Natural killer T cells and methods of using same

Abstract

Disclosed herein are modified NKTs expressing human IL-12, compositions comprising the modified NKTs, and therapeutic methods using the modified NKTs. The modified NKTs described herein are reprogrammed NKTs that demonstrate long-term persistence when expression of IL-12 is coupled with expression of a CAR. These NKTs acquire the long-term ability to eliminate tumor cells and the long-term persistence of NKTs expressing IL-12. Also disclosed herein are modified cells transformed to express a CAR and an exogenous membrane-bound moiety (such as IL-12).

Description

[Technical field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Application No. 63 / 304,556, filed January 28, 2022. The entire teachings of the above application are incorporated herein by reference.

[0002] Government Subsidy This invention was made with Government support under CA243543 awarded by the National Institutes of Health (NIH). The United States Government has certain rights in this invention. [Background technology]

[0003] Natural killer T cells (NKT or NKT cells), a specific subset of T cells demonstrated to respond both as innate and memory-like cells, bridge the innate and adaptive immune responses and have shown promise as a platform for adoptive T cell therapy in cancer patients. Engineered NKT expressing chimeric antigen receptors (CARs) represent a class of immunotherapeutic agents that have shown promising results in preclinical and clinical cancer research and treatment.

[0004] NKT CARs can promote indirect antitumor activity through induction of dendritic cell (DC) maturation and secretion of NK cell and CD8+ T cell activating cytokines. However, the numerous immunosuppressive modifications that malignant cells make to their extracellular microenvironment and their own surface protein expression have thus far limited the effectiveness and scope of CAR therapy for certain cancers. There remains a need for NKT cells modified to retain long-term persistence and the ability to eliminate tumor cells and protect against tumor re-challenge. Summary of the Invention

[0005] Disclosed herein are modified natural killer T (NKT) cells. In some embodiments, the NKT expresses transgenic IL-12.

[0006] In some embodiments, the NKT cells comprise a chimeric antigen receptor (CAR). The CAR can be GD2.CAR or CD19.CAR.

[0007] In one embodiment, the NKT cells are human NKT cells. In one embodiment, the NKT cells are non-human NKT cells (e.g., mouse NKT cells). In some embodiments, the NKT cells are isolated from peripheral blood.

[0008] The modified NKT cells described herein can be produced using any method known to those skilled in the art, including, for example, transduction or transfection. In one embodiment, the NKT cells expressing transgenic IL-12 are produced by transducing NKT cells with a retroviral supernatant containing IL12. In one embodiment, the NKT cells expressing transgenic IL-12 are produced using lentiviral vector (LV) transduction. In one embodiment, the NKT cells expressing transgenic IL-12 are produced using electroporation of mRNA.

[0009] In some embodiments, transgenic IL-12 is bound to the NKT cell membrane via a CD4 or CD8 stalk. In one embodiment, transgenic IL-12 is bound to the NKT cell membrane via a CD8 stalk (e.g., a CD8a stalk). The CD8 stalk may comprise the hinge region and transmembrane domain of CD8. In some embodiments, the CD8 stalk is modified to remove one or more cysteine ​​residues from the hinge region, and in some aspects, the CD8 stalk is modified by replacing one or more cysteine ​​residues with serine residues. In one embodiment, the CD8 stalk comprises a hinge region of the amino acid sequence of SEQ ID NO: 12. In one embodiment, transgenic IL-12 is bound to the NKT cell membrane via a CD4 stalk. The CD4 stalk may comprise the hinge region and transmembrane domain of CD4. In some embodiments, the CD4 stalk is modified to remove one or more cysteine ​​residues from the hinge region, and in some aspects, the CD4 stalk is modified by replacing one or more cysteine ​​residues with serine residues. In one embodiment, the CD4 stalk comprises a hinge region of the amino acid sequence of SEQ ID NO: 13.

[0010] In some embodiments, the NKT cells exhibit one or more features including enhanced tumor control and improved survival compared to control cells, control of tumor growth upon tumor rechallenge compared to control cells, increased anti-tumor activity, increased expression of CD62L, long-term persistence compared to control cells, enhanced cytotoxic activity upon repeated exposure to tumor cells in vitro, and / or reduced risk of causing graft-versus-host disease compared to control cells.

[0011] In one embodiment, the NKT cells do not express CD62L.

[0012] Also disclosed herein is a population of modified NKT cells as disclosed herein. Also disclosed herein is a population of genetically modified NKT cells isolated from peripheral blood, wherein the NKT express transgenic IL-12.

[0013] In some embodiments, the NKT population comprises a chimeric antigen receptor (CAR), such as GD2.CAR or CD19.CAR, hi some embodiments, the NKT population is transduced with a CAR and IL-12.

[0014] In one embodiment, the NKT does not express CD62L.

[0015] Disclosed herein is a method for producing the NKT disclosed herein by transducing the NKT with a retroviral vector. In some embodiments, the retroviral vector comprises IL-12.

[0016] In some embodiments, the retroviral vector further comprises a green fluorescent protein (GFP). In some embodiments, the retroviral vector further comprises an internal ribosome entry site (IRES). In some embodiments, the retroviral vector further comprises a CAR (e.g., GD2.CAR or CD19.CAR). In some embodiments, the CAR is a B7H3 CAR or a CSPG4 CAR.

[0017] Also disclosed herein are methods of treating cancer by administering to a subject the modified NKT cells disclosed herein.

[0018] In some embodiments, the subject is a mammal (e.g., a human or a mouse). In some embodiments, the modified NKT cells are administered intravenously to the subject.

[0019] Also disclosed herein are cells that have been transformed to express CAR and an exogenous membrane-binding moiety, where the exogenous membrane-binding moiety comprises a transmembrane domain that has been modified to remove one or more cysteine ​​residues that could form disulfide bonds with cysteine ​​residues otherwise present in the CAR. [Brief description of the drawings]

[0020] [Figure 1-1] IL12(i)GFP-transduced NKTs upregulate CD62L. A is a schematic timeline of the protocol used to select, transduce, and expand NKTs. B shows a schematic of the retroviral vector used to engineer human NKTs. The p40 and p35 subunits of IL-12 are connected by a flexible linker. [Figure 1-2] IL12(i)GFP transduced NKT upregulates CD62L. C shows representative flow cytometry plots of NKT purity in non-transduced NKT (NT), NKT transduced with control GFP vector (GFP), and NKT transduced with IL12(i)GFP vector. D provides representative flow cytometry plots of NKT transduction efficiency measured as the percentage of GFP+ cells in non-transduced NKT (NT), NKT transduced with control GFP vector (GFP), and NKT transduced with IL12(i)GFP vector. E shows representative flow cytometry plots of CD62L in non-transduced NKT (NT), NKT transduced with control GFP vector (GFP), and NKT transduced with IL12(i)GFP vector. [Figure 2A]We demonstrate that IL12(i)GFP-transduced NKT release IL12 in vitro and persist longer in vivo. Quantification of IL-12, IFN-γ, and IL-4 produced by NT, GFP-transduced NKT, and IL12(i)GFP-transduced NKT in resting conditions (unstimulated) or activated with anti-CD3-anti-CD28 antibodies is shown. Cytokines were measured in supernatants collected 24 hours after plating 1*106 cells / well in 24-well plates in 2 mL complete medium without cytokines. Means shown, n=4; *, P=0.0273; **, P=0.0022; ***, P=0.0006; ****, P<0.0001; two-way ANOVA. [Figure 2B] We demonstrate that IL12(i)GFP transduced NKT release IL12 in vitro and persist longer in vivo. We provide a volcano plot illustrating differential gene expression (alpha < 0.2) of RNAseq data between IL12(i)GFP NKT and GFP NKT. Positive LFC values ​​reflect overexpression in IL12(i)GFP NKT. [Figure 2C] We demonstrate that NKT transduced with IL12(i)GFP release IL12 in vitro and persist longer in vivo. A schematic representation of an in vivo experiment to assess NKT persistence in a xenograft NSG mouse model is shown. Mice were intravenously transplanted with 5*106 GFP NKT or IL12(i)GFP firefly luciferase-labeled NKT. Mice were imaged with an IVIS kinetic machine at days 0, 2, 4, and 7 after NKT injection, and euthanized at day 10 to collect peripheral blood, liver, and spleen. [Figure 2D] We demonstrate that NKT transduced with IL12(i)GFP release IL12 in vitro and persist longer in vivo. Bioluminescence imaging (BLI) of representative tumors is shown (measured as total flux p / s). [Figure 2E]We demonstrate that NKT transduced with IL12(i)GFP release IL12 in vitro and persist longer in vivo. Quantification of human NKT (iNKT+CD45+) in peripheral blood, liver, and spleen samples collected 10 days after NKT infusion is shown. Means shown, n=10; *, P=0.0161, **, P=0.0020, ***, P=0.0006, unpaired t-test. [Figure 3A] NKTs transduced with GD2.CAR and IL-12 demonstrate that they express CAR and upregulate CD62L. A diagram of the retroviral vectors used to transduce NKTs is shown. The scFv FMC.63 was used for CD19.CAR and the 1A7 scFv was used for GD2.CAR. [Figure 3B] Demonstration that NKTs transduced with GD2.CAR and IL-12 express CAR and upregulate CD62L. Representative flow cytometry plots of CAR expression in control NKTs (NT) and CAR-transduced NKTs assessed on day 10 of culture are shown. [Figure 3C] Demonstration that NKTs transduced with GD2.CAR and IL-12 express CAR and upregulate CD62L. Representative flow cytometry plots of CD62L expression in control NKTs (NT) and CAR-transduced NKTs assessed on day 10 of culture are shown. [Figure 4A] We demonstrate that IL-12 producing GD2.CAR NKT has enhanced cytotoxic activity upon repeated exposure to tumor cells in vitro and mediates long-term tumor control. NKT were co-cultured with CHLA-225 (E:T=1:1) and then harvested and stained with anti-iTCR (Vβ11) and anti-GD2 to identify NKT and neuroblastoma cells, respectively, by flow cytometry. Summary of quantification of remaining tumor cells after each cycle is shown. Mean is shown, n=4. [Figure 4B]We demonstrate that IL-12 producing GD2.CAR NKT has enhanced cytotoxic activity upon repeated exposure to tumor cells in vitro and mediates long-term tumor control. A schematic representation of a xenograft metastatic neuroblastoma model is shown. Mice were implanted intravenously with 2*106 CHLA-225 firefly luciferase labeled neuroblastoma tumor cells. Ten days later, mice were administered 5*106 CAR+ NKT intravenously. Mice were imaged weekly by IVIS kinetic machine and euthanized on day 80 to collect peripheral blood, liver, and spleen. [Figure 4C] We demonstrate that IL-12 producing GD2.CAR NKT has enhanced cytotoxic activity upon repeated exposure to tumor cells in vitro and mediates long-term tumor control. Representative tumor BLI (measured as total flux p / s) is shown. [Figure 4D] We demonstrate that IL-12-producing GD2.CAR NKT has enhanced cytotoxic activity upon repeated exposure to tumor cells in vitro and mediates long-term tumor control. Representative flow cytometry plots of human NKT (iNKT+CD45+) in peripheral blood, liver, and spleen samples collected 80 days after NKT infusion are shown. [Figure 5A] We demonstrate that NKTs transduced with GD2.CAR and membrane-bound IL-12 express CAR and upregulate CD62L. We provide a diagram of the retroviral vector used to transduce NKTs. 1A7 scFv was used for GD2.CAR. Modified CD8a stalk and transmembrane domains were used to anchor IL-12 to the membrane of NKTs. [Figure 5B] Demonstrates that NKTs transduced with GD2.CAR and membrane-bound IL-12 express CAR and upregulate CD62L. Provides representative flow cytometry plots of CAR expression in control CAR-transduced NKTs assessed on day 10 of culture. [Figure 5C]Demonstrates that NKTs transduced with GD2.CAR and membrane-bound IL-12 express CAR and upregulate CD62L. Provides representative flow cytometry plots of CD62L expression in control CAR-transduced NKTs assessed on day 10 of culture. [Figure 6A] We demonstrate that membrane-bound IL-12 is detected on the cell surface and induces STAT4 phosphorylation. Representative flow cytometry plots of IL-12 expression detected on the cell surface of control NKT (NT) or CAR-transduced NKT assessed on day 10 of culture are provided. [Figure 6B] We demonstrate that membrane-bound IL-12 is detected on the cell surface and induces STAT4 phosphorylation. Representative flow cytometry plots of IL-12 receptor beta (IL-12RB) expression detected on the cell surface of control NKT (NT) or CAR-transduced NKT assessed on day 10 of culture are provided. [Figure 6C] We demonstrate that membrane-bound IL-12 is detected on the cell surface and induces STAT4 phosphorylation. Representative Western blots illustrating STAT4 phosphorylation in NT, IL12(i)GFP NKT, and CAR.GD2(i)IL12TM NKT at day 14 of culture are provided. [Figure 7A] We demonstrate that GD2.CAR NKT with membrane-bound IL-12 has comparable cytotoxic activity to soluble IL-12 upon repeated exposure to tumor cells in vitro. NKT were co-cultured with CHLA-225 (E:T=1:1) and then harvested and stained with anti-iTCR (Vβ11) and anti-B7-H3 to identify NKT and neuroblastoma cells by flow cytometry, respectively. Representative flow plots of quantification of remaining tumor cells after each cycle are provided. [Figure 7B]We demonstrate that GD2.CAR NKT with membrane-bound IL-12 has comparable cytotoxic activity to soluble IL-12 upon repeated exposure to tumor cells in vitro. Quantification of IFN-γ and IL-12pr produced by control NKTS (NT) or CAR-transduced NKT co-cultured with CHLA-225 at an E:T ratio of 1:1 is shown. Cytokines were measured in supernatants collected 24 hours after plating 2.5×105 NKT cells / well and 2.5×105 CHLA-225 / well in 24-well plates in 2 mL complete medium without cytokines. Mean and standard deviation are shown, n=2. [Figure 7C] We demonstrate that GD2.CAR NKT with membrane-bound IL-12 has comparable cytotoxic activity to soluble IL-12 upon repeated exposure to tumor cells in vitro. We show the persistence of GD2.CAR(i)IL12TM in vivo. NSG mice were injected with 10*106 GD2.CAR(i)IL12TM NKT. After 27 days, NKT were detected in peripheral blood and maintained high expression of CD62L. [Figure 8-1] 1 provides amino acid sequences for various IL-12 constructs. [Figure 8-2] Same as above. [Figure 8-3] Same as above. [Figure 9] 1 provides the amino acid sequences for CD19.CAR and GD2.CAR. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] Natural killer T cells (NKT or NKT cells) were isolated from peripheral blood and genetically engineered to express human IL-12. These NKTs were shown to acquire high expression levels of CD62L in immunodeficient mice after adoptive transfer and persist for a long time without causing graft-versus-host disease. Surprisingly, it was found that transgenic IL-12 can be used to reprogram and improve IL-12 expression of NKT cells, rather than simply as a soluble cytokine to expand NKT cells. It was further shown that IL-12 is sufficient by itself to support long-term engraftment of NKT cells. In some embodiments, CD62L may not be necessary to support the effect of transgenic IL-12.

[0022] Described herein are modified NKT cells, compositions comprising the modified NKT cells, and therapeutic methods utilizing the modified NKT cells. The modified NKT cells described herein exhibit long-term persistence, and when expression of IL-12 is coupled with expression of a CAR, these NKT cells acquire long-term ability to eliminate tumor cells and long-term persistence.

[0023] Qualified NKT Aspects of the disclosure relate to modified T cells (e.g., modified or engineered natural killer T (NKT) cells). In some embodiments, the NKT cells are modified to express transgenic IL-12.

[0024] In some embodiments, the NKT cells comprise a chimeric antigen receptor (CAR). As is generally known to those skilled in the art, CAR T cells (e.g., CAR NKT cells) can be produced by obtaining NKT cells, such as from a subject in need of the CAR T cells or a donor subject, and manipulating the cells so that they comprise a chimeric antigen receptor (CAR). CARs provide the ability to target specific proteins to cancer cells and include an antigen recognition domain, an extracellular hinge region, a transmembrane domain, and an intracellular T cell signaling domain. CAR T cells can be classified as first generation, second generation, third generation, or fourth generation.

[0025] First generation CARs are engineered with only the CD3ζ domain. Second generation CARs are engineered with the CD3ζ domain and a costimulatory signaling domain (e.g., CD28 or 4-1BB). Third generation CARs are engineered to contain a CD3ζ domain in addition to two costimulatory signaling domains (e.g., both CD28 and CD137). Finally, fourth generation CARs (also referred to as T-cells redirected for universal cytokine-mediated killing (TRUCK)) are engineered to contain a CD3ζ domain, two costimulatory signaling domains (e.g., both CD28 and CD137), and some additional genetic modifications (addition of transgenes for cytokine secretion or additional costimulatory signaling domains). Any type of CAR can be used in the production of modified NKT cells. In some embodiments, the CAR is a second generation CAR. In some embodiments, the CAR is selected from the group consisting of GD2.CAR, CD19.CAR, B7H3.CAR, and CSPG4.CAR.

[0026] In some embodiments, the NKT cells comprise a T cell receptor (TCR). As is generally known to those skilled in the art, NKT cells comprising a T cell receptor can be produced by obtaining NKT cells, such as from a subject in need of the NKT cells or a donor subject, and manipulating the cells so that they comprise a T cell receptor (TCR). Any type of TCR can be used in the production of modified NKT cells.

[0027] In some embodiments, the NKT cells are human NKT cells or non-human NKT cells. In some embodiments, mammalian cells are used. In some embodiments, the mammalian cells are primate cells (human cells or non-human primate cells), rodent (e.g. mouse, rat, rabbit, hamster) cells, dog, cat, cow, or other mammalian cells. In some embodiments, avian cells are used. In some embodiments, the NKT cells are isolated from peripheral blood, bone marrow, lymph, or lymphoid organs. The NKT cells can be isolated by any method known to those skilled in the art.

[0028] In some embodiments, the NKT cells are autologous cells. In some embodiments, the NKT cells are not autologous cells. In some embodiments, the NKT cells are of the same species as the subject. In some embodiments, the NKT cells are of a different species than the subject. In some embodiments, the NKT cells are differentiated in vitro from stem or progenitor cells using differentiation protocols known in the art. In some embodiments, the population of NKT cells is expanded at least about 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 500-fold, or more to provide an increase in the number of NKT cells.

[0029] The modified NKT cells described herein exhibit one or more characteristics. Non-limiting examples of the characteristics of the modified NKT cells include enhanced tumor control and improved survival (compared to control cells), control of tumor growth upon tumor rechallenge (compared to control cells), increased anti-tumor activity, increased expression of CD62L, long-term persistence, enhanced cytotoxic activity upon repeated exposure to tumor cells in vitro (compared to control cells), reduced risk of causing graft-versus-host disease, and combinations thereof. In some embodiments, the modified NKT cells enhance tumor control and improved survival (compared to control cells). In some embodiments, the modified NKT cells control tumor growth upon tumor rechallenge (compared to control cells). In some embodiments, the modified NKT cells exhibit increased anti-tumor activity. In some embodiments, the modified NKT cells exhibit increased expression of CD62L. In some embodiments, the modified NKT cells exhibit long-term persistence. In some embodiments, the modified NKT cells exhibit enhanced cytotoxic activity upon repeated exposure to tumor cells in vitro compared to control cells. In some embodiments, the modified NKT cells exhibit a reduced risk of causing graft-versus-host disease when administered to a subject. In some embodiments, the modified NKT cells do not express CD62L.

[0030] In some embodiments, the modified NKT cells contain a membrane-bound cytokine attached to the cell surface (e.g., the cell surface of the NKT cell). In one embodiment, the membrane-bound cytokine is IL-12. In some aspects, the modified NKT cells secrete IL-12, which is then membrane-bound to the surface of the NKT cell. In some embodiments, the IL-12 is bound to the cell membrane (e.g., the NKT cell membrane) via a transmembrane domain. In some embodiments, the IL-12 is fused to a transmembrane domain. In some embodiments, the transmembrane domain has a sequence derived from a transmembrane domain of a molecule selected from the group consisting of the integrin family, CD4, CD8, CD44, glycophorin, MHC class I and II glycoproteins, EGF receptor, G protein-coupled receptor (GPCR) family, receptor tyrosine kinases (e.g., insulin-like growth factor 1 receptor (IGFR) and platelet-derived growth factor receptor (PDGFR)), the porin family, other transmembrane proteins known to those of skill in the art, and combinations thereof. In one embodiment, the transmembrane domain comprises the transmembrane domain of CD4. In one embodiment, the transmembrane domain comprises the transmembrane domain of CD8.

[0031] In some embodiments, the transgenic IL-12 is bound to the NKT cell membrane via a CD4 or CD8 stalk. In one embodiment, the transgenic IL-12 is bound to the NKT cell membrane via a CD8 stalk. In some embodiments, the CD8 stalk comprises a CD8 hinge region and transmembrane domain. Suitable CD8 hinge regions and transmembrane domains are known to those of skill in the art, and an illustrative example is provided in SEQ ID NO:6. In some embodiments, the CD8 stalk is modified to remove one or more cysteine ​​residues from the hinge region. In some embodiments, the CD8 stalk is modified to remove all cysteine ​​residues from the hinge region. In some embodiments, one or more cysteine ​​residues are removed by replacing one or more cysteine ​​residues with serine residues. In some embodiments, the CD8 stalk is modified by replacing all of the cysteine ​​residues in the hinge region with serine residues. In one embodiment, the CD8 stalk is a CD8a stalk. In some embodiments, the CD8 stalk comprises a hinge region of the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity thereto. In one embodiment, the CD8 stalk comprises, consists of, or consists essentially of a hinge region of the amino acid sequence of SEQ ID NO: 12.

[0032] In one embodiment, transgenic IL-12 is bound to the NKT cell membrane via the CD4 stalk. In some embodiments, the CD4 stalk comprises a hinge region and a transmembrane domain of CD4. Suitable hinge regions and transmembrane domains of CD4 are known to those of skill in the art, and an illustrative example is provided in SEQ ID NO:8. In some embodiments, the CD4 stalk is modified to remove one or more cysteine ​​residues from the hinge region. In some embodiments, the CD4 stalk is modified to remove all cysteine ​​residues from the hinge region. In some embodiments, one or more cysteine ​​residues are removed by replacing one or more cysteine ​​residues with serine residues. In some embodiments, the CD4 stalk is modified by replacing all cysteine ​​residues in the hinge region with serine residues. In some embodiments, the CD4 stalk comprises a hinge region of the amino acid sequence of SEQ ID NO: 13, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity thereto. In one embodiment, the CD4 stalk comprises, consists of, or consists essentially of a hinge region of the amino acid sequence of SEQ ID NO: 13.

[0033] In some embodiments, IL-12 is bound to the cell membrane via the CD4 or CD8a stalk. The CD4 or CD8 stalk may comprise a hinge and a transmembrane domain. In some embodiments, the hinge region (e.g., the CD8 hinge region) is modified to minimize or otherwise prevent dimerization with a co-expressed CAR. The hinge region may comprise one or more modifications (e.g., deletion and / or substitution of 1, 2, 3, 4, 5, or more amino acids). In one embodiment, the CD8 hinge region comprises a modification to a cysteine ​​(Cys) residue at position(s) 27 and / or 44 of the CD8a hinge region as shown in SEQ ID NO:6. In one embodiment, the CD8 hinge region comprises a modification to a cysteine ​​(Cys) residue at position(s) C571 and / or C588 of SEQ ID NO:6. In one embodiment, the CD4 hinge region comprises a modification to a Cys residue at position(s) 14 and / or 56 of the CD4 hinge region as set forth in SEQ ID NO: 8. In one embodiment, the CD4 hinge region comprises a modification to a Cys residue at position(s) C558 and / or C600 of SEQ ID NO: 8. In some aspects, one or more cysteine ​​residues are replaced with one or more serine (Ser) residues. In one embodiment, the transmembrane domain of the membrane bound cytokine comprises the sequence TTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFASD (SEQ ID NO: 12). In one embodiment, the transmembrane domain of the membrane bound cytokine comprises the sequence VVMRATQLQKNLTSEVWGPTSPKLMLSLKLENKEAKVSKREKAVWVLNPEAGMWQSLLSDSGQVLLESNIKVLPTWSTPVQP (SEQ ID NO: 13). Surprisingly, it was found that modifying the CD8a stalk to remove a cysteine ​​residue unexpectedly improved the efficacy of anchored IL-12 co-expressed CAR.

[0034] Without being bound by theory or by a particular mode of application, it is generally understood that cysteine ​​residues present in the membrane-binding portion (including the native sequence of the CD4 or CD8 stalk) likely form disulfide bonds with cysteine ​​residues present in the co-expressed CAR, thereby reducing the efficacy of the CAR. By replacing cysteine ​​residues predicted to form disulfide bonds between the membrane-bound cytokine (e.g., IL-12) and the CAR, dimerization of the membrane-bound cytokine with the CAR is minimized or otherwise prevented, thereby restoring the efficacy of the CAR. Thus, the present disclosure also extends to cells transformed to express a CAR and an exogenous membrane-binding portion, the exogenous membrane-binding portion comprising a transmembrane domain modified to remove one or more cysteine ​​residues that would otherwise be capable of forming disulfide bonds with cysteine ​​residues present in the CAR.

[0035] In some embodiments, the exogenous membrane-bound moiety is bound to the cell membrane via a CD4 or CD8 stalk. In one embodiment, the membrane-bound therapeutic moiety is bound to the cell membrane via a CD8 stalk. In some embodiments, the CD8 stalk comprises a CD8 hinge region and a transmembrane domain. Suitable CD8 hinge regions and transmembrane domains are known to those of skill in the art, and an illustrative example is shown in SEQ ID NO:6. In some embodiments, the CD8 stalk is modified to remove one or more cysteine ​​residues from the hinge region. In some embodiments, the CD8 stalk is modified to remove all cysteine ​​residues from the hinge region. In some embodiments, one or more cysteine ​​residues are removed by replacing one or more cysteine ​​residues with serine residues. In some embodiments, the CD8 stalk is modified by replacing all cysteine ​​residues in the hinge region with serine residues. In one embodiment, the CD8 stalk is a CD8a stalk. In some embodiments, the CD8 stalk comprises a hinge region of the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity thereto. In one embodiment, the CD8 stalk comprises, consists of, or consists essentially of a hinge region of the amino acid sequence of SEQ ID NO: 12.

[0036] In another embodiment, the exogenous membrane-binding moiety is attached to the cell membrane via a CD4 stalk. In some embodiments, the CD4 stalk comprises a CD4 hinge region and a transmembrane domain. Suitable CD4 hinge regions and transmembrane domains are known to those of skill in the art, and an illustrative example is shown in SEQ ID NO:8. In some embodiments, the CD4 stalk is modified to remove one or more cysteine ​​residues from the hinge region. In some embodiments, the CD4 stalk is modified to remove all cysteine ​​residues from the hinge region. In some embodiments, one or more cysteine ​​residues are removed by replacing one or more cysteine ​​residues with serine residues. In some embodiments, the CD4 stalk is modified by replacing all cysteine ​​residues in the hinge region with serine residues. In some embodiments, the CD4 stalk comprises a hinge region of the amino acid sequence of SEQ ID NO: 13, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity thereto. In one embodiment, the CD4 stalk comprises, consists of, or consists essentially of a hinge region of the amino acid sequence of SEQ ID NO: 13.

[0037] The present disclosure also extends to cells transformed to express a CAR and an exogenous membrane-binding portion, where the amino acid sequence of the CAR has been modified to remove one or more cysteine ​​residues that would otherwise be capable of forming disulfide bonds with cysteine ​​residues present in the transmembrane domain of the exogenous membrane-binding portion.

[0038] In some embodiments, the exogenous membrane-bound moiety is a therapeutic moiety. Suitable therapeutic moieties are known to those skilled in the art, and exemplary embodiments thereof include cytokines, integrin family members, CD4, CD8, CD44, glycophorin, MHC class I and II glycoproteins, EGF receptor, G protein-coupled receptor (GPCR) family members, receptor tyrosine kinases (e.g., insulin-like growth factor 1 receptor (IGFR) and platelet-derived growth factor receptor (PDGFR)), porin family members, and combinations of any of the above. In one embodiment, the therapeutic moiety is a cytokine.

[0039] In some embodiments, the cell is an immune cell.Suitable immune cells are known to those skilled in the art and include T cells.In some embodiments, the immune cell is a T cell or a NKT cell.

[0040] In another embodiment, one or more of the cysteine ​​residues of a membrane-bound CAR that are predicted to form disulfide bonds with cysteine ​​residues present in the hinge and / or transmembrane region of a membrane-bound cytokine (e.g., IL-12) may be modified (e.g., substituted for a serine residue), thereby minimizing or otherwise preventing dimerization of the membrane-bound cytokine with the CAR.

[0041] In some embodiments, the IL-12 fused to the transmembrane domain comprises a sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9. In some embodiments, the IL-12 fused to the CD8 transmembrane domain comprises a sequence of SEQ ID NO:6 or SEQ ID NO:7. In some embodiments, the IL-12 fused to the CD4 transmembrane domain comprises a sequence of SEQ ID NO:8 or SEQ ID NO:9. In some embodiments, the IL-12 fused to the mutated transmembrane domain comprises a sequence of SEQ ID NO:7 or SEQ ID NO:9. In one embodiment, the IL-12 fused to the transmembrane domain comprises a sequence of SEQ ID NO:6. In one embodiment, the IL-12 fused to the transmembrane domain comprises a sequence of SEQ ID NO:7. In one embodiment, the IL-12 fused to the transmembrane domain comprises a sequence of SEQ ID NO:8. In one embodiment, the IL-12 fused to the transmembrane domain comprises a sequence of SEQ ID NO:9.

[0042] In some embodiments, the membrane-bound or anchored IL-12 contains a linker. Linkers are generally known to those skilled in the art. In some embodiments, the linker comprises one or more amino acids. For example, the linker can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids. In some embodiments, the linker can be a flexible linker.

[0043] NKT cells can be modified to express IL-12, for example, using any method known to one of skill in the art. In some embodiments, modifications to NKT cells can be introduced by RNA targeting agents (such as RNAi, miRNA, or ribozymes). In some cases, modifications can be introduced via a gene editing system or components thereof. Examples of gene editing systems include CRISPR-Cas systems, zinc finger nuclease (ZFN) systems, TALE systems, TALEN systems, and meganuclease systems. In some embodiments, the gene editing system can be delivered using a vector delivery system. In some embodiments, the gene editing system can be delivered using a viral vector delivery system. In some embodiments, modifications to NKT cells can be introduced using transduction or transfection. Non-limiting examples of methods for modifying NKT cells include viral, non-viral, and hybrid (viral and non-viral) based methods. Methods of transduction can include the use of lentiviral vectors, adenoviral vectors, oncoretroviral vectors, and / or other vectors known to one of skill in the art. Transfection methods may include lipofection, nucleofection, particle bombardment, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, electroporation, sonoporation, magnetofection, gene microinjection, laser irradiation, and other methods known to those of skill in the art.

[0044] In some embodiments, transgenic IL-12-expressing NKT cells are produced by transducing NKT cells with a viral vector containing IL-12, for example, NKT cells can be transduced with a retroviral supernatant containing IL-12. In one embodiment, transgenic IL-12-expressing NKT cells are produced using lentiviral vector (LV) transduction. In one embodiment, transgenic IL-12-expressing NKT cells are produced using electroporation of mRNA.

[0045] In some embodiments, the NKT cells are modified to reduce or knock out expression of CD62L. In some embodiments, the NKT cells are modified using a gene editing system (such as a CRISPR-related system, e.g., CRISPR / Cas9). In some embodiments, the NKT cells are modified using CRISPR / Cas to reduce or knock out expression of one or more targets. In some embodiments, the NKT cells are modified using CRISPR / Cas to reduce or knock out expression of CD62L.

[0046] In some embodiments, NKT cells are isolated from a mammal and genetically modified (i.e., transduced or transfected in vitro) with IL-12 and, in some embodiments, with a CAR. In some embodiments, the NKT cells can be transduced with a viral vector (e.g., a retroviral vector) or transfected with a plasmid or nucleic acid construct. In some embodiments, the retroviral vector comprises IL-12. The IL-12 can have a p40 subunit and a p35 subunit connected via a flexible linker. In some embodiments, the retroviral vector further comprises SFG and / or GFP. In some embodiments, the retroviral vector further comprises an internal ribosome entry site (IRES). In some embodiments, the NKT cells are transduced with a CAR and IL-12.

[0047] For administration to a subject, the modified NKT cells produced by the methods disclosed herein can be administered to a subject, for example, in a pharma- ceutically acceptable composition. These pharma-ceutically acceptable compositions comprise a therapeutically effective amount of the modified NKT cells described above, formulated together with one or more pharma- ceutically acceptable carriers (additives) and / or diluents. In some embodiments, the pharmaceutical composition further comprises a diluent and / or other components, and / or other cytokines and / or cell populations.

[0048] As described herein, the pharmaceutical composition of the present invention can be formulated for administration in solid or liquid form, including: (1) oral administration, such as drenches (aqueous or non-aqueous solutions or suspensions), lozenges, dragees, capsules, pills, tablets (e.g. buccal, sublingual, and targeted for systemic absorption), boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, such as sterile solutions or suspensions or sustained release formulations, for example by subcutaneous, intramuscular, intravenous, or epidural injection; (3) topical application, such as creams, ointments, or controlled release patches or sprays applied to the skin; (4) vaginally or rectally, such as pessaries, creams, or foams; (5) sublingual; (6) intraocular; (7) transdermal; (8) transmucosal; or (9) nasally adapted. Additionally, the compound can be implanted into the patient or injected using a drug delivery system. See, e.g., Urquhart, et al., Ann. Rev. Pharmacol. Toxicol. 24:199-236 (1984); Lewis, ed. "Controlled Release of Pesticides and Pharmaceuticals" (Plenum Press, New York, 1981); U.S. Patent No. 3,773,919; and U.S. Patent No. 353,270,960. In some embodiments, direct administration to a tumor and / or body cavity, opening, and / or tissue containing a tumor may be desirable.

[0049] As used herein, the term "pharmacologically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are suitable, within the scope of sound medical judgment, for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0050] As used herein, the term "pharmaceutically acceptable carrier" refers to a pharma- ceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricating substances, magnesium talc, calcium or zinc stearate, or steric acid), or solvent encapsulating material, that is involved in carrying or transporting a compound of interest from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials that can serve as pharma- ceutically acceptable carriers include: (1) sugars (such as lactose, glucose, and sucrose); (2) starches (such as corn starch and potato starch); (3) cellulose and its derivatives (such as sodium carboxymethylcellulose, methylcellulose, ethylcellulose, microcrystalline cellulose, and cellulose acetate); (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants (such as magnesium stearate, sodium lauryl sulfate, and talc); (8) excipients (such as cocoa butter and suppository wax); (9) oils (such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil); (10) cellulose acetate; 0) glycols (such as propylene glycol); (11) polyols (such as glycerin, sorbitol, mannitol, and polyethylene glycol (PEG)); (12) esters (such as ethyl oleate and ethyl laurate); (13) agar; (14) buffers (such as magnesium hydroxide and aluminum hydroxide); (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffer solutions; (21) polyesters, polycarbonates, and / or polyanhydrides; (22) bulking agents (such as polypeptides and amino acids); (23) serum components (such as serum albumin, HDL, and LDL); (22) C2-C 12Alcohols (such as ethanol); and (23) other non-toxic compatible substances used in pharmaceutical formulations. Wetting agents, coloring agents, releasing agents, coating agents, sweetening agents, flavoring agents, fragrances, preservatives, and antioxidants may also be present in the formulation. The terms "excipient", "carrier", "pharmaceutical acceptable carrier", or the like, are used interchangeably herein.

[0051] Treatment methods Disclosed herein are methods of treating or preventing cancer in a subject in need thereof. In some embodiments, the methods include administering modified NKT cells that express transgenic IL-12 and, in some embodiments, comprise a chimeric antigen receptor (CAR) as described herein. In some embodiments, the methods include administering a therapeutically effective amount of modified NKT cells that express transgenic IL-12 and, in some embodiments, comprise a CAR.

[0052] Also disclosed herein are methods of expanding and / or generating a population of modified NKT cells in a subject (e.g., a subject diagnosed with cancer and / or otherwise in need of such a population). In some embodiments, the method comprises administering to the subject NKT cells that express transgenic IL-12. In some embodiments, the population of modified NKT cells persists in the subject for a period of time (e.g., at least 1 week, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 9 months, 1 year, 2 years, 5 years, etc.) following administration to the subject.

[0053] In some embodiments, the cells described herein (e.g., modified NKT cells) are transplantable (e.g., the modified NKT cells can be administered to a subject). In some embodiments, the subject to which the modified NKT cells are administered is the same subject from which the pre-modified NKT cells were obtained (e.g., for autologous cell therapy). In some embodiments, the subject is a different subject. In some embodiments, the subject is afflicted with cancer or is a normal subject. For example, the modified NKT cells for transplantation can be in a form suitable for transplantation.

[0054] The method may further include administering the modified NKT cells to a subject in need of said administration (e.g., a mammalian subject, e.g., a human subject). The source of the cells may be a mammal, preferably a human. The source or recipient of the cells may also be a non-human subject (e.g., an animal model). The term "mammal" encompasses organisms, including mice, rats, cows, sheep, pigs, rabbits, goats, horses, monkeys, dogs, cats, and preferably humans. Similarly, transplantable cells may be obtained from any of these organisms, including non-human transgenic organisms. In some embodiments, the source of the cells is the peripheral blood of a subject.

[0055] The composition comprising the modified NKT cells can be administered to a subject using an implantable device. Implantable devices and related technologies are known in the art and are useful as delivery systems when continuous or timed release delivery of the compounds or compositions delineated herein is desired. Additionally, implantable device delivery systems are useful for targeting specific points of delivery of the compounds or compositions (e.g., localized sites, organs). Negrin et al., Biomaterials, 22(6):563 (2001). Timed release technologies, including alternative delivery methods, can also be used in the present invention. For example, timed release formulations based on polymeric technologies, sustained release techniques, and encapsulation techniques (e.g., polymers, liposomes) can also be used for delivery of the compounds and compositions delineated herein.

[0056] As used herein, the term "administering" refers to placing a composition into a subject by a method or route that results in at least partial localization of the composition at a desired site such that a desired effect is produced. Suitable routes of administration for the methods of the present invention include both local and systemic administration. Generally, local administration results in greater delivery of the administered modified NKT cells to a specific location compared to the entire body of the subject, while systemic administration results in delivery of the modified NKT cells essentially to the entire body of the subject.

[0057] In the context of administration of modified NKT cells, the term "administration" also includes transplantation of such cells in a subject. As used herein, the term "transplantation" refers to the process of implanting or transferring at least one cell into a subject. The term "transplantation" includes, for example, autotransplantation (removal of cells from one location of a patient and transfer to the same or another location of the same patient), allotransplantation (transplantation between members of the same species), and xenotransplantation (transplantation between members of different species). Those skilled in the art are well aware of methods of implantation or transplantation of cells to treat cancer, which are suitable for the present invention.

[0058] The modified NKT cells or compositions comprising same may be administered by any suitable route known in the art, including, but not limited to, oral or parenteral routes, including intravenous, intramuscular, subcutaneous, transdermal, and airway (aerosol), pulmonary, nasal, rectal, and topical (including buccal and sublingual) administration.

[0059] Exemplary modes of administration include, but are not limited to, injection, infusion, infusion, inhalation, or ingestion. "Injection" includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injection and infusion. In a preferred embodiment, the composition is administered by intravenous infusion or injection.

[0060] As used herein, "subject" refers to a human or animal. Typically, an animal is a vertebrate, such as a primate, rodent, domestic animal, or game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, such as rhesus monkeys. Rodents include mice, rats, wood mice, ferrets, rabbits, and hamsters. Domestic and game animals include cattle, horses, pigs, deer, bison, buffalo, feline species, such as domestic cats, canine species, such as dogs, foxes, wolves, avian species, such as chickens, emus, ostriches, and fish, such as trout, catfish, and salmon. Patients or subjects include any subset of the above, such as all of the above, but excluding one or more groups or species, such as humans, primates, or rodents. In certain embodiments of the aspects described herein, the subject is a mammal (e.g., a primate, e.g., a human). The terms "patient" and "subject" are used interchangeably herein. The terms "patient" and "subject" are used interchangeably herein. The subject can be male or female.

[0061] Preferably, the subject is a mammal. The mammal may be, but is not limited to, a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow. In addition, the methods and compositions described herein may be used to treat domestic animals and / or pets.

[0062] In some embodiments, a subject is considered to be "at risk" of having or developing cancer or of having a recurrence of cancer. Whether a subject is at risk of having or developing cancer or of having a recurrence of cancer is a decision that can be within the discretion of a person skilled in the art who cares for the subject. Any suitable diagnostic test and / or criteria can be used. For example, a subject can be determined to be "at risk" of having or developing cancer if (i) the subject has a mutation, genetic polymorphism, gene or protein expression profile, and / or the presence of a specific substance in the blood that is associated with an increased risk of developing or having cancer compared to other members of the general population who do not have the mutation or genetic polymorphism; (ii) the subject has one or more risk factors (having a family history of cancer, being exposed to carcinogens or tumor-promoting agents or conditions (e.g., asbestos, tobacco smoke, aflatoxin, radiation, chronic infection / inflammation, etc.), aging, etc.); (iii) the subject has one or more symptoms of cancer, (iv) the subject has a known medical condition that increases the likelihood of cancer, etc.

[0063] As used herein, the type of cancer is not limited. The term "cancer" as used herein is defined as the hyperproliferation of cells whose unique traits (loss of normal control) result in unregulated growth, lack of differentiation, local tissue invasion, and metastasis. In the context of the method of the present invention, the cancer can be any cancer, including acute lymphocytic cancer, acute myeloid leukemia, adenocarcinoma, alveolar rhabdomyosarcoma, anal cancer, angiosarcoma, B-cell lymphoma, basal cell carcinoma, bladder cancer, bone cancer, brain tumor, breast cancer, anal, anal canal, or anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, cervical, gallbladder, or pleural cancer, nasal cavity, or middle ear cancer, oral cavity cancer, vulva cancer, chronic lymphocytic leukemia, chronic bone marrow cancer, colon cancer, colorectal cancer, esophageal cancer, cervical cancer, endometrial cancer, fibrosarcoma, gastrointestinal carcinoid tumor, hematopoietic neoplasm, Hodgkin's disease, and the like. The term "tumor" as used herein includes any of the following cancers: lymphoma, hypopharyngeal cancer, renal cancer, laryngeal cancer, leukemia, liquid tumor, liver cancer, lung cancer, lymphoma, malignant mesothelioma, mast cell tumor, melanoma, multiple myeloma, myeloma, nasopharyngeal cancer, non-Hodgkin's lymphoma, ovarian cancer, pancreatic cancer, peritoneal, omental and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, sarcoma, skin cancer, small intestine cancer, soft tissue cancer, solid tumor, squamous cell carcinoma, gastric cancer, T-cell lymphoma, testicular cancer, thymoma, thyroid cancer, ureteral cancer, bladder cancer, and uterine cancer.As used herein, the term "tumor" refers to the abnormal growth of malignant type of cells or tissue, and does not include benign type of tissue, unless otherwise specified.

[0064] As used herein, the terms "treat" and "treatment" refer to administering to a subject an effective amount of modified NKT cells modified ex vivo according to the methods described herein, such that the subject has a reduction in at least one symptom of the disease or an improvement in the disease (e.g., a beneficial or desired clinical outcome). For purposes of the present invention, a beneficial or desired clinical outcome includes, but is not limited to, alleviation of one or more symptoms, whether detectable or undetectable, a reduction in the extent of the disease, a stabilized (i.e., not worsening) disease state, a delay or slowdown in disease progression, a palliation or mitigation of the disease state, and remission (whether partial or complete). Treating may refer to prolonging survival compared to expected survival in the absence of treatment. Thus, one of skill in the art will understand that treatment may improve disease pathology, but may not result in a complete cure of the disease. As used herein, the term "treatment" encompasses prophylaxis. Alternatively, treatment is "effective" if the progression of the disease is reduced or stopped. "Treatment" may also mean prolonging survival compared to expected survival in the absence of treatment. Those in need of treatment include those already diagnosed with a disorder associated with expression of the polynucleotide sequence, as well as those at risk of developing such a disorder due to genetic susceptibility or other factors.

[0065] By "treating," "preventing," or "ameliorating" a disease or disorder is meant delaying or preventing the onset of such disease or disorder, ameliorating, ameliorating, inhibiting, slowing, or halting the progression, worsening, or aggravation of the progression or severity of the pathology associated with such disease or disorder. In one embodiment, symptoms of the disease or disorder are alleviated by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%.

[0066] The dosage, administration schedule, and method of administration of the modified NKT cells are not limited. The dosage depends on a variety of factors, including other treatments, number of doses, and individual patient parameters including age, physical condition, size, and weight. These are factors well known to those of skill in the art and can be addressed with no more than routine experimentation. In some embodiments, the maximum tolerated dose can be used according to sound medical judgment (i.e., the safest and most tolerable dose). In some embodiments, the pharmaceutical composition comprising the modified NKT cells is administered at a dose of about 10 3 ~about 10 10 The dosage may be administered in a range of cells / kg body weight, and in some embodiments, the dosage may include all integer values ​​within those ranges (e.g., 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 ) containing about 10 5 ~about 10 6 It may be cells / kg body weight.

[0067] The dose used may be the maximum tolerated dose or a sub-therapeutic dose, or any dose therebetween. In some embodiments, the modified NKT cells are administered in combination with one or more agents. In some embodiments, the modified NKT cells and / or one or more agents are administered according to a defined administration schedule. Multiple doses are contemplated. In some embodiments, when the modified NKT cells and one or more agents are administered in combination, one or more sub-therapeutic dosages of the agents may be used. "Sub-therapeutic dose," as used herein, refers to a dosage that is less than that dosage that would produce a therapeutic result in a subject if administered in the absence of other agents. In some embodiments, a sub-therapeutic dose of an anti-cancer agent is one that would not produce a useful therapeutic result in a subject in the absence of administration of the modified NKT cells described herein. Therapeutic doses of anti-cancer agents are well known in the art of medicines for the treatment of cancer.

[0068] As used herein, a pharmaceutical composition comprises one or more agents or compositions having a therapeutic utility and a pharma- ceutically acceptable carrier (e.g., a carrier that facilitates delivery of the agent or composition). The agents and pharmaceutical compositions disclosed herein can be administered by any suitable means, such as orally, intranasally, subcutaneously, intramuscularly, intravenously, intraarterially, parenterally, intraperitoneally, intrathecally, intratracheally, intraocularly, sublingually, vaginally, rectally, transdermally, or as an aerosol. Depending on the type of condition being treated (e.g., cancer), the compounds of the present invention can be administered, for example, by oral ingestion, inhalation, or systemic routes. Thus, a variety of modes or routes of administration are available. The particular mode selected will typically depend on factors such as the particular compound selected, the particular condition being treated, and the dosage required for therapeutic efficacy. The methods described herein can be practiced using any medically acceptable mode of administration, which generally means any mode that produces an acceptable level of efficacy without causing clinically unacceptable side effects. Preferred modes of administration are parenteral and oral routes. The term "parenteral" encompasses subcutaneous, intravenous, intramuscular, intraperitoneal, and intrasternal injection or infusion techniques. In some embodiments, inhaled medications are particularly useful, as they are delivered directly to the lungs, for example in lung cancer patients. Several types of metered dose inhalers are commonly used for administration by inhalation. These types of devices include metered dose inhalers (MDIs), breath-actuated MDIs, dry powder inhalers (DPIs), spacer / holding chambers in combination with MDIs, and nebulizers. In some embodiments, the medicament is delivered by pulmonary aerosol. Other suitable routes will be apparent to those skilled in the art.

[0069] Toxicity and therapeutic efficacy of administration of compositions comprising modified NKT cells can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, for determination of the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). Compositions comprising modified NKT cells that exhibit large therapeutic indices are preferred.

[0070] The amount of a composition comprising modified NKT cells can be tested using several well-established animal models.

[0071] In some embodiments, data obtained from cell culture assays and animal studies can be used to formulate a range of dosages for use in humans. The dosage of such compounds is preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. Dosages can vary within this range depending on the dosage form used and the route of administration utilized.

[0072] The therapeutically effective dose of a composition comprising modified NKT cells can also be estimated initially from cell culture assays. Alternatively, the effect of any particular dosage can be monitored by a suitable bioassay.

[0073] With regard to duration and frequency of treatment, it is typical for a skilled clinician to monitor the subject to determine when the treatment provides therapeutic benefit, and to decide whether to increase or decrease the dosage, increase or decrease the frequency of administration, discontinue treatment, resume treatment, or make other changes to the treatment regimen. Dosing schedules can vary from once a week to daily, depending on a number of clinical factors. The desired dose can be administered once or divided into subdoses (e.g., 2-4 subdoses) and administered over a period of time (e.g., at appropriate intervals throughout the day, or on other suitable schedules). Such subdoses can be administered as unit dosage forms. In some embodiments, administration is chronic (e.g., one or more doses daily over a period of weeks or months). Examples of dosing schedules are daily, twice daily, three times daily, or four or more times daily administration for a period of 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months or more.

[0074] In another aspect of the invention, the method provides for the use of an isolated population of NKT cells. In one embodiment of the invention, the isolated population of modified NKT cells disclosed herein can be used for the production of a pharmaceutical composition for use in transplantation into a subject in need of treatment (e.g., a subject having cancer or at risk of developing cancer). Examples include subjects with melanoma or pancreatic cancer. In some embodiments, the isolated population of modified NKT cells disclosed herein can be autologous and / or allogeneic. In some embodiments, the subject is a mammal, and in other embodiments, the mammal is a human.

[0075] One embodiment of the present invention relates to a method of treating cancer in a subject comprising administering to a subject having cancer an effective amount of a composition comprising the modified NKT cells disclosed herein. Another embodiment relates to a method of treating a tumor in a subject comprising administering to a subject having a tumor an effective amount of a composition comprising the modified NKT cells disclosed herein.

[0076] In some embodiments, the modified NKT cells disclosed herein are administered to a subject with cancer in combination with a second therapeutic treatment (e.g., chemotherapy, radiation, immunosuppressants (such as cyclosporine, azathioprine, methotrexate, mycophenolate, and FK506), antibodies, or other immune ablative agents (such as CAMPATH, anti-CD3 antibodies or other antibody therapies, cytotoxins, fludaribine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and / or irradiation, etc.).

[0077] In some embodiments, the modified NKT cells are administered to the patient in combination with (e.g., prior to, concurrently with, and / or following) bone marrow transplant, T cell ablative therapy, using either chemotherapeutic agents (such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, etc.) or antibodies (such as OKT3 or CAMPATH). In another embodiment, the modified NKT cells are administered following B cell ablative therapy (such as agents that react with CD20, e.g., Rituxan). For example, in one embodiment, the subject may undergo standard of care with high dose chemotherapy followed by peripheral blood stem cell transplant. In certain embodiments, following transplant, the subject may receive an infusion of expanded modified NKT cells. In additional embodiments, the expanded cells may be administered prior to and / or following surgery.

[0078] In treating cancer or tumors, the modified NKT cells may optionally be administered with other different cytotoxic agents (e.g., chemotherapeutic or anti-neoplastic compounds) useful in treating the disorders or conditions described herein, such as radiation therapy. The other compounds may be administered prior to, simultaneously with, and / or after administration of the modified NKT cells. As used herein, the word "concurrently" means sufficiently close in time to produce a combined effect (i.e., simultaneously may be simultaneous, or may be two or more administrations occurring before or after each other).

[0079] As used herein, the phrase "radiation therapy" includes, but is not limited to, X-rays or gamma rays delivered either from an externally applied source (such as a beam) or by implantation of small radioactive sources.

[0080] Non-limiting examples of suitable chemotherapeutic agents that may be administered by the modified NKT cells described herein include daunomycin, cisplatin, verapamil, cytosine arabinoside, aminopterin, democolcine, tamoxifen, actinomycin D, alkylating agents (including but not limited to nitrogen mustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas, and triazenes): uracil mustard, chlormethine, cyclophosphamide (Cytoxan®), ifosfamide, melphalan, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramine, busulfan, carmustine, lomustine, streptozocin, dacarbazine, and temozolomide; antimetabolites (folate antagonists, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors, including, but not limited to: methotrexate, 5-fluorouracil, floxuridine, cytarabine, 6-mercaptopurine, 6-thioguanine, fludarabine phosphate, pentostatin, and gemcitabine, natural products and their derivatives (e.g., vinca alkaloids, antitumor antibiotics, enzymes, lymphokines, and epipodophyllotoxins): vinblastine, vincristine, vindesine, bleomycin, dactinomycin, daunorubicin, doxorubicin, epirubicin, idarubicin, ara-C, paclitaxel (paclitaxel is commercially available as Taxol®), mithramycin, deoxyco-formycin, mitomycin C, L-asparaginase, interferons (especially IFN-a), etoposide, and teniposide. Other antiproliferative cytotoxic agents are navelbene, CPT-11, anastrazole, letrazole, capecitabine, reloxafine, cyclophosphamide, ifosamide, and droloxafine.Additional antiproliferative cytotoxic agents include, but are not limited to, melphalan, hexamethylmelamine, thiotepa, cytarabine, idatrexate, trimetrexate, dacarbazine, L-asparaginase, camptothecin, topotecan, bicalutamide, flutamide, leuprolide, pyridobenzoindole derivatives, interferons, and interleukins.Preferred classes of antiproliferative cytotoxic agents are EGFR inhibitors, Her-2 inhibitors, CDK inhibitors, and Herceptin® (trastuzumab) (see, for example, U.S. Patent Nos. 6,537,988 and 6,420,377).Such compounds can be given according to currently known techniques for their administration.

[0081] In some embodiments, the modified NKT cells disclosed herein may be administered in any physiologically acceptable excipient where the modified NKT cells may find a suitable site for replication, proliferation, and / or engraftment. In some embodiments, the modified NKT cells disclosed herein may be introduced by injection, catheter, or the like. In some embodiments, the modified NKT cells disclosed herein may be frozen at liquid nitrogen temperature and stored for long periods of time and may be thawed and used. Once frozen, the modified NKT cells will typically be stored in 10% DMSO, 50% FCS, 40% RPMI 1640 medium. Once thawed, the cells may be expanded by the use of growth factors and / or feeder cells associated with the culture of NKT cells.

[0082] In some embodiments, the modified NKT cells disclosed herein may be provided in the form of a pharmaceutical composition comprising an isotonic excipient prepared under sufficiently sterile conditions for human administration. For general principles in pharmaceutical preparations, the reader is referred to Cell Therapy: Stem Cell Transplantation, Gene Therapy, and Cellular Immunotherapy, by G. Morstyn & W. Sheridan eds, Cambridge University Press, 1996; and Hematopoietic Stem Cell Therapy, E. D. Ball, J. Lister & P. ​​Law, Churchill Livingstone, 2000. The selection of the cellular excipient and any associated elements of the composition comprising the modified NKT cells disclosed herein will be adapted to the route and device used for administration. In some embodiments, the composition comprising the modified NKT cells may also comprise or be associated with one or more other components that facilitate engraftment or functional mobilization of the modified NKT cells. Suitable components include matrix proteins that support or enhance adhesion of modified NKT cells or complementary cell types. In another embodiment, the composition may include a resorbable or biodegradable matrix scaffold.

[0083] In some embodiments, the modified NKT cells may be administered and dosed according to the principles of good medical practice, taking into account the clinical condition of the individual patient, the site and method of administration, the scheduling of administration, the age, sex, weight of the patient, and other factors known to medical practitioners. The pharmacologic "effective amount" for purposes herein is therefore determined by such considerations as are known in the art. The amount must be effective to achieve improvement, including but not limited to improved survival or faster recovery, or improvement or disappearance of symptoms, and other indicators selected as appropriate measures by the skilled artisan. The modified NKT cells may be administered to a subject in the following locations: clinics, doctors' offices, emergency departments, hospital wards, intensive care units, operating rooms, catheterization suites, and radiologic suites.

[0084] In other embodiments, the modified NKT cells are stored for later implantation / infusion. The modified NKT cells may be divided into two or more aliquots or units, such that a portion of the modified NKT cells is retained for later application and a portion is immediately applied to the subject. Medium to long-term storage of all or a portion of the cells in a cell bank is also within the scope of the present invention and is disclosed in U.S. Patent Publication No. 2003 / 0054331 and Patent Publication No. WO 03 / 024215, which are incorporated herein by reference in their entireties. At the end of processing, the enriched cells may be loaded by any means known to one of skill in the art into a delivery device (such as a syringe) for placement into the recipient.

[0085] Pharmaceutical compositions comprising an effective amount of NKT cells are also contemplated by the present invention.These compositions comprise an effective number of modified NKT cells, optionally in combination with a pharma- ceutically acceptable carrier, additive, or excipient.Systemic administration of modified NKT cells to a subject may be preferred in certain indications, while direct administration at or near the site of a tumor may be preferred in other indications.

[0086] In some embodiments, the modified NKT cells may be optionally packaged in a suitable container with written instructions for a desired purpose, such as reconstitution or thawing (if frozen) of the modified NKT cells prior to administration to a subject.

[0087] Those skilled in the art will readily recognize that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The details and examples described herein are representative of certain embodiments, are illustrative, and are not intended as limitations on the scope of the invention. Modifications and other uses herein will occur to those skilled in the art. These modifications are encompassed within the spirit of the invention. It will be readily apparent to those skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.

[0088] The articles "a" and "an" as used herein should be understood to include plural referents unless clearly indicated to the contrary in the specification and claims. A claim or description containing "or" between one or more members of a group is deemed satisfied if one, more than one, or all of the members of the group are present, used, or otherwise relevant in a given product or process, unless indicated to the contrary or otherwise clear from the context. The invention includes embodiments in which exactly one member of a group is present, used, or otherwise relevant in a given product or process. The invention also includes embodiments in which more than one member or all members of a group are present, used, or otherwise relevant in a given product or process. Moreover, it should be understood that the present invention provides for all variations, combinations, and permutations, wherein one or more limitations, elements, clauses, descriptive terms, etc. from one or more of the listed claims are introduced into another claim, relying on the same base claim (or any other claim, if relevant), unless otherwise indicated or unless it is obvious to one skilled in the art that a contradiction or inconsistency would result. All embodiments described herein are contemplated as applicable to all different aspects of the present invention, where appropriate. It is also contemplated that any of the embodiments or aspects may be freely combined with one or more other such embodiments or aspects, where appropriate. When elements are presented as a list, for example in a Markush group or similar format, it should be understood that each subgroup of the elements is also disclosed and that any element(s) may be removed from the group. In general, when the invention, or aspects of the invention, are referred to as including certain elements, features, etc., it should be understood that a certain embodiment of the invention or aspect of the invention consists of or consists essentially of such elements, features, etc. For purposes of simplicity, these embodiments have not in all instances been specifically described in great detail herein.It should also be understood that any embodiment or aspect of the invention may be explicitly excluded from the claims, regardless of whether a specific exclusion is recited in the specification. For example, any one or more active agents, additives, ingredients, optional agents, organism types, disorders, subjects, or combinations thereof may be excluded.

[0089] Where a claim or description is directed to a composition of matter, it should be understood that methods of making or using the subject compositions according to any of the methods disclosed herein, and methods of using the subject compositions for any of the purposes disclosed herein, are aspects of the invention, unless otherwise indicated or it is apparent to one of ordinary skill in the art that a contradiction or inconsistency would arise. Where a claim or description is directed to a method, it should be understood that methods of making compositions useful, for example, for carrying out the method, and products produced according to the method, are aspects of the invention, unless otherwise indicated or it is apparent to one of ordinary skill in the art that a contradiction or inconsistency would arise.

[0090] When ranges are given herein, the invention encompasses embodiments in which the endpoints are included, in which both endpoints are excluded, and in which one endpoint is included and the other endpoint is excluded. It should be assumed that both endpoints are included unless otherwise indicated. Furthermore, unless otherwise indicated or otherwise evident from the context and the understanding of one of ordinary skill in the art, values ​​expressed as ranges should be understood to include any particular value or subrange within the range set forth in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. It is also understood that when a series of numerical values ​​is set forth herein, the invention encompasses embodiments related to any intervening value or range defined by any two values ​​in the series, where the minimum value may be considered as the minimum and the maximum value may be considered as the maximum. Numeric values, as used herein, include values ​​expressed as a percentage. For any embodiment of the invention in which a numerical value is prefaced by "about" or "approximately," the invention encompasses the embodiment in which the exact value is recited. For any embodiment of the invention where a numerical value is not prefaced by "about" or "approximately," the invention includes embodiments where the value is prefaced by "about" or "approximately."

[0091] As used herein, "A and / or B," where A and B are different claim terms, generally means at least one of A, B, or both A and B. For example, a sequence that is complementary and / or hybridizes to another sequence includes (i) a sequence that is complementary to the other sequence, even if the one sequence does not necessarily hybridize to the other sequence under all conditions, (ii) a sequence that hybridizes to the other sequence, even if the one sequence is not fully complementary to the other sequence, and (iii) a sequence that is both complementary and hybridizes to the other sequence.

[0092] "Approximately" or "about" generally includes numbers that fall within 1% of a number, or in some embodiments, within 5% of a number, or in some embodiments, within 10% of a number, in either direction (greater or less than that number), unless otherwise stated or otherwise clear from the context (unless such number unacceptably exceeds 100% of the possible values). Unless expressly indicated to the contrary, in any method claimed herein that includes two or more acts, the order of the method acts is not necessarily limited to the order in which the method acts are recited, but it should be understood that the invention includes embodiments in which the order is so limited. It should also be understood that any product or composition described herein may be deemed to be "isolated" unless otherwise indicated or clear from the context.

[0093] As used herein, the terms "comprising" or "comprising" are used in reference to compositions, methods, and their respective component(s) that are essential to the present invention, and allow further room for the incorporation of non-specified elements, whether or not essential.

[0094] As used herein, the term "consisting essentially of" refers to elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of the embodiment of the invention.

[0095] The term "consisting of" as used herein refers to compositions, methods, and their respective components, and excludes any element not recited within that description of an embodiment. EXAMPLES

[0096] Modified NKT cells are optionally obtained via the method outlined in FIG. 1A. On day 0, NKT cells are isolated; on days 1-4, the population of NKT cells is expanded with IL-2; on day 5, the NKT cells are transduced with at least one retroviral vector; on days 6-13, the population of NKT cells is expanded with IL-2; on day 14, the NKT cells are assessed using one or more functional assays. Transduction of NKT cells on day 5 occurs with one or more retroviral vectors (FIG. 1B). One retroviral vector is SFG(i)GFP and the second retroviral vector is SFG.IL12(i)GFP. Both vectors contain an IRES and GFP. The SFG.IL12(i)GRP vector contains the p40 and p35 subunits of IL-12 connected via a flexible linker.

[0097] After cytokines were administered, the modified NKT cells were assessed. Flow cytometry plots showed NKT cell purity, NKT cell transduction efficiency as a percentage of GFP+ cells, and CD62L expression in non-transduced NKT cells (NT), NKT cells transduced with a control GFP vector (GFP), and NKT cells transduced with an IL12(i)GFP vector (Figures 1C-1E). Figure 1E clearly shows that CD62L is significantly upregulated in NKT cells transduced with IL12(i)GFP.

[0098] Various cytokines including IL-12, IFN-γ, and IL-4 produced by NKT cells (NT cells, GFP NKT cells, and IL12(i)GFP NKT cells) were measured and quantified (Figure 2A). NKT cells were either resting (unstimulated) or activated (treated with anti-CD3 and anti-CD28 antibodies). NKT cell persistence was then assessed in a xenograft NSG mouse model in which mice were transplanted with GFP-labeled NKT cells or IL12(i)GFP firefly luciferase-labeled NKT cells (Figure 2C). Mice were imaged by an IVIS kinetic machine on days 0, 2, 4, and 7 after NKT cell injection, and then euthanized on day 10. Tissue collection included peripheral blood, liver, and spleen. Bioluminescence imaging (BLI) of tumors was shown for GFP and IL12(i)GFP mice on days 0, 2, 4, and 7 (Figure 2D). In addition, quantification of human NKT cells in peripheral blood, liver, and spleen was also provided for samples collected 10 days after NKT cell infusion (Figure 2E).

[0099] To fully characterize the transcriptomic effects of IL-12 on NKT, IL12(i)GFP NKT and GFP NKT were compared by performing RNASeq. Increased expression of IL-12A / B introduced by retroviral vector was observed in IL12(i)GFP NKT. In addition, differential expression of approximately 380 genes was observed between IL12(i)GFP NKT and GFP NKT (Figure 2B), including HAVRC2, SELL, and IFNG, and proinflammatory genes. In addition, high expression of CD62L was identified compared to GFP NKT, which is consistent with human NKT with high proliferation capacity in vivo. Furthermore, after activation via iTCR or CAR, IL-12 NKT acquired a proinflammatory phenotype, as IL-12 NKT produced more IFN-g and less IL-4 compared to control NKT.

[0100] Various retroviral vectors were assessed for transduction of NKT cells with CAR. The first retroviral vector was SFG.CAR.CD19(i)IL12; the second retroviral vector was SFG.CAR.GD2; the third retroviral vector was SFG.CAR.GD2(i)IL12; and the fourth retroviral vector was SFG.CAR.GD2(i)IL12TM (Figures 3A and 5A). CD19.CAR used scFv FMC.63 and GD2.CAR used 1A7 scFv. Representative flow cytometry plots show CAR expression and CD62L expression in control (NT) and CAR-transduced NKT cells assessed at day 10 of culture (Figures 3B-3C and 5B-5C).

[0101] NKT cells transduced with the three CAR retroviral vectors discussed above from Figure 3A were co-cultured with CHLA-225 (E:T=1:1). Residual tumor cells were collected and stained with anti-iTCR (Vβ11) and anti-GD2 to identify and quantify NKT cells and neuroblastoma cells by flow cytometry, respectively (Figure 4A). Mice were inoculated with 2*10 6 Mice were implanted intravenously with 5x10 CHLA-225 firefly luciferase-labeled neuroblastoma tumor cells. Ten days later, mice were 6 CAR+ NKT cells were given intravenously (as described in FIG. 3A). Mice were imaged weekly by an IVIS kinetic machine and euthanized on day 80. Samples were collected from peripheral blood, liver, and spleen (FIG. 4B). In addition, tumor BLI was provided on days 0, 14, 21, 35, 56, and 79 (FIG. 4C) to reveal human NKT cells (iNKT cells) in peripheral blood, liver, and spleen. + CD45 + ) was measured and plotted (Figure 4D).

[0102] NKT cells were transduced with the three CAR retroviral vectors discussed above from Figure 5A. As shown in Figure 5B, NKT cells unexpectedly transduced with a retroviral vector encoding GD2 CAR and membrane-bound IL-12 showed greater levels of GD2 CAR expression (SFG.CAR.GD2(i)IL12TM; 78%) when compared to NKT cells transduced with a retroviral vector encoding GD2 CAR and soluble IL-12 (SFG.CAR.GD2(i)IL12TM; 52%). NKT cells transduced with the three CAR retroviral vectors discussed above from Figure 5A were co-cultured with CHLA-225 (E:T=1:1). Residual tumor cells were collected and stained with anti-iTCR (Vβ11) and anti-GD2 to identify and quantify NKT cells and neuroblastoma cells by flow cytometry, respectively. As shown in Figure 7A, upon repeated exposure to tumor cells in vitro, GD2.CAR NKTs with membrane-bound IL-12 exhibited comparable cytotoxic activity to GD2.CAR NKTs expressing soluble IL-12. Although IFN-γ production by GD2.CAR NKTs exceeded that of control NKTs (NT), there was no significant difference in the level of IFN-γ production between GD2.CAR NKTs with membrane-bound IL-12 and GD2.CAR NKTs with soluble IL-12 (Figure 7B). As expected, GD2.CAR NKTs modified to express soluble IL-12 showed greater IL-12 production when compared to GD2.CAR NKTs with NT and membrane-bound IL-12 (Figure 7B). As shown in Figure 7C, NKTs transduced with GD2.CAR(i)IL12TM retroviral vectors exhibited persistence in vivo. Mice will be implanted intravenously with CHLA-225 firefly luciferase-labeled neuroblastoma tumor cells. Ten days later, mice will receive CAR+ NKT cells intravenously (as described in FIG. 5A). Mice will be imaged weekly by an IVIS kinetic machine and euthanized on day 80. Samples will be collected from peripheral blood, liver, and spleen.In addition, tumor BLI was performed at various points in time to assess the expression of human NKT cells (iNKT cells) in peripheral blood, liver, and spleen. + CD45 + ) will be measured and plotted.

[0103] Future mouse models will involve the use of NKT cells transduced with a CAR retroviral vector, as described above and in Figures 3A and 5A, where the NKT cells will be further modified to reduce or eliminate expression of CD62L. Expression of CD62L can be reduced or eliminated using any technique known to one of skill in the art.

[0104] Based on the data from the above experiments, it was surprisingly found that IL-12 reprograms NKT for long-term persistence.In addition, when NKT cells are transduced to co-express CAR, NKT unexpectedly acquires the long-term ability to eliminate tumor cells and protect mice from tumor rechallenge, further demonstrating the long-term persistence of IL-12-expressing NKT.

[0105] method Cell Lines. The tumor cell line CHLA-225 was maintained in culture in RPMI 1640 (Gibco) supplemented with 10% FBS (Sigma), 1% L-glutamine (Gibco), and 1% penicillin-streptomycin (Gibco) at 37°C in a humidified atmosphere containing 5% CO2. The CHLA-225 cell line was transduced with a SFGγ retroviral vector encoding the firefly luciferase gene (FFluc). Cells were maintained in culture for less than 2 continuous months, after which aliquots from the original amplified vial were used. All tumor cell lines were routinely tested to exclude mycoplasma contamination and were assessed for tumor marker expression by flow cytometry to confirm identity.

[0106] Generation of retroviral constructs and retroviral supernatants. Retroviral supernatants were prepared by transient transfection of 293T cells and used to transduce NKTs (isolated as described below). Sequences of the p40 and p35 subunits of IL-12 were obtained from the NCBI website and linked via overlap PCR together with flexible linkers as described from Anderson R. et al. (Forward p40: TATCCATGGGTCACCAGCAGTTGG (SEQ ID NO: 1); Reverse p40: CCACCGCCGCTTCCGCCACCGCCGCTTCCACCGCCACCACTGCAGGGCACAGATGC (SEQ ID NO: 2); Forward p35: GTGGCGGAAGCGGCGGTGGCGGCAGCGGCGGTGGCAGCAGAAACCTCCCCGTGGC (SEQ ID NO: 3); Reverse p35: GACGCATGCTTAGGAAGCATTCAGATAGCTCATCACTC (SEQ ID NO: 4)). The entire IL-12 cassette was cloned into the SFG retroviral vector containing an IRES and GFP. As a control, the vector SFG containing only an IRES and GFP was used. Cassettes encoding the GD2-specific scFv (scFv14G2a), the stalk and transmembrane domains of CD8a, the CD28 intracellular domain, and the CD3z chain were previously cloned into the SFG backbone (GD2.CAR). IL-12 was then cloned in the GD2.CAR cassette separated by an IRES element (GD2.CAR(i)IL12). Finally, for selected experiments, the scFv14G2a in the GD2.CAR(i)IL12 vector was exchanged for a CD19-specific scFv (scFv FMC.63) to obtain the CD19.CAR(i)IL12 vector.

[0107] To generate the GD2.CAR(i)IL12TM construct, the stop codon was removed from GD2.CAR(i)IL12 at the end of the p35 subunit and the stalk and transmembrane domains of CD8a were added.

[0108] Isolation, transduction, and in vitro expansion of NKT cells. Buffy coats from healthy volunteer blood donors were purchased from Gulf Coast Regional Blood Center (Houston, Texas, USA). Peripheral blood mononuclear cells (PBMCs) were isolated by Lymphoprep (Accurate Chemical and Scientific Corporation) density gradient centrifugation. NKTs were purified from PBMCs using anti-iNKT microbeads (Miltenyi Biotech). NKTs were cultured in complete medium consisting of 45% Click medium (Irvine Scientific), 45% RPMI 1640 (Hyclone), 10% FBS (Hyclone), 1% L-glutammine (Gibco), and 1% penicillin-streptomycin (Gibco). For NKT selection, the negative fraction was used as a feeder after irradiation (40 Gy) in the presence of α-galactosylceramide (αGalCer, 100 ng / mL, Diagnocine LLC) and IL-2 (200 IU / mL, Stem Cell Factor) at a PBMC:NKT ratio of 10:1. NKT were transduced on day 5 in retronectin-coated plates and further expanded for 10 days in the presence of IL-2 and then used for functional assays.

[0109] Immunophenotypic analysis: NKT cells were stained with Abs against CD3 (APC-H7, clone SK7), CD62L (BV605, clone DREG-56), CD4 (PE-Cy7, clone SK3), CD8 (Alexa Fluor, clone RPA-T8 700), and CD45 (APC) from BD Biosciences, and IL-12 (p70, APC) and CD212 (IL12R b2, APC) from Miltenyi Biotech. Tumor cells were stained with Abs against GD2 (PE) and CD276 (B7-H3, BV421, clone 7-517) from BD Biosciences. NKT purity was assessed by staining cells with PE-conjugated Ab specific for TCR Va24 chain (anti-iNKT, clone 6B11 from BD Biosciences) or Ab specific for TCR β11 chain (FITC, Beckman Coulter). To detect GD2.CAR expression in transduced NKT cells, 1A7 anti-idiotypic mAb specific for 14G2a.scFv and PE- or APC-conjugated rat anti-mouse secondary mAb were used (BD Biosciences). Data acquisition was performed on a BD FACSCanto II or BD LSRFortessa using BD FACS-Diva software. Data analysis was performed with FlowJo software.

[0110] Western blot: Protein lysates were separated on 4%-15% SDS polyacrylamide gel electrophoresis gels (SDS-PAGE, Bio-Rad). After protein transfer onto polyvinylidene difluoride membranes (Bio-Rad), membranes were blocked in 5% skim milk in TBS-T and incubated with primary and secondary Abs in 1% milk and TBS-T. The following Abs were used: α-Stat4 (C46B10, dilution 1:1000) and α-phospho-Stat4 (Tyr693, dilution 1:1000) from Cell Signaling; horseradish peroxidase (HRP)-conjugated α-CD3ζ (dilution 1:1000) from Santa Cruz; HRP-conjugated secondary Ab (goat-α-rabbit #32460, dilution 1:500) from Thermo Scientific. Incubation with primary Ab was performed overnight at 4°C, while incubation with secondary Ab was performed for 1 h at room temperature. Membranes were developed with Clarity Max Western ECL Substrate (Bio-Rad) or SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific) on a gel station (Bio-Rad).

[0111] ELISA:NKT(5*10 5 Cells / well) were cultured in 24-well plates coated with anti-CD3 (1 mg / mL, Miltenyi Biotech) and anti-CD28 (1 mg / mL, BD Biosciences). Uncoated wells were used as negative controls. Supernatants were collected after 24 hours of culture from each cycle of co-culture. IFN-γ, IL-4, and IL-12 were measured using specific ELISAs (R&D System).

[0112] Repeated co-culture: NKT(2.5*10 510 cells / well) were co-cultured with CHLA-225 at an E:T ratio of 1:1 in 24-well plates in the absence of cytokines (Cycle I). On day 3, all cells were cultured at 2.5*10 5 On day 6, all cells were transferred into new wells pre-seeded with 2.5*10 neuroblastoma cells (cycle II). 5 Neuroblastoma cells were pre-seeded into new wells (cycle III). At the end of every cycle, cells were harvested and stained for CD3 or TCRβ11 and GD2 or B7-H3 mAbs to detect NKT and tumor cells, respectively. The number of tumor cells remaining in culture was counted by flow cytometry using CountBright absolute counting beads (Invitrogen).

[0113] Xenogeneic neuroblastoma model: In experiments assessing NKT persistence in vivo, female and male NSG mice (7-9 weeks old, obtained from the UNC Animal Core) were administered 5*10 6 Mice were injected intravenously (iv) with FFluc-labeled GFP NKT or IL12(i)GFP NKT. NKT persistence was monitored for 7 days by bioluminescence (BLI; total flux, photons / sec) using an IVIS kinetic in vivo imaging system (PerkinElmer). Mice were euthanized on day 10, peripheral blood was collected from the heart and spleen, and livers were crushed on a cell strainer and washed with 2 mL of PBS. Peripheral blood, spleen, and liver were analyzed to detect the presence of NKT [stained with Abs against CD3 (APC-H7, clone SK7) and CD45 (APC, clone 2D1)] by flow cytometry, and the percentage of human NKT was enumerated. In experiments assessing the antitumor activity of NKT in vivo, female and male NSG mice (7-9 weeks old, obtained from UNC Animal Core) were injected with 2*10 IgG via tail injection. 6 FFluc-labeled CHLA-225 cells were intravenously (iv) injected. Seven days after tumor cell injection, mice were 6Mice were iv-injected with CAR.CD19(i)IL12, CAR.GD2, or CAR.GD2(i)IL12-transduced NKT. Neuroblastoma tumor growth was monitored weekly by bioluminescence (BLI; total flux, photons / sec) using an IVIS Kinetic In Vivo Imaging System (PerkinElmer). Mice were sacrificed according to pre-determined guidelines for signs of tumor growth and discomfort or to terminate the experiment. When mice were euthanized, peripheral blood was collected from the heart and spleen, and the liver was crushed on a cell strainer and washed with 2 mL of PBS. Peripheral blood, spleen, and liver were analyzed to detect the presence of NKT [stained with Abs against CD3 (APC-H7, clone SK7) and CD45 (APC, clone 2D1)] by flow cytometry to enumerate the percentage of human NKT.

Claims

1. A modified natural killer T (NKT) cell, wherein the NKT cell expresses transgenic IL-12.

2. The modified NKT cell of claim 1 , comprising a chimeric antigen receptor (CAR).

3. The modified NKT cell of claim 1, wherein the CAR is GD2.CAR.

4. The modified NKT cell of claim 1, wherein the CAR is a CD19 CAR.

5. The modified NKT cell of claim 1 , wherein the NKT cell is a human NKT cell.

6. The modified NKT cell of claim 1 , wherein the NKT cell is a non-human NKT cell.

7. The modified NKT cell of claim 1 , wherein the NKT cell is a mouse NKT cell.

8. The modified NKT cells of claim 1, wherein the NKT cells are isolated from peripheral blood.

9. The modified NKT cell of claim 1, wherein the NKT cell expressing transgenic IL-12 is produced by transduction of the NKT cell with a retroviral supernatant containing IL12.

10. The modified NKT cell of claim 1, wherein the transgenic IL-12 is bound to the NKT cell membrane.

11. The modified NKT cell of claim 10, wherein the transgenic IL-12 is bound to the NKT cell membrane via a CD4 or CD8 stalk.

12. The modified NKT cell of claim 10, wherein the transgenic IL-12 is bound to the NKT cell membrane via a CD8 stalk.

13. The modified NKT cell of claim 12, wherein the CD8 stalk comprises the hinge region and transmembrane domain of CD8.

14. 14. The modified NKT cell of claim 13, wherein the CD8 stalk is modified to remove one or more cysteine ​​residues from the hinge region.

15. 14. The modified NKT cell of claim 13, wherein the CD8 stalk is modified to replace one or more cysteine ​​residues from the hinge region with serine residues.

16. The modified NKT cell according to any one of claims 12 to 15, wherein the CD8 stalk is a CD8a stalk.

17. The modified NKT cell of claim 16, wherein the CD8 stalk comprises a hinge region having the amino acid sequence of SEQ ID NO:

12.

18. The modified NKT cell of claim 10, wherein the transgenic IL-12 is bound to the NKT cell membrane via a CD4 stalk.

19. 19. The modified NKT cell of claim 18, wherein the CD4 stalk comprises the hinge region and transmembrane domain of CD4.

20. 20. The modified NKT cell of claim 19, wherein the CD4 stalk is modified to remove one or more cysteine ​​residues from the hinge region.

21. 20. The modified NKT cell of claim 19, wherein the CD4 stalk is modified to replace one or more cysteine ​​residues from the hinge region with serine residues.

22. The modified NKT cell of claim 18, wherein the CD4 stalk comprises a hinge region having the amino acid sequence of SEQ ID NO:

13.

23. The modified NKT cells of claim 1, wherein the modified NKT cells enhance tumor control and improve survival compared to control cells.

24. The modified NKT cell of claim 1, wherein the NKT cell controls tumor growth upon tumor re-challenge compared to control cells.

25. The modified NKT cell of claim 1 , wherein the NKT cell exhibits increased anti-tumor activity.

26. The modified NKT cell of claim 1, wherein the NKT cell exhibits increased expression of CD62L.

27. The modified NKT cell of claim 1 , wherein the NKT cell exhibits long-term persistence.

28. 2. The modified NKT cell of claim 1, wherein the NKT cell exhibits enhanced cytotoxic activity upon repeated exposure to tumor cells in vitro compared to control cells.

29. The modified NKT cell of claim 1 , wherein the NKT cell exhibits a reduced risk of causing graft-versus-host disease.

30. The modified NKT cell of claim 1 , wherein the NKT cell does not express CD62L.

31. A population of modified NKT cells according to claim 1.

32. A population of genetically modified natural killer T cells (NKTs) isolated from peripheral blood, wherein said NKTs express transgenic IL-12.

33. 33. The population of claim 32, wherein the NKT comprises a chimeric antigen receptor (CAR).

34. 34. The population of claim 33, wherein the CAR is a GD2.CAR.

35. 34. The population of claim 33, wherein the CAR is a CD19 CAR.

36. 33. The population of claim 32, wherein the NKTs are transduced with a CAR and IL-12.

37. The population of claim 32, wherein the NKTs do not express CD62L.

38. 2. A method for producing the modified NKT cells of claim 1, comprising transducing the NKT cells with a retroviral vector.

39. 39. The method of claim 38, wherein the retroviral vector comprises IL-12.

40. 39. The method of claim 38, wherein the retroviral vector comprises a green fluorescent protein (GFP).

41. 39. The method of claim 38, wherein the retroviral vector comprises an internal ribosome entry site (IRES).

42. 39. The method of claim 38, wherein the retroviral vector comprises a CAR.

43. 39. The method of claim 38, wherein the retroviral vector comprises a CD19 CAR.

44. 39. The method of claim 38, wherein the retroviral vector comprises GD2.CAR.

45. A composition for treating cancer in a subject, comprising the modified NKT cells of claim 1.

46. 46. ​​The composition of claim 45, wherein the subject is a mammal.

47. 46. ​​The composition of claim 45, wherein the subject is a human.

48. 46. ​​The composition of claim 45, wherein the composition is administered intravenously to the subject.

49. A cell transformed to express a CAR and an exogenous membrane-binding portion, wherein the exogenous membrane-binding portion comprises a transmembrane domain modified to remove one or more cysteine ​​residues.

50. 50. The cell of claim 49, wherein the one or more removed cysteine ​​residues are capable of forming disulfide bonds with cysteine ​​residues otherwise present in the CAR.

51. 50. The cell of claim 49, wherein the exogenous membrane-bound moiety is IL-12.

52. 50. The cell of claim 49, wherein the exogenous membrane-associated moiety is attached to the cell membrane via a CD4 or CD8 stalk.

53. 50. The cell of claim 49, wherein the transmembrane domain comprises a CD8 stalk.

54. 54. The cell of claim 53, wherein the CD8 stalk comprises a CD8 hinge region.

55. 50. The cell of claim 49, wherein the transmembrane domain is modified to replace the one or more cysteine ​​residues with serine residues.

56. 50. The cell of claim 49, wherein the transmembrane domain comprises a CD8a hinge region having the amino acid sequence of SEQ ID NO:

12.

57. 50. The cell of claim 49, wherein the exogenous membrane-binding moiety is bound to the cell membrane via a CD4 stalk.

58. 58. The cell of claim 57, wherein the CD4 stalk comprises a CD4 hinge region.

59. 50. The cell of claim 49, wherein the transmembrane domain comprises a CD4 hinge region having the amino acid sequence of SEQ ID NO: 13.

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