NKT cell subsets for in vivo survival, therapeutic activity, and proliferation

Abstract

Methods and compositions are provided for generating natural killer T (NKT) cells that are effective in immunotherapy, as well as methods and compositions for providing an effective amount of NKT cells to an individual in need of immunotherapy. The NKT cells are CD62L+ and maintain CD62L expression upon exposure to one or more costimulatory factors. Optionally, the NKT cells may be engineered to incorporate a chimeric antigen receptor.

Description

[Technical Field]

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

[0002] Statements regarding federally supported research or development. This invention was made with government support under RO1CA116548 and P50CA126752, awarded by the National Institutes of Health. The government has specific rights to this invention.

[0003] Field of Invention Embodiments of this disclosure encompass at least areas of medicine, including cell biology, molecular biology, immunology, and 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 TCRα chain Vα24-Jα18 and respond to self- or microbial 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 immunotherapy has been demonstrated in multiple mouse models of cancer and in early clinical trials of 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, NKTs can effectively migrate to tumor sites and intervene in antitumor responses through direct killing of CD1d+ tumor cells, inhibition of tumor-supporting macrophages, or transactivation of NK cells (Metelitsa, 2011). Several studies have shown a strong and clear association between the number of tumor-infiltrating or circulating NKTs and improved disease outcomes in patients with diverse 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 in malignant tumor cells (Dhodapkar et al., 2003). To counteract these tumor evasion mechanisms, a method has been developed to proliferate primary human NKT cells in vitro to clinical scale and redirect their cytotoxicity towards tumor cells through the 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 xenotumor models and the in vivo persistence of CAR NKT cell products (Heczey et al., 2014).However, the mechanisms governing the in vitro proliferation and subsequent in vivo survival of human NKT cells remain largely unknown, hindering the rational design of NKT cell-based cancer immunotherapies.

[0005] Recent global transcriptional profiling studies have demonstrated that NKTs, while sharing characteristics with T cells and NK cells, are a distinctly different population of lymphocytes (Cohen et al., 2013). In mice, the developmental program and functional differentiation of NKTs have been fairly extensively characterized in the last decade, as summarized in recent reviews (Kim et al., 2015; Contantinides and Bendelac, 2013). Several key features of mouse NKTs have also been confirmed in their corresponding human counterparts. In both mice and humans, NKTs branch off from T cells at the CD4+CD8+ (double-positive, DP) thymocyte stage. Unlike T cells, which are positively selected by thymic epithelial cells, NKTs are selected by CD1d-expressing DP thymocytes (Gapin et al., 2001). The expression of promyelocytic leukemia zinc finger transcription factor (PLZF) immediately after positive selection enables the proliferation of NKT cells in the thymus and their effector / memory-like differentiation (Savage et al., 2008). Peripheral NKT cells are long-lived lymphocytes, and their postthymic maintenance largely depends on slow, constitutive 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 little constitutive division in adults compared to the CD4- subset (Baev et al., 2004), suggesting that CD4+ NKT cells may contribute to the long-term persistence of adoptively introduced therapeutic NKT cells under certain conditions. However, for example, in vitro proliferation of human NKT cells in response to antigen stimulation with α-galactosylceramide (αGalCer) produces a similar number of CD4+ and DN NKT cells (28). NKT cells also exhibit NK-like lineage differentiation through the acquisition of CD161 and subsequently CD56 expression. Similar to 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 (Sallusto et al., 2004), which have a well-established developmental hierarchy from naive to central memory, then to effector memory, and finally to ultimate effector cells, NKT cells are widely described as cells with an "activated / memory" phenotype that lacks 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 along with CD62L and CCR7, which lack 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 (despite significant inter-individual variability). Adult NKT cells lack a clear boundary between "memory" and "effector" states because they express altered memory markers and possess rapid-acting 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 revealed that CD62L+ central memory T cells possess stem cell characteristics 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 in NKT remains unknown. In this disclosure, the CD62L+ subset is required for the in vitro proliferation and in vivo persistence of NKT cells. Importantly, when manipulated to express a CD19-specific CAR (CAR.CD19), persistent tumor regression of CD62L+ CAR.CD19 NKT cells, rather than CD62L- NKT cells, occurred in a B-cell lymphoma model in NSG mice. CD62L+ NKT cells can be maintained during in vitro proliferation when provided with a specific costimulatory ligand. This knowledge allows for the manipulation 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. [Overview of the project]

[0008] The methods and compositions of this disclosure relate to immunotherapies for individuals requiring them. In some embodiments, individuals require therapies that target specific antigen-possessing cells, such as cancer cells, for destruction. This disclosure generally provides the use of NKT cells for immunotherapy based on improvements in methods for generating NKT cells in clinically useful quantities and with efficacy.

[0009] Embodiments of this disclosure provide CD62L+ NKT cells with superior in vivo viability and antitumor activity. Embodiments of this disclosure enable the effective proliferation of NKT cells so that they can be used in therapeutic applications. The NKTs of this disclosure have improved viability and proliferation associated with CD62L expression. CD62L expression is present in NKT cells and maintained in the cells due to co-stimulation of NKT cells. Embodiments of this disclosure include co-stimulation of NKT cells by a method for maintaining CD62L expression. NKT cells are exposed to co-stimulation using one or more methods, such as exposure to artificial antigen-presenting cells expressing one or more cytokines (including at least IL-21), one or more agonist antibodies bound to a costimulatory receptor, and / or CD1d, and, for example, one or more costimulatory receptor ligands. Thus, in specific embodiments, artificial antigen-presenting cells can be utilized for the generation of CD62L-enriched NKTs for effective cancer immunotherapy.

[0010] In one embodiment, there is a method for preparing natural killer T (NKT) cells for use in immunotherapy, which includes the step of enriching a population of NKT cells for CD62L-positive NKT cells. In a specific embodiment, CD62L-positive NKT cells are activated by stimulation of a T cell receptor and co-stimulatory stimulation with a co-stimulatory receptor and / or cytokine. In some cases, the method further includes the step of delivering a therapeutically effective amount of cells to an individual in need of treatment. In certain embodiments, 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 a combination thereof.

[0011] In certain embodiments, there is a method for treating an individual for a medical condition using immunotherapy, which includes the steps of (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 the individual with a therapeutically effective amount of CD62L-positive NKT cells.

[0012] In one embodiment, there is a method of treating an individual for a disease condition using immunotherapy, which includes the steps of: enriching a population mixture of CD62+NKT cells and CD62-NKT cells by exposing them to one or more costimulatory factors to co-stimulate CD62+NKT cells and growing CD62+NKT cells from the population mixture to produce CD62+NKT cells; and providing the individual with a therapeutically effective amount of co-stimulated CD62+NKT cells. In a specific embodiment, the stimulatory factors and costimulatory factors include (a) one or more cytokines; (b) a substrate containing an agonist antibody or ligand for a T cell receptor (e.g., recombinant human CD1d with an agonist glycolipid such as OKT3mAb, 6B11mAb, or conjugated alpha-galactosylceramide) and one or more agonist antibodies targeting the costimulatory receptor; or (c) antigen-presenting cells containing CD1d expression and one or more ligands of 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 beads, plates, or gels. In specific embodiments, 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 combinations thereof. The ligand for the costimulatory receptor may be CD80, CD86, OX40L, 4-1BBL, ICOS ligand, CD154, CD30L, or combinations thereof.

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

[0014] In certain embodiments, there are methods for producing NKT cells for immunotherapy, which include the step of co-stimulating a population of NKT cells to maintain CD62L expression in at least some of the NKT cells. In some cases, the method further includes the step of providing a therapeutically effective amount of NKT cells to an individual in need.

[0015] In one embodiment, there is a method for producing NKT cells for immunotherapy, comprising the step of 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 co-stimulatory factors designed to intentionally enrich or retain CD62L+NKT cells. In some cases, the method further comprises the step of obtaining a mixed population. In specific embodiments, the mixed population is derived from individuals to which the enriched population will be delivered. In certain embodiments, the mixed population is derived from individuals different from those to which 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 a substance that includes unnatural cells expressing CD1d and expressing one or more ligands of one or more costimulatory receptors. [Brief explanation of the drawing]

[0017] [Figure 1A](1A~1D) Figures showing the accumulation of the CD62L+ subset in culture of primary NKT cells after antigen stimulation in vitro. (1A) CD62L expression was examined by FACS in primary NKT cells at day 12 after stimulation with αGalCer derived from newly isolated PBMCs (day 0) and growth in vitro in culture. (1B) Dynamics of CD62L expression in NKT cells at the indicated intervals after primary stimulation (as in 1A) derived 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 at day 0 and day 12. Representative figures from one of four donors (upper panel) or mean ± SD of MFI for all donors (lower panel). (1D) On day 12 after primary stimulation, NKT cells were magnetically separated into CD62L+ and CD62L- subsets. Subsequently, RNA was isolated using the Human Immunology Panel v2 and nCounter analysis systems, and gene expression was analyzed. The heatmap shows log-2 changes in genes (CD62+ / CD62L-) with p-values ​​less than 0.02 and mean multiplicative changes greater than 2. Data were generated from six NKT cell donors (12 pairs of 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) Figures showing the functional characterization of CD62L+ and CD62L- NKT cells. (2A) CD1d+ DAOY cells transfected with luciferase were pulsed overnight with PBS (control) or αGalCer, and then co-cultured with magnetically sorted CD62L+ or CD62L- NKT cells. Cytotoxicity was analyzed 4 hours later by measuring luciferase intensity with a plate reader. The left figure is representative of three donors with no difference in cytotoxicity between NKT subsets. The right figure is representative of three donors with a significant difference in cytotoxicity between NKT subsets. (2B) Concentrations of IFNγ and IL-4 were measured in the 24-hour supernatants of CD62L+ or CD62L- NKT cells stimulated with αGalCer by Luminex assay in three independent experiments performed by NKT cells from three donors. (2C) NKT cells labeled with CFSE were magnetically sorted into CD62L+ and CD62L- subsets as confirmed by FACS after sorting (upper panel), and stimulated with irradiated APCs pulsed with αGalCer. Three days after stimulation, after gating on CFSE-positive events, staining for annexin V and 7-AAD on NKT cells was analyzed by FACS. The results are representative of five donors examined (middle panel). The corresponding bar graph (lower panel) shows the mean ± SD of the percentage of annexin V+ NKT on day 3 (N = 5). (2D) Cell proliferation was evaluated on day 6 after stimulation as measured by CFSE dilution. The results are representative of five donors examined (upper panel), and are the mean ± SD of the MFI of CFSE for all five donors (lower panel). (2E) Total cell counts were performed at the indicated time intervals after stimulation of NKT cells. Shown are the mean ± SD of viable cells of a representative donor (upper panel) or the fold change for each of the five donors examined on day 6 after stimulation. ***P < 0.001, corresponding 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) Figures showing that CD62L+ NKT cells have excellent in vivo viability and antitumor activity. (3A) Transduced NKT cells with luciferase were sorted into CD62L+ and CD62L- subsets and injected into NSG mice. In vivo viability of NKT cells was monitored by bioluminescence imaging. (3B) Mean ± SD (P=0.008, paired t-test) of bioluminescence photon counts at the number of days after injection of CD62L+ or CD62L- NKT cells. (3C) Each mouse was given an intravenous injection of 2 × 10⁵ Daudi lymphoma cells transduced with luciferase (day 0), followed by an intravenous injection of 10⁷ CAR.CD19 transduced NKT cells with IL-2 (1000 U / mouse) or PBS as a control (day 4). Tumor growth was monitored weekly using bioluminescence imaging. The survival probability was analyzed using the (3D) Kaplan-Meier method (10 mice per group). Then, a log-rank test was used to compare the differences in survival. [Figure 3B] Same as above. [Figure 3C] Same as above. [Figure 3D] Same as above.

[0020] [Figure 4A](4A~4D) This figure shows that simultaneous stimulation maintains CD62L+NKT cells and prevents depletion. (4A) NKT cells were sorted into CD62L+ and CD62L- subsets, stimulated with αGalCer, and the expression of 4-1BB and OX40 was examined by FACS before stimulation and 3 days later. The figures shown are representative from four donors. (4B) CD62L+NKT cells were stimulated using plates coated with the agonist mAb shown. The figures show the mean ± SD (N=4) change in the absolute number of NKT cells at day 7 after stimulation compared to day 0. P<0.001, one-way ANOVA. (4C) CD62L+NKT cells were stimulated in the same manner as in B, and the expression of CD62L (black) compared to the isotype control (gray) was analyzed at day 7. The figures show representative overlay histograms (upper panel) and the mean ± SD (N=4) percentage of CD62L+ cells. (4D)CD62L+NKT cells were stimulated in the same manner as in cell B, and PD-1 expression (black) was analyzed on day 12 in comparison to isotype control (gray). The representative overlay histograms (upper panel) and the mean ± SD of the percentage of PD-1+ cells are shown. **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) Figures show the phenotypic analysis of newly isolated and in vitro grown NKT cells. (5A) Expression of CD4 and CD62L was examined by FACS in newly isolated primary NKT cells (gated to a subset of CD3+Vα24-Jα18+) in umbilical cord blood mononuclear cells (CBMCs). The figure is from representatives of 5 CBMC donors. (5B) Expression of CD4 and CD62L was examined in primary NKT cells before αGalCer stimulation and in vitro growth (day 0) and after 12 days, after being gated as shown in A. The figure is from representatives of 10 PBMC donors. (5C) Expression of CCR7, CD27, and CD28 in relation to CD62L expression in primary NKT cells before αGalCer stimulation and in vitro growth (day 0) and after 12 days, after being gated as shown in A. The figure is from representatives of 6 PBMC donors. (5D) Expression of CD161, CD56, and IL7Rα in relation to CD62L expression 12 days after stimulation with αGalCer and in vitro proliferation. Figures are from representatives of three PBMC donors. (5E) Expression of PLZF, LEF1, and GATA3 in relation to CD62L expression, as well as co-expression of LEF1 and GATA3, was analyzed using intracellular flow cytometry 12 days after stimulation with αGalCer and in vitro proliferation. Figures are from representatives 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) This figure compares the purity and absolute number of NKT cells after proliferation with CD3 / CD28 agonist mAbs compared to PBMCs pulsed with aGalCer and irradiated with radioactive materials (PBMCs). NKT cells were isolated from PBMCs of four individuals. Half were stimulated using autologous PBMCs pulsed with aGalCer and irradiated with radioactive materials, and the other half were stimulated using plates coated with CD3 / CD28 mAbs. In both cases, cells were grown in culture with IL-2 (200 U / ml) added every other day. The cultures were analyzed on day 12. (6A) The purity of NKT cells was determined by flow cytometry as the percentage of cells expressing CD3 and iTCRα. (6B) The absolute cell count of NKT cells was performed using a trypan blue exclusion assay in triplicates. *P<0.05, data were analyzed after Log(2) transformation using paired t-tests. [Figure 6A-2] Same as above. [Figure 6B] Same as above.

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

[0024] [Figure 8A](8A~8B) Figures show the expression of costimulatory receptors on quiescent and activated NKT cells. (8A) FACS analysis of CD4-related OX40 and 4-1BB expression in quiescent NKT cells (12 days after primary stimulation) and NKT cells 3 days after restimulation with αGalCer. Figures are from representatives of 6 PBMC donors. (8B) Analysis of CD4-related OX40 and 4-1BB expression in magnetically sorted CD62L+ and CD62L- NKT cells 3 days after restimulation with αGalCer. Figures are from representatives of 4 PBMC donors. [Figure 8B] Same as above.

[0025] [Figure 9] This figure compares the proliferation of NKT cells using plates bound to low and high concentrations of OKT3mAb. NKT cells, grown in vitro and then resting, were stimulated either alone with 20 ng / ml or 1 μg / ml of anti-CD3 OKT3mAb, or with 500 ng / ml of anti-CD28 CD28.2mAb. Cells were cultured with IL-2 (200 U / ml) added every other day. On day 12, the absolute cell count of NKT cells was performed using a triplicate trypan blue exclusion assay and separated by the number of cells introduced on day 0. Data are M±SD, N=4. **P=0.01, paired t-test.

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

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

[0028] [Figure 12-1] This figure shows an example of CD1d and co-stimulatory molecule expression in Ramos cells. [Figure 12-2] Same as above.

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

[0030] [Figure 14] This figure shows that Ramos cells proliferate NKT cells upon secondary stimulation accompanied by significant retention of CD62L expression. [Modes for carrying out the invention]

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

[0032] As used herein, “a” or “an” may mean one or more. As used herein in claims, when used in conjunction with the word “comprising,” the word “a” or “an” may mean one or more than one. As used herein, “another” may mean at least two or more. In specific embodiments, aspects of the present invention may, for example, “essentially consist” of one or more arrangements of the present invention. Some embodiments of the present invention may, or essentially consist of one or more elements, process steps and / or methods of the present invention. It is considered that any method or composition described herein may be carried out in connection with other methods or compositions described herein. The scope of this application is not intended to be limited to specific embodiments of the processes, machines, manufactures, composition of substances, means, methods and processes described herein.

[0033] I. General Embodiments This disclosure provides NKT cells suitable for use in immunotherapy because they can proliferate to sufficient levels and achieve sustained therapeutic effects at sufficient levels in vivo. The NKT cells of this disclosure are engineered to express and maintain CD62L, which gives them at least some degree of high therapeutic applicability. Such maintenance of CD62L expression in NKT cells occurs at least to some extent during co-stimulation, including exposure to one or more co-stimulatory factors.

[0034] II. NKT cells and their simultaneous stimulation In certain embodiments, NKT cells are useful in therapeutic applications because they have improved in vitro proliferation and in vivo viability following exposure to one or more costimulators that enable the cells to maintain CD62L expression. The one or more costimulators may be of any kind, but in specific embodiments, they include (b) a substrate (e.g., beads, plates, etc.) containing one or more agonist antibodies targeting a costimulatory receptor; and / or (c) a cell such as an antigen-presenting cell that includes the expression of CD1d and the expression of one or more ligands of one or more costimulatory receptors. Where NKT cells are exposed to cytokines, the cytokines may be of a preferred type, but in specific cases, the cytokines are 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] When NKT cells are exposed to one or more costimulators, which are agonist antibodies (at least in some cases monoclonal) that immunologically recognize costimulatory receptors, the receptor may be a costimulatory receptor. However, in specific embodiments, the receptor may be, 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 so that a population of cells, for example, a population of NKT cells, is sufficiently exposed to the antibody. The antibody may be commercially available, obtained as a gift, or manufactured by means standard in the art.

[0036] When NKT cells are exposed to a therapeutically effective amount of cells having antigen-presenting cell activity, such as artificial antigen-presenting cells (e.g., non-natural cells with antigen-presenting cell activity), the cells may be transduced with one or more polynucleotides to express one or more ligands of one or more costimulatory receptors. The cells may be of any kind, as long as they express one or more ligands of one or more costimulatory receptors, but at least in some cases the cells also express CD1d. In certain embodiments, the cells are antigen-presenting cells. In specific cases, cells expressing CD1d and / or one or more ligands of one or more costimulatory receptors are naturally occurring and may be used in the manner incorporated herein. In other cases, cells that do not naturally express CD1d and / or do not naturally express one or more ligands of one or more costimulatory receptors are transduced to express each component and used in the manner incorporated herein. The ligand for the co-stimulatory receptor may be of any type, but in specific embodiments, the ligand may be CD80, CD86, 4-1BBL, OX40L, ICOSL, CD30L, GITRL, TIM4, LIGHT, etc. When cells having antigen-presenting cell activity are transduced with one or more polynucleotides, the polynucleotide may be contained in a vector that includes a viral vector or a non-viral vector (e.g., a plasmid). Examples of viral vectors include retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, etc. The vector will contain suitable regulatory elements for expression in cells having antigen-presenting cell activity. In some cases, the polynucleotide transduced into cells having antigen-presenting cell activity may encode two or more coding regions, for example, encoding two co-stimulatory receptor ligands. In such cases, separate coding regions may or may not be regulated by the same regulatory element.

[0037] In embodiments where CD62L expression in NKT cells is sustained due to co-stimulation of NKT cells, there may or may not be a general sequence in the steps for preparing 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., sorting with magnetic beads or FACS) and exist as a mixed population of cells. Subsequently, NKT cells are activated via stimulation of the TCR using a substrate containing an agonist antibody or ligand against the T cell receptor (e.g., recombinant human CD1d with a conjugated agonist glycolipid such as OKT3mAb, 6B11mAb, or alpha-galactosylceramide), or antigen-presenting cells expressing CD1d and a conjugated agonist glycolipid such as alpha-galactosylceramide. TCR-activated NKT cells may be exposed to co-stimulation to produce a population of CD62L+ NKT cells at a higher level than that produced in the absence of co-stimulation. In some embodiments, prior to delivery to an individual requiring it and following exposure to one or more costimulators, NKT cells are manipulated by recombinant means to incorporate one or more features, for example, expressing one or more therapeutic agents or therapeutic entities that make the NKT cells therapeutic. In specific embodiments, cells are genetically engineered to give them the ability to target antigen-carrying cells. In specific embodiments, the manipulation involves transduction of NKT cells to express one or more chimeric antigen receptors and / or T cell receptors, or those that target a specific antigen of interest. In specific embodiments, the antigen is a tumor antigen.

[0038] NKT cells used to treat a disease in an individual may originate from the individual to which they are administered, from another individual, or they may be obtained from a cell deposit. In a specific embodiment, the NKT cells are type 1 NKT cells.

[0039] NKT cells may or may not be sorted prior to delivery to an individual. In specific embodiments, CD62L-positive NKT cells are not sorted from CD62L-negative NKT cells, but in alternative embodiments, they may be sorted. For example, if cells are not sorted based on whether they express CD2L, and the cells are not sorted by physical separation, they can be enriched using co-stimulation of cells that result in the maintenance of CD62L expression.

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

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

[0042] Genetic manipulation of NKT cells can be performed by humans, and in certain embodiments, the genetic manipulation allows the cells to specifically target one or more cancer cells, for example, cancer cells expressing a particular antigen. In specific embodiments, the manipulation provides NKT cells with a specific non-native receptor for a particular cancer cell. The receptor may be of any kind, 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 for MHC / peptide complexes derived from unmutated tumor-associated antigens (e.g., Survivin, MYCN, NY-ESO1, MAGE, PRAME, WT1, etc.) or patient-specific mutated tumor antigens, as revealed by tumor DNA sequencing.

[0044] In some cases, NKT cells are engineered to express CARs. Genetic engineering of NKT cells to express tumor-directed chimeric antigen receptors (CARs) can produce anti-tumor effector cells that circumvent tumor immune evasion mechanisms, which 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 this disclosure, there are NKT cells engineered to contain at least one CAR. In specific embodiments, certain NKT cells contain the expression of two or more CARs.

[0045] This 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 embodiments of this disclosure, it is specific to a cancer antigen. The 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, NKT cells are chimeric, non-natural, and have been manipulated at least to some extent by human hands, and contain a CAR directed to a specific cancer antigen of interest. In certain cases, the manipulated CAR has one, two, three, four or more components, and in some embodiments, one or more components promote the targeting or binding of NKT cells to cancer cells containing the cancer antigen. In specific embodiments, the CAR includes an antibody against the cancer antigen, some or all of a cytoplasmic signaling domain, and / or one or more costimulatory molecules, e.g., some or all of the intracellular domain of a costimulatory molecule. In specific embodiments, the antibody is a single-chain variable fragment (scFv). In certain embodiments, the antibody is directed, for example, to the cancer antigen on the cell surface of a cancer cell expressing the antigen of interest. In certain embodiments, a cytoplasmic signaling domain, such as one derived from the T cell receptor ζ chain, is employed as at least part of the chimeric receptor to generate a stimulating signal for NKT cell proliferation and effector function following binding of the chimeric receptor to the target antigen. Examples 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 mediated by CAR after antigen binding. In specific embodiments, 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 that links the antigen-recognition domain to the transmembrane domain. The antigen-recognition domain will generally contain a single-chain variable fragment (scFv) specific to a particular cancer antigen. However, if there are two or more CARs in the same cell, the second CAR may contain an scFv specific to another particular antigen. Examples of cancer antigens include, for example, melanoma-associated antigen (MAGE), melanoma-preferentially expressed antigen (PRAME), CD19, CD20, CD22, κ-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, and HLA-A2. Examples include NY-ESO-1, PSC1, folate receptor-a, CD44v6, CD44v7 / 8, 8H9, NCAM, VEGF receptor, 5T4, fetal AchR, NKG2D ligand, or one of the following: CD44v6.

[0048] Examples of hinge regions for extracellular domains include the CH2CH3 region of immunoglobulins, the hinge region derived from IgG1, and parts of CD3. In some cases, the transmembrane region is CD28, but it can be of any kind.

[0049] Generally, the intracellular domains of the CARs of this disclosure are utilized for signal transduction in cell and receptor clusters after antigen recognition. The most commonly used intracellular domain component is the 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 the CD3 zeta in combination with CD28, 4-1BB, and / or OX40.

[0050] IV. General Cells The cells of this disclosure include both CD62L-positive NKT cells co-stimulated by one or more costimulators, as well as specific costimulators (which may be called non-native cells having antigen-presenting cell activity) that are antigen-presenting cells themselves. In some embodiments, there is a composition of substance which is a non-native cell expressing CD1d and an artificial antigen-presenting cell expressing one or more ligands of one or more costimulatory receptors.

[0051] When used herein, the terms “cell,” “cell line,” and “cell culture” may be used interchangeably. All of these terms also include their offspring, which are any and all subsequent generations. It is understood that not all offspring may be identical due to intentional or unintentional mutations. In the context of expressing heterologous nucleic acid sequences, “host cell” may refer to a prokaryotic or eukaryotic cell, including transformable organisms that can replicate a vector and / or express the heterologous gene encoded by the vector. Host cells can and are used as recipients for vectors. A host cell may be “transfected” or “transformed,” which refers to the process by which an exogenous nucleic acid may be transfected or introduced into a host cell. Transformed cells include primary target cells and their offspring. When 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. Recombinant cells can thus be distinguished from naturally occurring cells that do not contain recombinantly introduced nucleic acids.

[0052] In certain embodiments, it is considered that the RNA or protein sequence may be co-expressed with other selected RNA or protein sequences in the same host cell. Co-expression may be achieved by simultaneously translocating the host cell with two or more different recombinant vectors. Alternatively, a single recombinant vector may be constructed to contain multiple different coding regions for the RNA, and then expressed in the host cell translocated with the single vector.

[0053] Some vectors may employ regulatory sequences to ensure they are replicated and / or expressed in both prokaryotic and eukaryotic cells. Those skilled in the art will further understand the conditions under which all of the aforementioned host cells can be incubated and maintained, and which allow for vector replication. Also understood and known are techniques and conditions that enable the production of nucleic acids encoded by vectors and their related polypeptides, proteins, or peptides, as well as the large-scale production of vectors.

[0054] Although prokaryotic cells may be employed for the recombination operations of vectors or DNA incorporated into vectors, the cells used in this disclosure are eukaryotic cells, including mammalian cells. The cells are particularly human, but may relate to the animals of interest, especially livestock such as horses, cattle, mice, sheep, dogs, cats, etc., for use in each animal.

[0055] Cells can be, for example, self-cells, syngeneic cells, allogeneic cells, and, in some cases, heterogeneic cells, in relation to the individual receiving the cells. Cells may be manipulated by altering the properties of their major histocompatibility complex (MHC), such as by inactivating β2-microglobulin to prevent the formation of functional class I MHC molecules, thereby inactivating class II molecules, providing expression of one or more MHC molecules, or enhancing or inactivating cytotoxic performance by increasing or inhibiting the expression of genes related to cytotoxic activity.

[0056] An expression vector encoding a CAR can be introduced into cells as one or more DNA molecules or constructs, which may include at least one marker to allow selection of host cells containing the construct. The construct can be prepared by conventional methods, in which the gene and regulatory regions may be isolated, ligated by restriction or sequencing or other means as appropriate, cloned into a suitable cloning host, and analyzed. In particular, individual fragments containing all or part of the functional unit may be isolated by PCR, in which one or more mutations may be introduced using primer repair, ligation, or in vitro mutagenesis as appropriate. Once completed and demonstrated to have a suitable sequence, the construct may then be introduced into a CTL by conventional means. For cell infection or transduction, the construct may be incorporated into or packaged in a non-replicating incomplete viral genome, such as an adenovirus, adeno-associated virus (AAV), or herpes simplex virus (HSV), including a retroviral vector. The construct may optionally contain a viral sequence for transduction. Alternatively, the construct may be introduced by fusion, electroporation, gene gun, transfusion, lipofectin, etc. Host cells may be cultured and grown before the introduction of the construct, and then appropriate treatment for construct introduction and integration is performed. The cells are then grown and screened for markers present in the construct. Various markers that may be used effectively include HPRT, neomycin resistance, thymidine kinase, and hygromycin resistance.

[0057] In many situations, it may be desirable to be able to kill manipulated cells, in which case the person would want to terminate the treatment, and in studies, the absence of the cells after their presence is the target or other event, where the cells become tumorous. For this purpose, a person can provide the expression of a specific gene product that allows the person to kill manipulated cells under controlled conditions. Suicide gene products such as caspase-9 are examples of such products.

[0058] As an example, patients with cancer, or those susceptible to cancer, or suspected of having cancer, may be treated as follows: Manipulated cells as described herein may be administered to the patient and retained for an extended period. The individual may receive one or more doses of cells. The cells will be manipulated and provided to individuals who need them.

[0059] V. Polynucleotide This disclosure also includes compositions comprising nucleic acid sequences encoding antigen-specific CARs as defined herein, and cells containing such nucleic acid sequences. In certain embodiments, the nucleic acid sequence is a recombinant nucleic acid sequence and may be synthetic. It may comprise DNA, RNA, as well as PNA (peptide nucleic acid), and may be a hybrid thereof.

[0060] Furthermore, for further purposes, it is conceivable that the nucleic acid molecule may contain, for example, thioester bonds and / or nucleotide analogs. The operation may be useful for stabilizing the nucleic acid molecule against endonucleases and / or exonucleases in cells. The nucleic acid molecule may be transcribed in cells by a suitable vector containing a chimeric gene that enables transcription of the nucleic acid molecule. In this regard, it should be understood that such polynucleotides may also 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 and include, for example, Southern blotting and Northern blotting, PCR, or primer extension. This embodiment may be useful as a screening method for verifying the successful introduction of the above-mentioned nucleic acid molecule among gene therapy approaches.

[0061] The nucleic acid molecule may be a recombinant chimeric nucleic acid molecule containing any of the aforementioned nucleic acid molecules, either individually or in combination. In specific embodiments, the nucleic acid molecule is part of the vector.

[0062] Accordingly, this disclosure also relates to compositions comprising vectors containing nucleic acid molecules described herein.

[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, 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 this disclosure can be reconstituted into liposomes for delivery to target cells. Individual sequences of DNA may be isolated using cloning vectors. Sequences relevant to expression vectors to which the expression of a specific polypeptide is desired can be transferred. Typical cloning vectors include pBluescript SK, pGEM, pUC9, pBR322, and pGBT9. Typical expression vectors include pTRE, pCAL-n-EK, pESP-1, and pOP13CAT.

[0064] In specific embodiments, there are vectors containing a nucleic acid sequence that is a regulatory sequence operably ligated to a nucleic acid sequence encoding an antigen-specific CAR as defined herein. Such regulatory sequences (control elements) are known to experts and may include promoters, splice cassettes, translation start codons, and translation and insertion sites for introducing inserts into the vector. In specific embodiments, the nucleic acid is operably ligated to the expression regulatory sequence that enables expression in eukaryotic or prokaryotic cells.

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

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

[0067] The term “operably ligated” refers to a juxtaposition in which components described in this way are related in a way that enables them to function in the intended manner. A control sequence “operably ligated” to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequence. It will be obvious to those skilled in the art that double-stranded nucleic acids are preferably used when the control sequence is a promoter.

[0068] Therefore, the vector cited is, in certain embodiments, an expression vector. An "expression vector" is a construct that can be used to transform a selected host and provide expression of a coding sequence in the selected host. An expression vector can be, for example, a cloning vector, a binary vector, or an embedded vector. Expression involves transcription of a nucleic acid molecule into preferably translatable mRNA. Regulatory elements that ensure expression in prokaryotic and / or eukaryotic cells are well known to those skilled in the art. In the case of eukaryotic cells, they typically include a promoter that ensures the initiation of transcription and, optionally, a poly(A) signal that ensures the termination of transcription and stabilization of the transcript. Possible regulatory elements that enable expression in prokaryotic host cells include, for example, P in E. coli. L Examples of regulatory elements that enable expression in eukaryotic host cells, including lac, trp, or tac promoters, are the AOX1 or GAL1 promoter in yeast, or the CMV-, SV40-, RSV- promoters (Roussarcoma virus), CMV-enhancer, SV40-enhancer, or globin introns in mammalian and other animal cells.

[0069] For example, elements involved in the initiation of transcription, such as regulatory elements, may also include transcription termination signals downstream of the polynucleotide, such as the SV40-poly-A site or the tk-poly-A site. Furthermore, depending on the expression system used, a leader sequence that can direct the polynucleotide to a cellular compartment or secrete it into the culture medium may be added to the coding sequence of the cited nucleic acid sequence, as is well known in the art. The leader sequence is prepared in a suitable phase with translation start and termination sequences, and preferably the leader sequence can direct the secretion of the translated protein or a portion thereof into the pericellular lumen or extracellular culture medium. Optionally, heterologous sequences may encode a fusion protein containing an N-terminal specific peptide that confers a desired feature, such as stabilization or simplified purification of the expressed recombinant product. (See above.) In this context, suitable expression vectors known in this technique include, for example, 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 regulatory sequence is a eukaryotic promoter system in a vector capable of transforming or translocating a eukaryotic host cell, although a prokaryotic host regulatory sequence may also be used. Once the vector is incorporated into a suitable host, the host is maintained under conditions favorable for high levels of nucleotide sequence expression, and recovery and purification of the polypeptide of the Disclosure may follow as desired. In certain embodiments, one or more encodingable sequences are regulated by expression regulatory sequences that are responsive to a hypoxic environment.

[0071] Additional regulatory elements may include translational enhancers as well as transcriptional enhancers. Advantageously, the vectors described above in this disclosure include selectable and / or scoreable 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 a basis for selection of dhfr (Reiss, Plant Physiol. (Life-Sci. Adv.) 13 (1994), 143-149) for conferring resistance to methotrexate, npt (Herrera-Estrella, EMBO J. 2 (1983), 987-995) for conferring resistance to the aminoglycosides neomycin, kanamycin, and paromycin, and hygro (Marsh, Gene, 32 (1984), 481-485) for conferring resistance to hygromycin. Additional selectable genes have been described, namely trpB, which causes cells to utilize indole instead of tryptophan; hisD (Hartman, Proc. Natl. Acad. Sci. USA, 85(1988), 8047), which causes cells to utilize histinol instead of histidine; mannose-6-phosphate isomerase (WO94 / 20627), which causes cells to utilize mannose; and ornithine decarboxylase inhibitors such as ODC (ornithine decarboxylase) (McConlogue, 1987, In: Current Communications in Molecular Biology, Cold Spring Harbor Laboratory ed), which confers resistance to DFMO, or deaminase derived from Aspergillus terreus (Tamura, Biosci. Biotechnol. Biochem. 59(1995), 2336-2338), which confers resistance to blasticidine S.

[0072] Useful scoreable markers are known to those skilled in the art and are commercially available. Advantageously, these markers are genes 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 vector.

[0073] As described above, the cited nucleic acid molecules can be used in cells alone 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 one of the specific CAR constructs is then introduced into a cell that produces the target polypeptide. The cited nucleic acid molecules and vectors may be designed for direct introduction into cells or for introduction via liposomes or viral vectors (e.g., adenovirus, retrovirus).

[0074] As described above, this disclosure relates to methods for moving vectors, particularly plasmids, cosmids, viruses, and bacteriophages, conventionally used in genetic engineering, including nucleic acid molecules encoding antigen-specific CAR polypeptide sequences as 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, herpesviruses, or bovine papillomavirus may be used to deliver the cited polynucleotides or vectors to a targeted cell population. Recombinant vectors can be constructed using methods well known to those skilled in the art; see, for example, Sambrook et al. (loc cit.), Ausubel (1989, loc cit.) or other standard textbooks. Alternatively, the cited nucleic acid molecules and vectors can be reconstituted into liposomes for delivery to target cells. Vectors containing the nucleic acid molecules of this disclosure can be transferred to host cells by well known methods, which vary depending on the type of cell host. For example, calcium chloride translocation is commonly used for prokaryotic cells, while calcium phosphate treatment or electroporation may be used for other cell hosts; see Smbrook, above.

[0075] XII. Pharmaceutical Compositions According to this disclosure, the term “pharmaceutical composition” refers to a composition for administration to an individual. In certain embodiments of this disclosure, the pharmaceutical composition comprises a plurality of NKT cells. In preferred embodiments, the pharmaceutical composition comprises a composition for parenteral, transdermal, intracavitary, intra-arterial, subarachnoid or intravenous administration, or direct injection into cancer. It is particularly assumed that the pharmaceutical composition is administered to an individual via infusion or injection. Administration of a preferred composition may be achieved by a variety of methods, for example, by intravenous, subcutaneous, intraperitoneal, intramuscular, topical or intradermal administration.

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

[0077] The administration plan will be determined by the attending physician and clinical factors. As is well known in medical technology, the dosage for one patient depends on numerous 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-operated NKT may be administered via intravenous infusion. The dose is 1 × 10⁻⁶. 7 / m 2 ~2×10 8 / m 2 It can be within the range, and in specific embodiments, 10 9 You may use up to one cell.

[0078] The compositions of this disclosure may be administered topically or systemically. Administration is generally parenteral, for example, intravenously; DNA may also be administered directly to a target site, for example, by gene gun delivery to an internal or external target site, or by catheterization to an intra-arterial site. In preferred embodiments, the pharmaceutical composition is administered subcutaneously, and in even more preferred embodiments, 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 organic esters for injection such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions containing saline and buffer media. Parenteral vehicles include sodium chloride solutions, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution, or fixative oils. Intravenous vehicles include fluid and nutrient replacement solutions, electrolyte replacement solutions (for example, those based on Ringer's dextrose), etc. For example, preservatives such as antimicrobial agents, antioxidants, chelating agents, and inert gases, as well as other additives, may be present. In addition, the pharmaceutical compositions of this disclosure may contain protein carriers, for example, preferably human serum albumin or immunoglobulin. In addition to the CAR construct or nucleic acid molecules or vectors encoding it (as described in this disclosure), the pharmaceutical compositions of this disclosure may further contain biologically active activators depending on the application of the pharmaceutical composition.

[0079] XII. Therapeutic use of NKT cells For example, patients with cancer, or those susceptible to cancer, or suspected of having cancer, may be treated as described herein. NKT cells engineered as described herein may be administered to an individual and retained for an extended period. An individual may receive one or more doses of cells. In some embodiments, genetically engineered cells are encapsulated to inhibit immune recognition and positioned at the tumor site.

[0080] In various embodiments, expression constructs, vectors containing them, host cells, and / or pharmaceutical compositions are used to prevent, treat, or improve cancerous diseases such as neoplastic diseases. In certain embodiments, the pharmaceutical compositions of this disclosure may be particularly useful in preventing, improving, and / or treating cancers, for example, cancers having solid tumors.

[0081] As used herein, “treatment” or “to treat” includes beneficial or desirable effects on the symptoms or condition of a disease or pathological state, and may include even a minimal reduction in one or more measurable markers of the disease or condition being treated, such as cancer. Treatment may optionally involve the reduction or improvement of the symptoms of the disease or condition, or the delay of the progression of the disease or condition. “Treatment” does not necessarily imply the complete elimination 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 to prevent, suppress, or reduce the likelihood of a disease or condition, such as cancer, developing or recurring. It also means 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 its onset or recurrence.

[0083] In certain embodiments, the Disclosure considers to some extent cells containing expression constructs, nucleic acid molecules, and / or vectors that can be administered, either alone or in combination with other therapeutic agents, in at least some aspects, together with pharmaceutically acceptable carriers or excipients. In certain embodiments, the nucleic acid molecules or vectors may be stably incorporated into the cell genome prior to administration of the cells. In specific embodiments, viral vectors that are specific to a particular cell or tissue and persist in said cells may be used. Suitable pharmaceutical carriers and excipients are well known in the art. Compositions prepared in accordance with the Disclosure can be used for the prevention, treatment, or delay of the diseases specified above.

[0084] Furthermore, this disclosure relates to a method for preventing, treating or improving cancerous (including neoplastic) diseases, comprising the step of administering to a subject requiring such treatment an effective amount of cells containing an antigen-recognizing moiety molecule and a chemotherapy resistance molecule, a nucleic acid sequence encoding the same, and a vector encoding the same, manufactured by a process as described herein and / or described herein.

[0085] Possible indications for the administration of example compositions of manipulated immune cells include cancers of the breast, prostate, lung, and colon, or epithelial cancers / carcinomas, such as mammary gland cancer, breast cancer, colon cancer, prostate cancer, head and neck cancer, skin cancer, cancers of the urinary / genital tract, such as ovarian cancer, endometrial cancer, cervical cancer, and kidney cancer, lung cancer, gastric 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. Example indications for the administration of compositions of cells include cancers, such as malignancies expressing specific antigens. In addition, this includes malignancies abnormally expressing other tumor antigens, which may also be targeted. Administration of the compositions of this disclosure is useful for cancer at all stages and of all types, including minimal residual disease, early-stage cancer, advanced cancer, and / or metastatic cancer and / or refractory cancer.

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

[0087] Embodiments relate to a kit comprising one or more NKT cells as described herein, a nucleic acid sequence as described herein, a vector as described herein, and / or a host as described herein. The kits of this disclosure may also be considered to include the pharmaceutical compositions described herein alone or in combination with further drugs administered to an individual requiring pharmacotherapy or intervention.

[0088] NKT cells manipulated with the construct are then grown in culture under selective conditions, and the cells selected to possess the construct are then grown and analyzed, for example, using polymerase chain reactions to determine the presence of the construct in host cells. Once the manipulated host cells are identified, they may then be used, as planned, for example, to grow in culture or to be introduced into a host organism.

[0089] Depending on the properties of the cells, they may be introduced into a host organism, such as a mammal, in a wide variety of ways. In alternative embodiments, the cells are manipulated to progress to cancer or to become cancerous, but in specific embodiments, the cells may be introduced into the tumor site. The number of cells employed will depend on numerous factors, the purpose of introduction, the lifespan of the cells, the protocol used, such as the number of doses, the ability of the cells to proliferate, and the stability of the recombinant construct. The cells may be applied as a dispersion and generally injected into or near the site of interest. The cells may be placed in a physiologically acceptable culture medium.

[0090] DNA introduction does not always result in integration. In some situations, temporary maintenance of the introduced DNA may be sufficient. Thus, a short-term effect may be achieved, in which case the cells may be introduced into the host and then stimulated after a predetermined time, for example, after the cells have been able to home in to a specific site.

[0091] Cells may be administered as desired. Depending on the desired response, the method of administration, cell lifespan, number of cells present, and various protocols may be employed. The number of doses will depend, at least to some extent, on the factors described above.

[0092] It should be fully understood that the system depends on numerous variables, such as the cellular response to the ligand, the efficiency of expression, and, as appropriate, the level of secretion, the activity of the expression product, the patient's specific needs which may vary over time and circumstances, the rate of loss of cellular activity as a result of cell loss, or the expression activity of individual cells. Therefore, for each individual patient, even if there is a universal cell that can be administered to the population as a whole, it is expected that each patient will be monitored for an appropriate dosage for their individual needs, and such patient monitoring practices are routine in this technology.

[0093] VI. Kits of this Disclosure Any of the compositions described herein may be included in the kit. In non-limiting examples, cells or reagents for manipulating cells may be included in the kit. In certain embodiments, NKT cells or populations of cells containing NKT cells may be included in the kit. Such a kit may have 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 cytokines themselves may be included in the kit. Proteins such as antibodies containing cytokines or agonist monoclonal antibodies may be included in the kit. Substrates containing antibodies or bare substrates themselves may be included in the kit, and in some embodiments, reagents for generating antibody-containing substrates may be included in the kit. Substrates may be of any kind, including beads or plates. Cells containing 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 embodiments, the kit includes the cell therapy and other cancer therapies of this disclosure. In some cases, in addition to embodiments of cell therapy, the kit also includes a second cancer therapy, such as chemotherapy, hormone therapy, and immunotherapy. The kit may be tailored for a particular cancer in an individual and may include each second cancer therapy for an individual.

[0095] The kit may contain preferably equally divided compositions of the present invention. The components of the kit may be packaged in an aqueous medium or in a lyophilized form. The container means of the kit would generally include at least one vial, test tube, flask, bottle, syringe or other container means in which the components may be placed, preferably in which the components are preferably equally divided. If the kit contains more than one component, the kit would also generally include a second, third, or other additional container in which the additional components may be placed separately. However, various combinations of components may be contained in a single vial. The kit of the present invention would also typically include means for containing the compositions under strict control for commercial sale and other reagent containers. Such containers would include injection-molded or blow-molded containers in which the desired vials are held.

[0096] If the components of the kit are provided in one and / or more solutions, the liquid is an aqueous solution, and a sterile aqueous solution is particularly preferred. In that case, the container means may be itself, or other 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 other components of the kit. However, the components of the kit may be provided as a dry powder. If the reagents and / or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. The solvent may also be provided in a separate container means, but this is assumed. [Examples]

[0097] Preferred embodiments of the present invention will be demonstrated with the following examples. Those skilled in the art will understand that the techniques disclosed in the subsequent examples represent techniques that the inventors have found to work well in the practice of the present invention and can therefore be considered to constitute a preferred mode for its practice. However, those skilled in the art will understand that, from the perspective of the present invention, numerous modifications can be made to the specific embodiments disclosed without departing from the spirit and scope of the invention, and similar or comparable results can still be obtained. Example 1 Identifying NKT cell subsets involved in their survival in vivo, and defining their therapeutic activity and the conditions required for the proliferation of these subsets in culture.

[0098] Adoptive cell invariant natural killer (NKT) cells are being developed as a promising treatment for immunotherapy of cancer, autoimmune diseases, and other diseases. Because NKT cells are low in frequency in human peripheral blood, such therapies require the mass proliferation of primary NKT cells in vitro while preserving their longevity and function. However, the cellular and molecular mechanisms involved in the maintenance of NKT cells, either in vivo or in vitro, remain largely unknown. Here, we have revealed that antigen-induced proliferation of primary human NKT cells in vitro, regardless of the initial frequency of the subset, is associated with the progressive accumulation of CD62L-positive subsets in all five individuals studied. Following magnetic sorting of NKT cells into CD62L-positive and CD62L-negative subsets, only CD62L-positive cells survived and proliferated in response to TCR stimulation, while approximately 90% of CD62L-negative cells underwent apoptosis within 3 days. Furthermore, after adoptive cell introduction into NSG mice, CD62L-positive NKT cells persisted five times longer than CD62L-negative ones. Importantly, CD62L-positive NKT cells exhibited significantly higher therapeutic activity and considerably longer survival in mice in xenolymphoma models. Proliferating CD62L-positive cells downregulated or maintained CD62L expression when activated by the TCR alone or in conjunction with its costimulatory receptor, respectively. In particular, specific combinations of agonist mAbs against CD3, CD28, and / or 4-1BB enable stable CD62L expression in in vitro-stimulated NKT cells, associated with their maximum proliferation rate and subsequent in vivo survival. Thus, the results reveal a previously unpredicted functional hierarchy in human NKT cells that can be leveraged for their effective in vitro proliferation for cell therapy applications.

[0099] Therefore, what is identified herein is CD62L as a marker for human NKT cells with high proliferative capacity and excellent therapeutic activity. In vitro stimulation conditions that prevent downregulation of CD62L on NKT cells during in vitro proliferation, which is useful for generating NKT cell products with high therapeutic activity, have been demonstrated. Example 2 CD62L+NKT cells possess excellent in vivo survival and antitumor activity.

[0100] CD62L+ cells accumulate in culture upon antigen stimulation of primary NKT cells. Previous studies comparing the phenotype of human NKT cells in adult peripheral blood with that in umbilical cord blood have shown a much higher proportion of CD4+ and CD62L+ NKT cells in neonates (Baev et al., 2004; D'Andrea et al., 2000; Eger et al., 2006) (Figure 5A). The dominance of CD4+CD62L+ NKT cells in umbilical cord blood suggests that CD4 and / or CD62L expression points to a subset of NKT cells that possess superior developmental potential and can support in vitro proliferation of NKT cells for therapeutic applications. To investigate this embodiment, immunophenotyping of primary NKT cells was performed at various time intervals in culture immediately after isolation from peripheral blood and after stimulation with αGalCer, consistently resulting in higher frequency and absolute numbers of NKT cells compared to the use of a T cell proliferation protocol based on CD3 / CD28 stimulation (Figure 6). Despite significant inter-individual variability in CD62L expression in newly isolated NKT cells, there was a remarkable accumulation of the CD62L+ fraction in NKT cells, from 33.63%±27.62% in newly isolated NKT cells to 69.92%±10.57% at day 12 of culture (P<0.001, Figures 1A, 1B). Although CD62L was expressed more frequently on CD4+ NKT cells both before and after culture, the accumulation of CD62L+ cells could not be explained by the preferential proliferation of CD4+ NKT cells. Indeed, at the end of the 12-day culture period, the frequencies of CD62L+, CD4+, and CD62L+CD4+ NKT cells increased 3.9±1.8, 1.9±0.7, and 3.8±1.9 times, respectively. Consistent with these results, there was enrichment of the CD62L+CD4- subset at day 12 compared to day 0 (Figure 5B). Further multi-parameter characterization of NKTs revealed that CD62L is often co-expressed with CCR7 pre-culture, but CCR7 expression is gradually lost during culture (Figure 5C). Pre-culture, almost all NKTs expressed both CD27 and CD28. Regardless of the CD62L status, CD27 was downregulated in nearly half of the NKTs by day 12 of culture, while CD28 expression remained unchanged (Figure 5C).

[0101] CD62L- cells expressed high levels of CD161 and CD56 (NK-like differentiation) but low levels of IL-7Rα (Figure 5D). Newly isolated NKT cells rarely expressed depletion markers (PD-1, LAG-3, or 6TIM-3) in either the CD62L+ or CD62L- subset, whereas at day 12 of NKT cell culture, the CD62L- subset preferentially expressed PD-1 and TIM3 (P=0.0043, 0.0184, Figure 5C). Furthermore, expression analysis of immune-related genes in CD62L+ and CD62L-NKT cells selected on day 12 (using the nCounter® platform) revealed upregulation of mRNA for genes related to T cell survival / memory in CD62L+NKT cells (e.g., LEF1, S1PR1, IL-7Rα, IL21R) and genes related to depletion / terminal differentiation in CD62L-NKT cells (PD-1, LAG-3, TIM-3, CD244, CD161, CD56, Figure 1D). Lymphocyte enhancer factor 1 (LEF1), a transcription factor, was the highest-ranking immune-related gene overexpressed in CD62L+ cells compared to CD62L-NKT cells. Intracellular flow cytometry analysis revealed that CD62L+NKT cells uniformly expressed LEF1, while the major fraction of CD62L- cells was LEF1-negative. Since LEF1 has recently been shown to mediate the proliferation of mouse NKT cell progenitor cells, partly through transcriptional activation of GATA3 gene expression (Carr et al., 2015), we analyzed the levels of GATA3 protein in human NKT cells in relation to LEF1 and CD62L levels. GATA3 expression was strongly correlated with LEF1 and CD62L expression in human NKT cells (Figure 5E). Interestingly, CD62L+ and CD62L-NKT cells expressed PLZF, a major transcriptional regulator of functional differentiation of NKT cells, at the same level (Cohen et al., 2013). Therefore, the CD62L+ subset predominantly accumulated in culture upon antigen stimulation of primary NKT cells, and loss of CD62L expression is associated with NK-like terminal differentiation, depletion, and downregulation of proliferative transcriptional regulators.

[0102] CD62L+NKT cells are Th-O-like cells capable of numerical proliferation. Next, NKT cells were magnetically sorted into CD62L+ and CD62L- subsets from primary culture, and their functional characteristics were investigated. Figure 2A shows that when target cells were pulsed with αGalCer, both subsets were equally cytotoxic to CD1d+DAOY medulloblastoma (3 out of 6 donors), or CD62L-NKT cells were more cytotoxic than CD62L+NKT cells (3 out of 6 donors). Analysis of cytokine production in αGalCer-stimulated NKT cells revealed that CD62L+ cells produced significantly higher levels of both IFNγ and IL-4 compared to the CD62L- subset (P<0.001, Figure 2B). CD62L+ cells exhibited Th-O--like polarization (balanced production of IFNγ and IL-4, typical of the entire peripheral blood NKT population), whereas the polarization profile of CD62L- cells could not be clearly determined due to the low absolute levels of each cytokine. Despite strong upregulation of IL-23R mRNA expression in the CD62L- subset (Figure 1D, potential Th17 polarization), as determined by nCounter analysis, neither IL-23R protein expression on the cell surface of NKT cells by FACS nor IL-17 production upon TCR stimulation by ELISA was detected.

[0103] To investigate whether the accumulation of CD62L+NKT during in vitro proliferation was due to its preferential survival or proliferation in response to antigen stimulation, selected cells were stimulated with αGalCer-pulsed antigen-presenting cells, and the rates of cell death and proliferation were measured. On day 3 post-stimulation, 31%±21% and 74%±7.5% of CD62L+ and CD62L- cells, respectively, underwent apoptosis (Figure 2C). On day 6, the CD62L+ subset showed a much larger rate of proliferation compared to the CD62L- subset, as measured by CFSE dilution (Figure 2D). Furthermore, cells that survived and proliferated in the CD62L- group... majorityThe expression of CD62L suggests that these cells were descendants of a small subset of CD62L+ cells in the original CD62L- fraction. Consistent with these results, there was a significant difference in the number of NKT cells generated after 6 days of culture with IL-2 alone or with TCR stimulation in the selected CD62L+ and CD62L- NKT cells. In fact, Figure 2E (upper panel) shows that CD62L+ cells underwent 2.5-fold and 8-fold numerical proliferation with IL-2 alone and with TCR stimulation, respectively. In contrast, CD62L-NKT cells did not proliferate under either condition. Although the degree of NKT cell proliferation in response to antigen stimulation varied among donors, the CD62L- subset contributed little to no to NKT cell proliferation in all five donors examined (Figure 2E, lower panel). Therefore, CD62L+ NKT cells survive, proliferate in response to antigen stimulation, and contribute to the numerical proliferation of NKT cells in culture.

[0104] The CD62L+ subset is involved in the in vivo survival and therapeutic activity of NKT cells. To determine the role of the CD62L+ subset in the in vivo survival of adoptive NKT cells, firefly luciferase was transduced into NKT cells, and these were magnetically sorted into CD62L+ and CD62L- subsets. The inventors then introduced the sorted cells into NSG mice. Longitudinal bioluminescence imaging demonstrated that signals from CD62L- cells could be detected up to day 2, while CD62L+ cells remained detectable up to day 10 (P<0.001, Figure 3A, B). Next, the in vivo therapeutic activity of CD62L+ and CD62L- NKT cell subsets was compared in a model of CAR redirected immunotherapy for lymphoma. CD19-specific CARs containing a 4-1BB co-stimulatory intracellular domain (CAR.CD19, Figure 7) were transduced into NKT cells, and then sorted into CD62L+ and CD62L- subsets. NOD / SCID / IL2Rγ (null) (NSG) mice were intravenously injected with CD19+ Daudi lymphoma cells transduced with luciferase, and after 4 days, the mice were divided into two groups: one receiving CD62L+ or the other CD62L- CAR.CD19NKT cells. Both CD62L+ and CD62L- CAR.CD19NKT cells extended the survival of treated animals compared to untreated controls (P<0.001). Importantly, only CD62L+ CAR.NKT cells induced sustained tumor regression in 7 out of 9 treated animals that survived, and 5 of these animals were tumor-free for at least 3 months. In contrast, all 10 mice treated with CD62L-CAR.NKT succumbed to tumor progression (P<0.001, Figure 3C, D). Therefore, CD62L+NKT exhibits extended in vivo survival and superior therapeutic efficacy compared to CD62L-NKT.

[0105] Co-stimulation maintains CD62L+ NKT and prevents depletion. There is growing evidence that co-stimulation plays a role in NKT activation, survival, and proliferation (van den Heuvel et al., 2011). Restless NKTs express CD28 (Figure 5C), while they express little to no late-stage costimulatory receptors such as 4-1BB and OX40 (Figure 8A). However, stimulation of NKTs with autologous PBMCs pulsed with αGalCer resulted in rapid induction of 4-1BB in all NKTs and OX40 in CD4+ NKTs (Figure 8A). CD62L is temporarily downregulated within the first 24-48 hours of TCR stimulation (data not shown), so the dynamics of 4-1BB and OX40 expression were analyzed in CD62L+ and CD62L- NKTs selected prior to stimulation. It was found that 71.13% ± 18.66% and 51.98% ± 18.83% of CD62L+ and CD62L- NKTs upregulated OX40 within 72 hours after stimulation (P=0.0072, Figure 4A). Similarly, stimulated CD62L+ NKTs expressed higher levels of 4-1BB compared to CD62L- NKTs (P=0.011, Figure 4A). OX40 was preferentially upregulated in either the CD62L+ or CD62L- NKT CD4+ subsets, while 4-1BB was upregulated in all NKTs (Figure 8B).

[0106] Next, we investigated whether co-stimulation could counteract the depletion of NKT cells grown in vitro. NKT cells selected for CD62L+ were stimulated on plates coated with the anti-CD3 agonist mAb OKT3 alone, or in combination with CD28, 4-1BB, or both. Since the inventors were unable to obtain an anti-OX40 mAb with agonist activity, OX40 was not investigated in these settings. First, co-stimulation with anti-CD28, anti-4-1BB, or both increased the number of NKT cells generated within 7 days of culture compared to stimulation with anti-CD3 alone at 20 ng / ml (P<0.001, Figure 4B). In the absence of co-stimulation, increasing the concentration of OKT3 from 20 ng / ml to 1 μg / ml did not affect the number of NKT cells generated, whereas co-stimulation was effective in increasing the number of NKT cells only when combined with low concentrations of OKT3 (Figure 9). Importantly, less than half of the NKTs were positive for CD62L at day 7 after stimulation with OKT3 alone. Addition of anti-CD28, anti-4-1BB, or anti-CD28 with anti-4-1BBmAb resulted in retention of CD62L expression in 58%±7.1% (P=0.026), 73%±9.2% (P=0.0036), and 73%±6.1% (P=0.0002), respectively, of NKTs (Figure 4C). In conjunction with retention of CD62L expression, NKTs provided with co-stimulatory signals expressed significantly less PD-1 (P<0.05, Figure 4D). Therefore, the binding of the co-stimulatory receptor between antigen stimulation supports CD62L expression in proliferating NKTs and prevents its depletion.

[0107] Significance of Specific Embodiments of This Disclosure

[0108] A critical gap in knowledge of human NKT cell biology has slowed the development of effective NKT cell-based cancer immunotherapy. Numerical proliferation of NKT cells in vitro and subsequent in vivo survival, essential requirements for effective NKT cell-based cell and gene therapy applications, depend on a subset of CD62L+ in peripheral blood NKT cells. In response to repeated TCR stimulation, only CD62L+ NKT cells survive and proliferate, while CD62L- cells experience early depletion and cell death. While continuous NKT stimulation is associated with loss of CD62L expression, activation of co-stimulatory receptors between TCR stimulations can counteract this process. Specifically, we experimentally determined a unique combination of CD86, 4-1BBL, and OX40L molecules, as well as the level of co-expression on the aAPC surface that maximizes CD62L expression protection and enables highly efficient proliferation of clinical-scale NKT cells. In certain embodiments, CAR-NKTs generated using aAPC as encompassed by this disclosure exhibit long-term in vivo persistence and excellent therapeutic activity against in vivo models of lymphoma and neuroblastoma (examples of cancer types).

[0109] The CD62L+ subset is involved in the numerical proliferation of NKT cells in vitro upon antigen stimulation. The following findings support the above conclusion: (i) the fraction of CD62L+ cells increases dramatically after stimulation of primary NKT cells; (ii) selected CD62L+ cells proliferate in response to the same stimulation, while the majority of selected CD62L- cells undergo apoptosis; (iii) CD62L-NKT cells rapidly acquire a depletion phenotype upon in vitro stimulation, as evidenced by signs of terminal differentiation (expression of CD161 and CD56) in newly isolated PBMCs and marked upregulation of PD-1 and TIM-3 expression and decreased ability to produce cytokines. When similar features are observed in therapeutic products of T cells, they are associated with persistence or subsequent lack of objective response after adoption into cancer patients (Gattinoni et al., 2005; Klebanoff et al., 2005).

[0110] Proliferating NKT cells ultimately downregulate CD62L expression and acquire a depletion phenotype during in vitro culture. This finding is thought to reflect the ontogeny of human peripheral blood NKT, given that the frequency of CD62L+NKT is lower in adult peripheral blood compared to umbilical cord blood (Eger et al., 2006; Der Vliet et al., 2000). One report found that umbilical cord blood NKT cells do not express CD62L (D'Andrea et al., 2000). The reason for the inconsistency between that report and others, and between current results, is technical in certain embodiments. M. Constantinides et al. identified a very rare naive-like population of CD1d-restricted T cells with high levels of CD62L in peripheral blood (Constantinides et al., 2011). However, these cells do not express the invariant Vα24 chain and have low levels of PLZF expression compared to conventional NKT cells. Both CD62L+ and CD62L- NKT cells 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 characteristics; they can proliferate on their own while giving rise to effector / memory and effector T cells (Graef et al., 2014). In a very recent published study, D. Sommermeyer et al. demonstrated that CD8 or CD4 T cells expressing human CAR.CD19, generated from naive and central memory subsets, are more effective against Raji lymphoma xenografts than those generated from effector memory subsets (Sommermeyer et al., 2015). The authors also found that combining subsets expressing the most potent CD4+ and CD8+ CARs produces synergistic antitumor activity in vivo. Since NKT activation has been shown to result in transactivation of NK cells and CD8 T cells in mouse models and human clinical trials (Dhodapkar et al., 2009; Vivier et al., 2012), the combination of CAR NKT with other defined subsets of CAR-expressing lymphocytes may be a useful therapeutic strategy.

[0112] LEF1 and IL-7Rα were the most overexpressed immune-related genes in CD62L+ compared to CD62L-NKT. 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 thymic development, mediated by direct transcriptional activation of CD127 and expression of the c-myc gene (Carr et al., 2015). Consistent with the findings of harmonized expression of LEF1 and GATA3 in human NKT, these researchers also found 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, where CD62L+ cells have been found to preferentially express GATA3 and produce high levels of both cytokines. Accepting the findings from Carr et al.'s study on mouse NKT and human NKT, in specific embodiments, LEF1 plays a crucial role in the early stages of NKT cell development, regulating their number and function. High levels of LEF1 expression in the CD62L+ subset of human NKT cells and its loss in the CD62L- subset are consistent with a linear progression model derived from the Th-O-like cytokine profile, which is directed towards less differentiated CD62L+ NKT cells with conserved proliferative capacity and terminally differentiated CD62L-NKT cells with impaired ability to proliferate and produce cytokines.

[0113] There is growing evidence that co-stimulation plays a crucial role in the development, activation, and functional response of NKT cells in mouse models (van den Heuvel et al., 2011; Uldrich et al., 2005). However, little is known about the expression of co-stimulatory receptors in human NKT cells. In this disclosure, we focused on a set of co-stimulatory receptors that exhibit significant pro-survivability properties in human T cells: CD28, 4-1BB, and OX40 (Acuto et al., 2003; Kroczek et al., 2004; Redmond et al., 2009). First, we showed that newly isolated human NKT cells express CD28 (34) and co-express CD27, thereby confirming that this is analogous to a corresponding stage in the differentiation of memory T cells with conserved functional potential (Okada et al., 2008). This disclosure is the first to characterize the baseline and post-stimulation dynamics of 4-1BB and OX40 in human NKT cells. Neither receptor is detectable in newly isolated NKT cells, but they are induced following TCR stimulation. Similar to T cells (Croft et al., 2010), human NKT cells preferentially upregulated OX40 in the CD4+ subset. Most NKT cells also upregulated 4-1BB, but this was preferentially upregulated in the CD8+ subset of T cells (Lynch, 2008). Importantly, simultaneous stimulation of individual co-stimulatory receptors inhibited the loss of CD62L expression, rescuing NKT cells from depletion. Simultaneous activation of CD28 and 4-1BB had an additive effect on maintaining CD62L+ NKT cells. 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 types of culture media were supplemented with 10% FBS (Hyclone) and 2 mM GlutaMAX-1 (Gibco-BRL).

[0115] Isolation, transduction, proliferation, 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, following protocols approved by the institutional ethics committees 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). Negative PBMC fractions were irradiated (40 Gy) and fractionated. NKT cells were stimulated with aliquots 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% thermoinactivated fetal bovine serum, and 2 mM Glutamax). NKT cells were grown for 10 days and then restimulated with autologous PBMCs (irradiated with 40 Gy) or, if instructed, Ramos cells (irradiated with 100 Gy) as APCs. Three days after restimulation, 24-well non-tissue culture plates were coated with retronectin (Takara Bio), washed, and 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 the stimulated NKT cells were added to the wells in complete medium and 200 U / ml of rhIL-2. After 48 hours, the cells were removed from the plates, washed, and 10 6The cells were resuspended in complete RPMI with 200 U / ml IL-2 at a concentration of cells / ml and seeded on plates for continuous growth. The number of NKT cells was determined by trypan blue (Life Technologies) counting. Where instructed, NKT or CAR-NKT cells were labeled with CD62L-PEmAb (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] Production of retrovirus constructs and retroviruses Constructs of CAR.CD19 and CAR.GD2 were prepared as previously described (Heczey et al., 2014; Pule et al., 2005), and they contained a 4-1BB signaling intracellular domain sequence fused to the ζ chain and a CD8α-derived transmembrane domain linked to a CD19-specific antibody FMC-63 or a GD2-specific antibody 14G2a via a short spacer derived from the hinge region of IgG1. Retroviral supernatants were produced by transtransfusion of 293T cells with a combination of chimeric antigen-containing plasmids, but as previously described (Vera et al., 2006), the RDF plasmid encoded the envelope of RD114 and the PegPam3 plasmid encoded the gag-pol of MoMLV.

[0117] Proliferation and apoptosis assays NKT cells were labeled with CFSE (Invitrogen) and stimulated by PBMCs pulsed with αGalCer, or by 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 examined on days 3 and 6 by measuring the dilution of CFSE 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) and subsequent flow cytometry.

[0118] Multiple cytokine assay NKT cells were stimulated for 24 hours using plates coated with APC or agonist antibody (clone 6B11, BD Biosciences). The supernatant was collected and analyzed using the Luminex® assay kit (Millipore) according to the manufacturer's instructions.

[0119] Flow cytometry Immunophenotyping was performed using the following mAbs for the following targets: 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 EP2030YmAb (ABCAM). BD or R&D-proposed mAbs with matching fluorescent dyes and isotypes were used as negative controls. CAR.CD19 expression on NKT 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 using LSR-II5 laser flow cytometry with BD FACSDiva software v.6.0 and FlowJo 7.2.5 (Tree Star) (BD Biosciences).

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

[0121] Analysis of gene expression Total RNA was recovered using TRIzol reagent (Qiagen). Gene expression was analyzed using the immunology panel v.2 (NanoString) with the BCM genome and RNA profiling core using the nCounter analysis system. The data was analyzed using nSolver2.0 software (NanoString).

[0122] In vivo experiments The NSG mouse colonies were originally obtained from the Jackson Laboratory and maintained in the BCM animal facility. Tumor growth was initiated by i.v. injection of 2×10 5 cells. On day 3, the mice were treated with 4 - 8×10 6 CAR-NKT cells, and then, every 3 days, the mice received an i.p. injection of IL-2 (1000 U / mouse). Tumor growth was evaluated twice a week by bioluminescence imaging (Small Animal Imaging Core facility, Texas Children’s Hospital). For the in vivo persistence experiment, NKT cells were co-transduced with CAR.CD19 and luciferase using a retroviral construct and injected i.v. into tumor-free or tumor-bearing mice, and monitored twice a week using bioluminescence imaging. Animal experiments were performed according to an IACUC-approved protocol.

[0123] Statistical data For in vitro and in vivo experiments, the inventors evaluated continuous variables of two groups using a paired two-sided t-test and continuous variables of more than two groups using one-way ANOVA with Bonferroni's post-test. Survival was analyzed by the Kaplan / Meier method and log-rank (Mantel-Cox) test to compare pairs of groups. Statistical data were calculated using GraphPad (trademark) Prism 5.0 (GraphPad Software). A p-value of less than 0.05 was considered significant.

[0124] Approval of the study Cord blood units were obtained from the MD Anderson Cancer Center Cord Blood Bank in accordance with protocols approved by the institutional ethics committees at MD Anderson Cancer Center (H-16320) and Baylor College of Medicine (H-20911). Written informed consent was obtained from all participating women prior to joining protocol H-16320. Cord blood units unsuitable for clinical use (usually due to low cell counts) were discarded or used for research purposes under protocol H-20911 at Baylor College of Medicine. Animal experiments were conducted in accordance with protocol AN-5194, approved by the institutional animal care committee at Baylor College of Medicine. Example 4 The effect of IL-21 on NKT cells

[0125] This example demonstrates that IL-21 has beneficial effects on NKT cells when cells are exposed to it. Figure 10 shows that IL-21 increases the frequency of CD62L+NKT cells during primary proliferation. NKT cells were isolated from three donors and stimulated for 12 days with aGalCer-loaded PBMCs and IL-2 (100 U / ml) or IL-2 (10 ng / ml). NKT cells were harvested, stained for CD62L, and then subjected to FACS analysis. Figure 11 clearly shows that IL-21 increases the frequency of CD62L+NKT cells during secondary proliferation. Following primary proliferation (Figure 10), NKT cells from three donors were re-stimulated for 12 days with aGalCer-loaded PBMCs and IL-2 (100 U / ml) or IL-2 (10 ng / ml). NKT cells were collected, stained for CD62L, and then subjected to 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 corresponding mAb and IgG controls. Ramos cells can proliferate primary NKT cells with high levels of CD62L expression (Figure 13). NKT cells were isolated from three donors and Ramos cells (2 × 10⁶) were loaded with aGalCer in a medium containing IL-2. 6 Ramos cells were stimulated for 10 days with 10 cells / well. NKT cells were harvested, counted, and stained for CD3, 6B11, and CD62L. Finally, Figure 14 shows that Ramos cells significantly retain CD62L expression and proliferate NKT cells upon secondary stimulation. Following primary proliferation with PBMCs (as shown in Figure 10), NKT cells (1 × 10⁻ / well) were grown in Ramos cells (2 × 10⁻⁶ / well) loaded with aGalCer in a medium containing IL-2. 6 The cells were re-stimulated for 10 days (individual cells / well). NKT cells were collected, counted, and stained for CD3, 6B11, and CD62L. References

[0126] All patents and publications referenced herein represent the level of skill of those skilled in the art in which the present invention relates. All patents and publications are incorporated herein by reference in the same manner as if each individual publication were directed to be incorporated by reference specifically and individually.

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Claims

1. A pharmaceutical composition comprising multiple genetically modified human type I natural killer T (NKT) cells that are positive for CD62L, LEF1, S1PR1, ICAM4, IL7R, AIRE, IL21R, CCR8, and CD8B, wherein the cells comprise at least one chimeric antigen receptor (CAR) expression construct, and the human type I natural killer T (NKT) cells constitute the majority of the human type I NKT cells in the pharmaceutical composition.

2. The pharmaceutical composition contains a certain dose of a plurality of genetically modified type I CD62L-positive type I natural killer T (NKT) cells, wherein the dose is 1 × 10 cells per square meter of the patient's body surface area. ７ From 2 x 10 ８ The pharmaceutical composition according to claim 1, wherein the composition is one in number.

3. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition contains a therapeutically effective amount of the CD62L-positive human type I natural killer T (NKT) cells.

4. The pharmaceutical composition according to claim 2, wherein the NKT cells are primary human CD62L-positive NKT cells that have been proliferated in vitro at least 12 days old.

5. The pharmaceutical composition according to claim 1, wherein the chimeric antigen receptor (CAR) expression construct comprises an antigen recognition domain directed to at least one tumor-associated antigen.

6. The at least one tumor-associated antigen is melanoma-associated antigen (MAGE), melanoma-expressing antigen (PRAME), CD19, CD20, CD22, κ-light chain, CD30, CD33, CD123, CD38, CD138, ROR1, ErbB2, ErbB3 / 4, EGFr vIII, 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 pharmaceutical composition according to claim 5, selected from the group consisting of NY-ESO-1, PSC1, folate receptor-a, CD44v6, CD44v7 / 8, 8H9, NCAM, VEGF receptor, 5T4, fetal AchR, NKG2D ligand, CD44v6, GPC3, CSPG4, CEA, or a combination thereof.

7. The pharmaceutical composition according to claim 6, wherein the at least one antigen is CD19.

8. The pharmaceutical composition according to claim 6, wherein the at least one antigen is GD2.

9. The pharmaceutical composition according to claim 6, wherein the at least one antigen is GPC3.

10. The pharmaceutical composition according to claim 4, wherein the chimeric antigen receptor (CAR) expression construct comprises an extracellular domain containing an antigen recognition domain and a transmembrane domain, the transmembrane domain ligating the antigen recognition domain to the transmembrane domain.

11. The pharmaceutical composition according to claim 10, further comprising a spacer that links the antigen recognition domain to the transmembrane domain, wherein the spacer is selected from the group consisting of the CH2CH3 region of immunoglobulin, the hinge region derived from IgG1, and at least a portion of CD3.

12. The pharmaceutical composition according to claim 11, wherein the transmembrane domain includes a CD28 transmembrane region.

13. The pharmaceutical composition according to claim 5, wherein the antigen recognition domain comprises a single-chain variable fragment (scFv).

14. The pharmaceutical composition according to claim 1, wherein the chimeric antigen receptor (CAR) expression construct comprises at least a portion of the cytoplasmic signaling domain.

15. The pharmaceutical composition according to claim 14, wherein the cytoplasmic signaling domain is derived from the CD3 zeta chain of a T cell receptor.

16. The pharmaceutical composition according to claim 1, wherein the chimeric antigen receptor (CAR) expression construct comprises a costimulatory intracellular domain.

17. The pharmaceutical composition according to claim 16, wherein the aforementioned co-stimulating intracellular domain is selected from CD28, OX40, 4-1BB, ICOS, CD40, CD30, CD27, or a combination thereof.

18. The pharmaceutical composition according to claim 17, wherein the aforementioned co-stimulating intracellular domain is 4-1BB.

19. The pharmaceutical composition according to claim 17, wherein the aforementioned co-stimulating intracellular domain is CD28.

20. The pharmaceutical composition according to claim 5, wherein the chimeric antigen receptor (CAR) expression construct comprises an scFv from a CD19-specific antibody FMC-63 that connects to a transmembrane domain derived from CD8a via a spacer derived from the IgG1 hinge region, and a 4-1BB signaling intracellular domain that fuses with the CD3 zeta chain.

21. A pharmaceutical composition for immunotherapy for a subject requiring immunotherapy, comprising a certain dose of genetically modified human type I natural killer T (NKT) cells that express at least one chimeric antigen receptor (CAR) expression construct and are positive for CD62L, LEF1, S1PR1, ICAM4, IL7R, AIRE, IL21R, CCR8, and CD8B, wherein the dose contains 1 × 10 cells per square meter of the subject's body surface area. ７ From 2 x 10 ８ A pharmaceutical composition comprising a number of NKT cells, wherein the NKT cells constitute a majority of type I NKT cells in the pharmaceutical composition.

22. A pharmaceutical composition relating to immunotherapy for a target requiring immunotherapy as described in claim 21, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

23. A pharmaceutical composition comprising a plurality of genetically modified human type I natural killer T (NKT) cells as described in claim 1, wherein the cells are CD19 ＋ A pharmaceutical composition that, when introduced into NOD / SCID / IL2Rγ (null) (NSG) mice carrying Daudi lymphoma cells, exhibits in vivo persistence and is detectable on day 10.

24. A pharmaceutical composition relating to immunotherapy for a target requiring immunotherapy, as described in claim 22, wherein the pharmaceutical composition is a composition for parenteral administration.

25. A pharmaceutical composition relating to immunotherapy for a target requiring immunotherapy, as described in claim 24, wherein the pharmaceutical composition is a composition for intravenous administration.

26. The pharmaceutical composition for immunotherapy for a subject requiring immunotherapy according to claim 24, wherein the pharmaceutically acceptable carrier is physiological saline or a buffer medium.

27. The subject is a cancer patient, and the pharmaceutical composition relates to immunotherapy for a subject requiring immunotherapy as described in claim 21.

28. A pharmaceutical composition relating to immunotherapy for a target requiring immunotherapy according to claim 27, wherein the cancer is neuroblastoma, lymphoma, leukemia, or liver cancer.

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