated that CD8 or CD4 T cells expressing human CAR.CD19 generated from naive and central memory subsets were more effective against Raji lymphoma xenografts than those generated from effector memory subsets (Sommermeyer et al., 2015). The authors also found that combining the most potent CD4+ and CD8+ CAR-expressing subsets resulted in synergistic antitumor activity in vivo. NKT activation has been shown to result in the transactivation of NK cells and CD8 T cells in mouse models and human clinical trials (Dhodapkar et al., 2009; Vivier et al., 2012), so the combination of CAR NKT with other defined subsets of lymphocytes expressing CARs may be a useful therapeutic strategy.

[0112] LEF1 and IL-7Rα were the most overexpressed immune-related genes in CD62L+ compared with CD62L-NKT cells. In a recent report, Carr et al. revealed a unique function of LEF1 in the proliferation of mouse Vα14 invariant (iNKT) cells during stage 2 of their thymic development through direct transcriptional activation of CD127 and c-myc gene expression (Carr et al., 2015). Consistent with the observation of coordinated expression of LEF1 and GATA3 in human NKT cells, these researchers also showed that LEF1 upregulates the transcription factor GATA3, which is required for the dual production of IL-4 and INFγ in iNKT1 cells as well as IL-4 production in iNKT2 cells. The latter cells closely resemble human peripheral blood NKT cells, where CD62L+ cells were found to preferentially express GATA3 and produce high levels of both cytokines. Consistent with the work of Carr et al. with mouse NKTs and results with human NKTs, in specific embodiments, LEF1 plays a critical role early in NKT cell development, regulating their number and function. The high level of LEF1 expression in the CD62L+ subset of human NKTs and its loss in the CD62L- subset is consistent with a model of linear progression resulting from a Th-0-like cytokine profile toward less differentiated CD62L+ NKTs with preserved proliferative potential and terminally differentiated CD62L- NKT cells with impaired ability to proliferate and produce cytokines.

[0113] Increasing evidence suggests that costimulation plays a crucial role in the development, activation, and functional responses of NKT cells in mouse models (van den Heuvel et al., 2011; Uldrich et al., 2005). However, little is known about the expression of costimulatory receptors in human NKT cells. In this study, we focused on a set of costimulatory receptors with prominent pro-survival properties in human T cells: CD28, 4-1BB, and OX40 (Acuto et al., 2003; Kroczek et al., 2004; Redmond et al., 2009). We first demonstrated that freshly isolated human NKT cells express CD28 (34) and coexpress CD27, thereby resembling the corresponding stage of memory T cell differentiation with preserved functional potential (Okada et al., 2008). This study is the first to characterize the baseline and post-stimulation kinetics of 4-1BB and OX40 in human NKT cells. Both receptors are undetectable in freshly isolated NKTs but are induced following TCR stimulation. Similar to T cells (Croft et al., 2010), human NKTs preferentially upregulated OX40 in the CD4+ subset. The majority of NKTs also upregulated 4-1BB, which is preferentially upregulated in the CD8+ subset of T cells (Lynch, 2008). Importantly, co-stimulation of individual costimulatory receptors was able to inhibit the loss of CD62L expression and rescue NKTs from exhaustion. Co-activation of CD28 and 4-1BB had an additive effect on the maintenance of CD62L+ NKTs. Example 3 Examples of methods and materials

[0114] Cell lines and culture conditions Daudi, Raji, DAOY, Ramos, and 293T cells were purchased from ATCC. Daudi, Raji, and Ramos cells were cultured in RPMI, while DAOY and 293T cells were maintained in IMDM (Invitrogen). Both media were supplemented with 10% FBS (Hyclone) and 2 mM GlutaMAX-1 (Gibco-BRL).

[0115] Isolation, transduction, expansion and selection of NKT cells To analyze NKT cells in umbilical cord blood, discarded cord blood units obtained from the MD Anderson Cancer Center Cord Blood Bank were used in accordance with protocols approved by the Institutional Review Boards of MD Anderson Cancer Center and Baylor College of Medicine. PBMCs from healthy donors (at least 18 years old) were isolated by gradient centrifugation from buffy coats purchased from the Gulf Coast Regional Blood Center. NKT cells were purified using anti-iNKT microbeads (Miltenyi Biotec). The negative PBMC fraction was irradiated (40 Gy) and aliquoted. NKT cells were stimulated with an aliquot of autologous PBMCs pulsed with 100 ng / mL αGalCer (Kyowa Hakko Kirin). Recombinant IL-2 (200 U / ml, National Cancer Institute Frederick) was added every other day to complete RPMI (HyClone RPMI 1640, 10% heat-inactivated fetal bovine serum, and 2 mM Glutamax). NKTs were expanded for 10 days and then restimulated with autologous PBMCs (irradiated at 40 Gy) or, where indicated, Ramos cells (irradiated at 100 Gy) as APCs. Three days after restimulation, 24-well non-tissue culture plates were coated with Retronectin (Takara Bio), washed, and then inoculated with 1 ml of retroviral supernatant containing CAR.CD19 and centrifuged at 4600 G for 60 minutes. The viral supernatant was then removed, and stimulated NKTs were added to the wells in complete medium and 200 U / ml rhIL-2. After 48 hours, cells were removed from the plates, washed, and cultured at 10 6The cells were resuspended in complete RPMI with 200 U / ml IL-2 at a concentration of 1000 cells / ml and plated for continued expansion. NKT cell numbers were determined by trypan blue counting (Life Technologies). Where indicated, NKT or CAR-NKT cells were labeled with CD62L-PE mAb (GREG-56, BD Biosciences) and anti-PE microbeads (Miltenyi) and then magnetically sorted into CD62L+ and CD62L- subsets according to the manufacturer's instructions. The phenotype of the sorted cells was determined by FACS.

[0116] Retroviral constructs and production of retroviruses The CAR.CD19 and CAR.GD2 constructs were generated as previously described (Heczey et al., 2014; Pule et al., 2005). They contained scFvs derived from the CD19-specific antibody FMC-63 or the GD2-specific antibody 14G2a, connected to a transmembrane domain derived from CD8α via a short spacer derived from the hinge region of IgG1, followed by the 4-1BB signaling intracellular domain sequence fused to the ζ chain. Retroviral supernatants were produced by transfecting 293T cells with combinations of plasmids containing chimeric antigens. The RDF plasmid encoded the RD114 envelope and the PegPam3 plasmid encoded the MoMLV gag-pol gene, as previously described (Vera et al., 2006).

[0117] Proliferation and apoptosis assays NKT cells were labeled with CFSE (Invitrogen) and stimulated with αGalCer-pulsed PBMCs or plates coated with 20 ng / ml anti-CD3 (OKT3) alone or in combination with 0.5 μg / ml anti-CD28 (CD28.2) and / or 1.5 μg / ml anti-4-1BB (h41BB-M127) (BD Biosciences). Cell proliferation was assessed on days 3 and 6 by measuring CFSE dilution using flow cytometry. In addition, early and late stage apoptosis was measured on day 3 by staining with Annexin-V and 7-AAD (BD Biosciences) followed by flow cytometry.

[0118] Multiplex cytokine assay NKTs were stimulated with APC or agonist antibody-coated plates (clone 6B11, BD Biosciences) for 24 hours. Supernatants were collected and analyzed with a Human Cytokine / Chemokine Assay Kit (Millipore) using the Luminex® assay according to the manufacturer's instructions.

[0119] Flow cytometry Immunophenotyping was performed using the following mAbs: HLA-C EMR8-5, CD1d CD1d42, CD86 2331, 4-1BBL C65-485, OX40L ik-1, CD3 OKT, Vα24-Jα18 6B11, CD4 SK3, CD62L DREG-56, CD134 ACT35, CD137 4B4-1, PD-1 EH12.1, GATA3 L50-823 (BD ​​Biosciences), LAG-3 polyclonal, TIM-3 344823 (R&D System), and rabbit anti-LEF1 EP2030Y mAb (ABCAM). Fluorochrome- and isotype-matched Abs proposed by BD or R&D were used as negative controls. Expression of CAR.CD19 on NKT cells was measured using anti-Id (clone 136.20.1) CD19-CAR-specific mAb (Torikai et al., 2013) and goat anti-mouse IgG (BD Biosciences). Analysis was performed on an LSR-II5 laser flow cytometer (BD Biosciences) using BD FACSDiva software v.6.0 and FlowJo 7.2.5 (Tree Star).

[0120] In vitro cytotoxicity assay The cytotoxicity of parental NKT and CAR.CD19 NKT against DAOY or Raji cells was assessed using a 4-hour luciferase assay as previously described ( Liu et al., 2013 ).

[0121] Gene expression analysis Total RNA was collected using TRIzol Reagent (Qiagen). Gene expression analysis was performed using the Immunology Panel v.2 (NanoString) at the BCM Genomic and RNA Profiling Core using the nCounter Analysis System. Data were analyzed using nSolver 2.0 software (NanoString).

[0122] In vivo experiments The NSG mouse colony was originally obtained from the Jackson Laboratory and maintained in the BCM animal care facility. Luciferase-transduced Raji lymphoma cells were transfected at 2 × 10 5 Tumor growth was initiated by intravenous injection of 4–8 × 10 cells. 6 Mice were treated with CAR-NKT cells and subsequently received intravenous injections of IL-2 (1000 U / mouse) every 3 days. Tumor growth was assessed twice weekly by bioluminescence imaging (Small Animal Imaging Core facility, Texas Children's Hospital). For in vivo persistence studies, NKT cells were co-transduced with CAR.CD19 and luciferase using a retroviral construct, injected intravenously into tumor-free or tumor-bearing mice, and monitored twice weekly by bioluminescence imaging. Animal experiments were performed in accordance with IACUC-approved protocols.

[0123] Statistics For in vitro and in vivo experiments, we used a paired two-tailed t-test to evaluate continuous variables between two groups, and one-way ANOVA with Bonferroni's post-hoc test to evaluate continuous variables between more than two groups. Survival was analyzed and pairs of groups were compared using the Kaplan / Meier method and the log-rank (Mantel-Cox) test. Statistical data were calculated using GraphPad™ Prism 5.0 (GraphPad Software). Differences were considered significant if p-values ​​were less than 0.05.

[0124] Testing Approval Umbilical cord blood units were obtained from the MD Anderson Cancer Center Cord Blood Bank in accordance with protocols approved by the Institutional Review Boards at MD Anderson Cancer Center (H-16320) and Baylor College of Medicine (H-20911). Written informed consent was received from all participating women prior to enrollment in protocol H-16320. Cord blood units not suitable for clinical use (usually due to low cell counts) were discarded or used for research purposes at Baylor College of Medicine under protocol H-20911. Animal experiments were performed in accordance with protocol AN-5194, approved by the Baylor College of Medicine Institutional Animal Care and Use Committee. Example 4 Effect of IL-21 on NKT cells

[0125] This example demonstrates that IL-21 has a beneficial effect on NKT cells when the cells are exposed to IL-21. Figure 10 shows that IL-21 increases the frequency of CD62L+ NKT cells during primary expansion. NKT cells were isolated from three donors and stimulated with aGalCer-loaded PBMCs and IL-2 (100 U / ml) or IL-2 with IL-21 (10 ng / ml) for 12 days. NKT cells were harvested, stained for CD62L, and then subjected to FACS analysis. Figure 11 demonstrates that IL-21 increases the frequency of CD62L+ NKT cells during secondary expansion. Following primary expansion (Figure 10), NKT cells from three donors were restimulated with aGalCer-loaded PBMCs and IL-2 (100 U / ml) or IL-2 with IL-21 (10 ng / ml) for 12 days. NKTs were harvested and stained for CD62L, followed by FACS analysis. Figure 12 shows the expression of CD1d and costimulatory molecules on Ramos cells. Ramos B-cell lymphoma cells were obtained from ATCC and analyzed by FACS for CD1d, CD86, 4-1BBL, and OX40L using the corresponding mAbs and IgG controls. Ramos cells can expand primary NKTs with high levels of CD62L expression (Figure 13). NKTs were isolated from three donors and cultured on aGalCer-loaded Ramos cells (2 x 10) in medium containing IL-2. 6 NKTs (1 x 10 / well) were stimulated with aGalCer-loaded Ramos cells (2 x 10 / well) in medium containing IL-2 for 10 days. NKTs were harvested, counted, and stained for CD3, 6B11, and CD62L. Finally, Figure 14 shows that Ramos cells significantly retain CD62L expression upon secondary stimulation to expand NKTs. Following primary expansion with PBMCs (as in Figure 10), NKTs (1 x 10 / well) were stimulated with aGalCer-loaded Ramos cells (2 x 10 / well) in medium containing IL-2. 6 The cells were then restimulated with 1000 cells / well for 10 days. NKT cells were harvested, counted, and stained for CD3, 6B11, and CD62L. References

[0126] All patents and publications mentioned in this specification are indicative of the levels of those skilled in the art to which this invention pertains. All patents and publications are herein incorporated by reference in their entireties to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

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Claims

1. A method for preparing human natural killer T (NKT) cells for use in immunotherapy, The steps include isolating human NKT cells to generate an NKT-selected cell fraction and an NKT-depleted cell fraction, at least: One or more cytokines selected from the group consisting of IL-2, IL-21, IL-7, IL-15, IL-12, TNF-alpha, and combinations thereof; and Alpha-galactosylceramide (aGalCer) The steps include: culturing in the presence of the NKT-selected cell fraction to increase the cell volume, Includes, A method wherein the majority of human type I NKT cells in the proliferated NKT-selected cell fraction are CD62L-positive human type I NKT cells.

2. The method according to claim 1, wherein the NKT-selected cell fraction is grown in the presence of irradiated NKT-depleted PBMCs loaded with aGalCer.

3. The method according to claim 1 or 2, wherein the NKT-selected cell fraction is grown in the presence of irradiated aAPCs loaded with aGalCer.

4. The method according to any one of claims 1 to 3, wherein the group of one or more cytokines includes IL-15, IL-21, and / or IL-2.

5. The method according to any one of claims 1 to 4, wherein the NKT-selected cell fraction is grown by culturing it in a medium containing irradiated NKT-depleted PBMCs loaded with IL-15, IL-2, and / or IL-21, and aGalCer.

6. The method according to any one of claims 1 to 5, wherein the one or more cytokines are IL-15.

7. The method according to any one of claims 1 to 6, wherein the irradiated NKT-depleted cells are self to the NKT-selected cell fraction.

8. The method according to any one of claims 1 to 7, wherein the NKT-selected cell fraction is manipulated to express at least one chimeric antigen receptor (CAR).

9. The method according to claim 8, wherein the CAR is specific to GD2.

10. The aforementioned proliferated NKT-selected cell fraction is CD62L ＋ The method according to any one of claims 1 to 9, comprising NKT cells.

11. The method according to any one of claims 1 to 10, wherein the NKT-selected cell fraction is cultured in a medium containing fetal bovine serum.

12. The method according to any one of claims 1 to 11, wherein the irradiated aAPC is a Ramos cell.

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