Novel method to generate "metabolically-fit" til for cancer immunotherapy

Abstract

Described herein are methods for generating clinical grade metabolically enhanced Tumor Infiltrating Lymphocytes (TILs) from a subject with a tumor. Described herein are compositions comprising the TILs and methods of their use in treating cancer.

Description

[0001] Attorney Docket No. 206085-0189-00WO

[0002] TITLE OF THE INVENTION

[0003] NOVEL METHOD TO GENERATE “METABOLIC ALLY-FIT” TIL FOR CANCER

[0004] IMMUNOTHERAPY

[0005] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0006] This invention was made with government support under CA239952 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0007] BACKGROUND OF THE INVENTION

[0008] Adoptive transfer of T cells that can recognize tumor and cause tumor cell death is a promising approach and has gained momentum after novel anti-tumor T cells could be engineered by viral transductions of tumor reactive T cell receptor (TCR), or chimeric antigen receptors (CAR). However, many confounding factors as susceptibility to immunosuppression, or T cell’s inability to persist and undergo activation induced cell death due to chronic antigen stimulation in a tumor microenvironment remains. In addition to engineering one or two TCRs or CARs on a T cell renders them dedicated specificity, which when lost, can lead to tumor relapse and ineffective outcomes. Thus, using tumor-infiltrating lymphocytes (TILs) presents an option where tumor reactive T cells with multiple specificities or even neo-antigens can be used to treat tumors without undergoing recombinant engineering processes However, reproducibly generating TILs from different solid tumors, especially cold ones where low TILs infiltrate and high immunosuppression, has been an uphill task and reason for low enthusiasm in TIL therapy.

[0009] Thus, there is a need in the art for improved compositions and methods for the adoptive transfer of T cells. This invention satisfies this unmet need.

[0010] SUMMARY OF THE INVENTION

[0011] In one embodiment, the present invention comprises methods for generating clinical grade metabolically enhanced Tumor Infiltrating Lymphocytes (TILs) from a subject with a tumor, wherein the method comprises: obtaining TILs from the subject’s tumor; and expanding the TILs.

[0012] In one embodiment, the obtaining comprises incubating small pieces of the tumor of the subject in a first media comprising at least one interleukin wherein cells in the small pieces become isolated as individual cells; removing the cells from the first media; placing the cells in a second media comprising at least one antibody; adding at least one conditioning cytokine to the second media; adding at least one interleukin to the second media; isolating TILs from the second media.

[0013] In one embodiment, expanding the TILs comprises mixing feeder cells with the TILs; separating the TILs from the feeder cells and culturing the TILs in a gas permeable cell culture flask in a third media wherein the third media comprises at least one interleukin and at least one antibody; transferring the programmed meTILs to a second gas permeable cell culture flask and culturing the TILs in a replenished third media; removing the TILs to obtain clinical grade metabolically enhanced TILs.

[0014] In one embodiment, the at least one interleukin of is IL-2. In one embodiment, the at least one antibody is selected from the group consisting of anti-CD3, anti-CD28, and a combination thereof. In one embodiment, the at least one antibody is immobilized on a bead.

[0015] In one embodiment, isolating TILs from the second media comprises isolation via at least one technique selected from the group consisting of cell sorting, immunoprecipitation, antibody staining, and any combination thereof. In one embodiment, the method further comprises engineering the TILs to express a gene or protein of interest. In one embodiment, the tumor comprises melanoma tumor, an ovarian tumor, a head and neck tumor, or a prostate tumor.

[0016] In one embodiment, the present invention provides compositions comprising the TILs generated by the methods as disclosed herein. In one embodiment, the compositions comprise a pharmaceutically acceptable carrier.

[0017] In one embodiment, the present invention provides methods of treating a cancer in a subject in need thereof, the comprising administering to the subject the compositions as disclosed herein. In one embodiment, the cancer comprises lung cancer, melanoma, ovarian cancer, head and neck cancer, or prostate cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The following detailed description of embodiments of the invention will be better understood when read in conjunction with the appended drawings. It should be understood that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0019] Figure 1 depicts a representative illustration of the G-Rex™ platform. The G- Rex™ platform enhances T-cell expansion by using a gas-permeable membrane for: increased oxygen delivery, enables large-scale, high-density cell growth, maintains high viability and functionality, etc.

[0020] Figure 2 depicts a representative schematic of the Two Phase Expansion Protocol. Method: Tumor Infiltrating Lymphocytes (TILs) are extracted from human patient melanoma and prostate tissue and then expanded following a 2-phase process where the first phase allows for reprogramming of TILs and the second phase allows for rapid expansion of the TILs.

[0021] Figure 3, comprising Figure 3A through Figure 3F, depicts representative data demonstrating that TILs can be reprogrammed with hybrid conditioning during expansion. Melanoma TILs expanded in IL2, or hybrid programming conditions were characterized three weeks post-expansion. The distribution of CD4 and CD8 cells (Fig. 3 A). TILs were either TCR stimulated, or co-cultured with human melanoma cells 624- MEL overnight before cytokine secretion was evaluated by Enzyme Linked Immunosorbent Assay (ELISA) (Fig. 3B and Fig. 3C) and tumor cell death analyzed by annexin V and 7AAD (Fig. 3D). Figure 3E depicts representative data characterizing the CD8+ fraction in human melanoma TILs. Figure 3F depicts representative data characterizing the CD4+ fraction in human melanoma TILs. Activation and homing markers CD103, CD62L, CD28, CD25, and CD38 and specificity of T cells to 624-mel was accessed using CD137, GzmB, and CD107a after overnight co-culture (Fig. 3E and Fig. 3F). TILs were either TCR stimulated with anti-CD3 and anti-28 antibodies (2 ug / ml each), or co-cultured with human melanoma cells 624-MEL overnight before cytokine secretion was evaluated in the supernatant (Figure 3B and Figure 3C). CTV labeled tumor cells 624-MEL co-cultured overnight with either IL2 or hybrid programmed TILs before tumor cell death was determined using Annexin V / 7ADD staining (Figure 3D). Expression of cell surface activation and homing markers CD103, CD62L, CD28, CD25, and CD38 was determined using FACS (Figure 3E and Figure 3F). The level of CD137, GzmB, and CD 107a was determined in CD8+ or CD4+ TILs fraction after overnight coculture with human melanoma 624-MEL.

[0022] Figure 4, comprising Figure 4A through Figure 4D, depicts representative data demonstrating that expanded TILs are viable and functional. Using the methods described herein, prostate TILs from 3 patients were expanded and functionality and viability of the TILs were accessed after 30-days of expansion. TILs met expansion criteria for infusing 4x106cells / kg patient weight (Fig. 4A) and TIL function was accessed by measuring IFNy release by ELISA after restimulating cells with either CD3 / CD28 overnight or PMA and lonomycin for 3hrs (Fig. 4B). p*<0.05, p** *<0.001.

[0023] Figure 5 depicts representative data demonstrating the optimization of expansion conditions using G-Rex. TILs were expanded using varying G-Rex platforms to optimize expansion conditions for generating billions of TILs.

[0024] Figure 6 depicts a representative illustration of Adoptive T cell Therapy.

[0025] Figure 7 depicts representative data demonstrating the change in tumor size over time after treatment with control, Thl, Thl7, and hybrid Thl / Thl7 cell.

[0026] Figure 8 presents a representative illustration depicting that the CD38-NAD+ axis regulates immunotherapeutic anti-tumor T cell response.

[0027] Figure 9, comprising Figure 9A through Figure 9C, depicts representative data demonstrating Tl / 17 programming of human CD4+ T cells and TILs. Purified CD4+ T cells were obtained from PBL of healthy donors and ex vivo programmed to Thl, Th 17, and hybrid Thl / 17 cells (Figure 9A). Three days after programming, these Th subsets were re-stimulated overnight with anti-CD3 / 28 antibody and the supernatant analyzed to determine cytokines IFNy and IL 17 by ELISA. N=2. TILs from a melanoma patient were obtained and re-programmed under the TO condition (i.e. only IL2), T17, and Tl / 17 and gene expression analysis was done using qPCR. N=2 (Figure 9B). TILs programmed with Tl / 17 hybrid conditions for three days or kept in conventional IL2 underwent REP with soluble OKT3 and high dose IL2 (600 lU / ml) with irradiated allogeneic feeder cells (Figure 9C). Data from FACS based analysis for cell surface markers, and IFNg secretion (after CD3 / aCD28 restimulation) performed on day 12th of REP is shown, p value *<0.05, **<0.01.

[0028] Figure 10 depicts representative data characterizing melanoma TILs expanded in IL2 or hybrid programming conditions three weeks post-expansion. Distribution of CD4 and CD8 was ascertained using the flurochrome conjugated antibodies.

[0029] Figure 11, comprising Figure 11A through Figure 1 IF, depicts representative data demonstrating the molecular signature of CD8 and CD4 fraction in melanoma TILs. Melanoma TILs expanded in IL2 or hybrid programming conditions were characterized three weeks post-expansion by performing single cell sequencing. The data is presented as: UMAP showing cluster distribution between CD4+ and CD8+ T cells (Figure 11 A). Violin plot depicting the expression level of T cells markers CD4 and CD8a (Figure 1 IB). UMAP showing the similar cluster distribution between Hybrid and IL-2 and similar numbers of each cell type in each group (Figure 11C). UpSet plot showing the number of overlapping DEGs between Hybrid and IL-2 in both CD4+ and CD8+ T cells (Figure 1 ID). DEGs are split by up and down regulation. Volcano plots depicting the DEGs between Hybrid Vs IL-2 in CD4+ and CD8+ T cells respectively (Figure 1 IE). X- axis depicts log2(Fold Change) whereas the Y-axis correspond to the -loglO(FDR). Bar plot showing significant functional enrichment of Hybrid Vs IL-2 DEGs in CD4+ and CD8+ T cells (Figure 1 IF). X-axis depicts the -logl0(FDR) for each functional category whereas the Y-axis correspond to gene ontology categories. DEGs are split by up (top) and down (bottom) regulated.

[0030] Figure 12, comprising Figure 12A through Figure 121, depicts representative data characterizing prostate meTILs. Prostate TILs expanded in IL2 or hybrid programming conditions were characterized three weeks post-expansion. Distribution of CD4 and CD8 was ascertained using the fluorochrome conjugated antibodies (Figure 12A). TILs were TCR stimulated with anti-CD3 and anti-28 antibodies (2 ug / ml each) overnight before cytokine secretion was evaluated in the supernatant (Figure 12B and Figure 12C). CD25 expression was evaluated after programming without restimulation (Figure 12D). The level of GzmB, CD 137, and CD 107a was determined in the TILs after overnight coculture with human prostate line 22Rvl (Figure 12E through Figure 12G). The expression level of CD25, CD44, CD137, and CD107a was determined in CD8+ or CD4+ TILs fraction after overnight coculture with human prostate cells C4-2 and 22Rvl, while human melanoma 624-MEL served as specificity control (Figure 12H and Figure 121). Data from one of four experiments with similar results is presented.

[0031] Figure 13 depicts a representative schematic of the process to generate meTILs. 1 : Surgery to remove the tumor from patient; 2: Cut the tumor into small pieces; 3: Add high concentration of IL-2 (6000u / ml) and incubate in 37C incubator; 4: Take out the tumor chunks next day from the media and add them to a new well, replenish the media. On day 2, take out cells from all wells for activation and programming; 5: Programming of TILs with aCD3 + aCD28 (2pg / ml each) for 4 days with conditioning cytokines. Add IL2 (300u / ml) to the culture for an additional 4 days; 6: Prepare the feeder cells by irradiating human PBMCs (from 3 donors) at 25 Gy; 7: Mix the feeder cells with programmed TILs at 1 :50-1 : 100 ratio; 8: Expand the TILs in a GREX-100 for 7 days in the presence of IL2 (300u / ml) and aCD3 (300ng / ml); 9: Transfer TILs to a GREX-500 and expand TILs for an additional 7 days in the presence of IL2 (300u / ml) and aCD3 (300ng / ml); 10: Harvest and analyze TILs for phenotype and function.

[0032] Figure 14 presents representative data depicting the 5-year relative survival for ovarian cancer.

[0033] Figure 15 presents a representative illustration depicting a two approach strategy: hybrid programming and anti-tumor T cell selection.

[0034] Figure 16 presents a representative illustration depicting a two approach strategy: hybrid programming and anti -turn or T cell selection including CD 137+ selection (left panel, CD 137 upregulation for antigen-specific T cells) and ovarian cancer survival based upon CD3 and CD 137 levels (right panel).

[0035] Figure 17 presents a representative illustration of the protocol to obtain metabolically programmed and CD 137+ selected ovarian TILs. 1 : Surgery to remove the tumor from patient; 2: Cut the tumor into small pieces; 3: Add high concentration of IL-2 (6000IU / ml) and incubate in 37C incubator; 4: Once cells reach ~30 million, take out ells from all wells for activation and programming; 5: Programming of TILs with aCD3 + aCD28 (5pg / ml each) for 4 days with conditioning cytokines. Add IL2 (3000IU / mL) to the culture for an additional 4 days; 6: Once TILS reach ~50 million, stain cells with CD137+ antibody and sort on CD137+ cells; 7: Prepare the feeder cells by irradiating human PBMCs (from 3 donors) at 25 Gy. Mix the feeder cells with programmed TTLs at 1 :50-1 :100 ratio; 8: Expand TILS in a GREX-100 for 10 days in the presence of IL2 (300IU / mL) and aCD3 (30ng / ml); 9: Transfer TILs to a GREX-500 and expand TILs for an additional 7 days in the presence of IL2 (300IU / mL) and aCD3 (30ng / ml); 10: Harvest and analyze TILs for phenotype and function.

[0036] Figure 18 presents a representative illustration of the hybrid (CD137 sorted) and NCI IL2hlprotocols. TILs are isolated from ovarian tumors (strategies to improve ovarian immunotherapy outcomes).

[0037] Figure 19 presents representative data depicting that hybrid (CD137+) TILs express reduced exhaustion markers (OVA-6: Post Chemotherapy). Flow cytometry phenotyping. Representative plots show the distribution of CD4+and CD8+T cells following Hybrid versus Conventional expansion. Hybrid expansion yielded a higher proportion of CD8+T cells (62.6%) compared to Conventional IL-2 expansion (6.2%), which was predominantly CD4+. Histograms of PD-1, CD38, and TCF1 / 7 demonstrate that Hybrid-expanded TILs exhibited reduced PD-1 and CD38 expression but increased TCF1 / 7 compared to Conventional TILs.

[0038] Figure 20 presents representative data depicting that hybrid (CD137+) TILs express reduced exhaustion markers (OVA-5: Post-chemotherapy). Flow cytometry phenotyping. Representative plots show the distribution of CD4+and CD8+T cells after expansion. Hybrid (CD 137- sorted) TILs yielded a predominantly CD8+population (82.7%) with fewer CD4+T cells (9.5%), whereas Conventional expansion produced a more balanced CD4+(56.9%) and CD8+(31.0%) profile. Histograms of PD-1, CD38, and TCF1 / 7 demonstrate that Hybrid-expanded TILs expressed lower PD-1 and CD38, suggesting reduced exhaustion compared to Conventional TILs.

[0039] Figure 21 presents representative data depicting that hybrid (CD137+) TILs express reduced exhaustion markers (OVA 10: Pre-chemotherapy). Flow cytometry phenotyping. Representative plots display CD4+and CD8+T cell distributions after ex vivo expansion. Hybrid (CD137-sorted) TILs were enriched for CD8+T cells (35%), while Conventional NCI IL2 high TILs expansion yielded predominantly CD4+cells (39.5%) with limited CD81representation (9.1%). Histograms depict expression of PD-1, CD38, and TCF1 / 7 in Hybrid versus Conventional expanded TILs. Conventional high dose IL-2 expanded TILs exhibited higher CD38 positivity, consistent with greater exhaustion. Hybrid-expanded TILs showed higher TCF1 / 7, suggestive of preserved stemlike phenotype.

[0040] Figure 22 presents representative data depicting that hybrid (CD137+) TILs express reduced exhaustion markers (OVA 9: Post-chemotherapy). Flow cytometry phenotyping. Representative plots display CD4+and CD8+T cell distributions after ex vivo expansion. Hybrid (CD137-sorted) TILs were enriched for CD8+T cells (54.2%), while Conventional NCI IL2 high TILs expansion yielded predominantly CD4+cells (89.4%) with limited CD8+representation (6.1%).

[0041] Figure 23A and Figure 23B present representative data depicting that hybrid and CD 137+ sorting preferentially expands CD8+ TILs (OVA 6: Post-chemo). Single-cell RNA-seq profding. UMAP visualization of single-cell transcriptomic profdes from patient-derived ovarian tumor and matched blood samples (Fig. 23 A). UMAP plots display clustering of immune subsets, including CD8+cytotoxic T lymphocytes (CD8 CTL 1-4), proliferating CD8+T cells (CD8_Prolif), CD4+naive T cells (CD4_Naive_l-2), CD4+regulatory T cells (CD4_Tregs), CD4+cytotoxic / exhausted T cells (CD4_CTL / Exh), natural killer T (NKT) cell subsets (NKT 1-4), mucosal- associated invariant T (MAIT) cells, Th2 cells, B cells, macrophages, and erythrocytes. The accompanying bar plot (Fig. 23B) quantifies cell-type composition, showing Hybrid TILs enriched for CD8 CTLs and proliferative subsets, while Conventional NCI-IL2Ahi expansion favored CD4 naive and Treg populations.

[0042] Figures 24A and Figure 24B present representative data depicting that hybrid and CD 137+ sorting preferentially expands CD8+ TILs (OVA 5: Post- chemo). Single-cell RNA-seq profiling. UMAP projections display clustering of immune subsets from DO tumor TILs, Hybrid (CD137-sorted), Hybrid (CD137-unsorted), and Conventional (NCI IL2 high) expansions. Subsets included CD8 CTL clusters (CD8 CTL 1-4), proliferating T cells (Prolif_TCells_l-2), exhausted CD8 T cells (CD8_Exh), CD4 naive, effector, and regulatory T cells, Th2 cells, HSP+T cells, NK cells, B cells, and macrophages. The accompanying bar plot quantifies relative frequencies across conditions: Hybrid CD137- sorted expansion enriched for CD8 CTLs and proliferative subsets; Hybrid-unsorted produced a broader mix of CD4 and effector subsets; Conventional expansion retained greater representation of CD4 and exhausted populations.

[0043] Figure 25A and Figure 25B present representative data depicting that metabolic programming and CD 137+ sorting hyperexpands select TCR clones, OVA-6 (Postchemo). TCR repertoire and functional assays. (Fig. 25A) Clonal homeostasis analysis showing the relative abundance of TCR clonotypes stratified into rare (0 < X < 1 x I O4), small (1 x 104< X < 1 x 10-3), medium (1 x 103< X < 1 x 102), large (1 x IQ-2< X < 0.1), and hyperexpanded (0.1 < X < 1) groups. Conventional NCI-expanded TILs retained a substantial pool of rare and small clones, while Hybrid-expanded TILs were relatively enriched in large and hyperexpanded clonotypes. (Top right) Clonal space occupancy analysis based on nucleotide-defined ranks ([1 : 1], [2:5], [6:10], [11 : 100], [101: 1000], [1001 : 10000]) demonstrated that Conventional TILs preserved top-ranked clonotypes but distributed repertoire space more evenly across intermediate- and lower-ranked clones, whereas Hybrid (CD137-sorted) expansion was dominated by a narrower set of high- frequency clonotypes. (Fig 25B) Functional recognition assay. IFN-y release was measured upon co-culture of expanded TILs with autologous tumor targets and ovarian tumor cell lines. Hybrid (CD137-sorted) TILs produced stronger IFN-y responses than Conventional NCI IL2 high TILs, indicating a higher frequency of tumor-reactive clonotypes in the Hybrid population.

[0044] Figure 26A and Figure 26B present representative data depicting that metabolic programming and CD 137+ sorting hyperexpands select TCR clones, OVA-5 (Postchemo). TCR repertoire analysis. Clonal homeostasis plots stratified TCR clonotypes into rare (0 < X < 0.001), small (0.001 < X < 0.01), medium (0.01 < X < 0.1), large (0.1 < X < 0.3), and hyperexpanded (0.3 < X < 1) categories (Fig 26A). Hybrid (CD137-sorted) TILs were enriched for large and hyperexpanded clonotypes, while Conventional and Hybrid-unsorted populations preserved a greater fraction of rare and small clones. Clonal space occupancy, assessed by nucleotide-defined rank groups ([1 :1], [2:5], [6: 10], [11 : 100], [101 : 1000], [1001 : 10000]), confirmed that Hybrid CD137-sorted repertoires (Fig 26B) were dominated by top-ranked clonotypes, whereas Conventional and Hybrid- unsorted TILs maintained broader clonal diversity with increased representation of intermediate and rare clones. Figure 27 A and Figure 27B present representative data depicting that metabolic programming and CD 137+ sorting hyperexpands select TCR clones (stratified by cell type). (Fig. 27A) OVA-6: Bar plots show TCR clone size distribution stratified by cell type for peripheral blood (Ova6P.blood), CD137-sorted hybrid expansion (Ova6P.CD137S), high IL-2 NCI protocol expansion (0va6P.R), and baseline tumor- derived (Ova6P. tumor) T cells. Clone sizes were defined as: hyperexpanded (100 < X < 3468), large (20 < X < 100), medium (5 < X < 20), small (1 < X < 5), and singletons (0 < X < 1). Peripheral blood and tumor samples contained predominantly small and singleton clones across multiple subsets, particularly CD4+T cells and Tregs. In contrast, CD137- sorted expansion led to marked enrichment of hyperexpanded clonotypes within CD8+effector subsets, highlighting the selective outgrowth of dominant clones. NCI high IL-2 expansion yielded a broader distribution with small-to-medium clonotypes alongside expanded effector CD8+T cells. Together, these data suggest that CD137-driven selection promotes the dominance of hyperexpanded CD8+clones, whereas high IL-2 expansion maintains greater repertoire diversity while still supporting effector cell outgrowth. (Fig 27B) OVA-5: bar plots depict TCR clone size distribution stratified by cell type for CD137-sorted (Ova5P.CD137S), high IL-2 NCI protocol-expanded (Ova5P.R), and baseline tumor-derived (Ova5P.tumor) T cells. Clone sizes were classified as: hyperexpanded (100 < X < 3508), large (20 < X < 100), medium (5 < X < 20), small (1 < X < 5), and singletons (0 < X < 1). Both CD137-sorted and unsorted hybrid expansions showed enrichment of hyperexpanded clonotypes within CD8+effector subsets, whereas the NCI high IL-2 expanded sample (Ova5P.R) demonstrated a broader representation of small-to-medium clonotypes alongside selective expansion of effector T cells. In contrast, tumor-derived T cells before expansion (Ova5P.tumor) contained a more restricted clonal distribution dominated by small clones and singletons. Together, these data suggest that CD137-based expansion promotes the dominance of hyperexpanded CD8+clonotypes, generating a potent but narrower effector repertoire, while NCI high IL-2 expansion preserves greater clonal diversity across the repertoire.

[0045] Figure 28 presents representative data depicting that hybrid (CD137+) TILs selected show enhanced IFN-y secretion. IFN-y release was measured upon co-culture of expanded TILs with autologous tumor targets and ovarian tumor cell lines. Hybrid (CD 137- sorted) TILs produced stronger IFN-y responses than Conventional NCI IL2 high TILs.

[0046] Figure 29 presents representative data depicting CD137+ selected TILs enhanced control of Platinum-Resistant PE-04 cells. To functionally compare these two TIL groups, IxlO6PE-04, a human platinum-resistant high-grade serous carcinoma cell line, were injected into an immunocompromised NSG mouse model. Once the tumors reached 100 mm3, NCI-IL2 hi or hybrid (CD 137+ sorted) TILs were tail vein injected followed by 50,000 IU IL2. IL2 were further injected on Day 1, 2 and 3 following TIL injection. Tumor size was monitored twice weekly to track the tumor growth. The Hybrid (CD137+ sorted) TILs demonstrated enhanced tumor control compared to untreated.

[0047] DETAILED DESCRIPTION

[0048] The present invention is based upon the observation that clinical grade metabolically enhanced tumor infiltrating lymphocytes (meTILs) are generated by the methods and compositions described herein.

[0049] Definitions

[0050] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0051] As used herein, each of the following terms has the meaning associated with it in this section.

[0052] The articles “a” and “an” are used herein to refer to one or to more than one (z.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0053] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0054] “Activation”, as used herein, refers to the state of a T cell that has been sufficiently stimulated to induce detectable cellular proliferation. Activation can also be associated with induced cytokine production, and detectable effector functions. The term “activated T cells” refers to, among other things, T cells that are undergoing cell division.

[0055] The term “antigen” or “Ag” as used herein is defined as a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA, which comprises a nucleotide sequences or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an “antigen” as that term is used herein. Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a biological fluid.

[0056] The term “anti-tumor effect” as used herein, refers to a biological effect which can be manifested by a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, or amelioration of various physiological symptoms associated with the cancerous condition. An “anti-tumor effect” can also be manifested by the ability of the peptides, polynucleotides, cells and antibodies of the invention in prevention of the occurrence of tumor in the first place.

[0057] The term “cancer” as used herein is defined as disease characterized by the aberrant proliferation and / or growth of cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Cancer as here herein includes both solid tumors and hematopoietic malignancies. “Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0058] The term “expression” as used herein is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0059] The term “expression vector” as used herein refers to a vector containing a nucleic acid sequence coding for at least part of a gene product capable of being transcribed. In some cases, RNA molecules are then translated into a protein, polypeptide, or peptide. In other cases, these sequences are not translated, for example, in the production of antisense molecules, siRNA, ribozymes, and the like. Expression vectors can contain a variety of control sequences, which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operatively linked coding sequence in a particular host organism. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well.

[0060] By “nucleic acid” is meant any nucleic acid, whether composed of deoxyribonucleosides or ribonucleosides, and whether composed of phosphodiester linkages or modified linkages such as phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, phosphorothioate, methylphosphonate, phosphorodithioate, bridged phosphorothioate or sulfone linkages, and combinations of such linkages. The term nucleic acid also specifically includes nucleic acids composed of bases other than the five biologically occurring bases (adenine, guanine, thymine, cytosine and uracil). The term “nucleic acid” typically refers to large polynucleotides.

[0061] Conventional notation is used herein to describe polynucleotide sequences: the left-hand end of a single- stranded polynucleotide sequence is the 5'-end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5'-direction.

[0062] The direction of 5' to 3' addition of nucleotides to nascent RNA transcripts is referred to as the transcription direction. The DNA strand having the same sequence as an mRNA is referred to as the “coding strand”; sequences on the DNA strand which are located 5' to a reference point on the DNA are referred to as “upstream sequences”; sequences on the DNA strand which are 3' to a reference point on the DNA are referred to as “downstream sequences.”

[0063] The phrase “inhibit,” as used herein, means to reduce a molecule, a reaction, an interaction, a gene, an mRNA, and / or a protein’s expression, stability, function or activity by a measurable amount or to prevent entirely. Inhibitors are compounds that, e.g., bind to, partially or totally block stimulation, decrease, prevent, delay activation, inactivate, desensitize, or down regulate a protein, a gene, and an mRNA stability, expression, function and activity, e.g., antagonists.

[0064] The terms “patient,” “subject,” “individual,” and the like are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. In certain non-limiting embodiments, the patient, subject or individual is a human.

[0065] Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range. The terms “treatment” and “therapeutic” refer to compositions for alleviating or preventing symptoms of a disease or disorder. The terms “treat” and “therapy” refer to methods of administering a treatment or therapeutic to a subject in need thereof, for example, a subject afflicted with a disease or disorder, or a subject who ultimately may acquire such a disease or disorder, in order to prevent, cure, delay, reduce the severity of, or ameliorate one or more signs or symptoms of the disease or disorder or recurring disease or disorder.

[0066] A “vector” is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” includes an autonomously replicating plasmid or a virus. The term should also be construed to include non-plasmid and non- viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and the like.

[0067] Description

[0068] In one aspect the present invention comprises methods and compositions for the generation of metabolically enhanced Tumor Infiltrating Lymphocytes (meTILs). In one aspect the present invention comprises methods and compositions for the generation of CD137+ TILs from a subject with a tumor. In one aspect the present invention comprises compositions comprising meTILs. In one aspect the present invention comprises compositions comprising CD137+ TILs. In one aspect the present invention comprises methods for the treatment of cancer in a subject with cancer comprising administering to the subject compositions comprising the meTILs or CD137+ TILs of the present invention.

[0069] Methods

[0070] Generating “metabolically-fit” TIL (meTILs) for cancer immunotherapy In one embodiment, the present invention comprises methods for generating clinical grade metabolically enhanced Tumor Infdtrating Lymphocytes (meTILs) from a subject with a tumor, wherein the method comprises obtaining TILs from the subject’s tumor, programming the TILs to obtain programmed meTILs, and expanding the programmed meTILs to obtain clinical grade meTILs. In one embodiment, the meTILs are generated from a subject with pancreatic cancer. In one embodiment, the meTILs are generated from a subject with melanoma.

[0071] Generally, the meTILs of the invention are generated by contact with at least one agent, wherein the at least one agent is anti-CD3 antibody, anti-CD28 antibody, or IL-2.

[0072] In one embodiment, the meTILs of the invention are generated by removing a tumor from a subject and cutting the tumor into small pieces. In some embodiments, the small pieces are incubated at 37°C in a first media comprising at least one interleukin for 12-24 hours. In some embodiments, the small pieces are incubated at 37°C in a first media comprising at least one interleukin for 12 hours. In some embodiments, the at least one interleukin is IL-2.

[0073] In some embodiments, the small pieces are removed from the first media and incubated in a replenished first media comprising IL-2.

[0074] In one embodiment, the IL-2 is at a concentration of 4000-4500 units / ml, 4500- 5000 units / ml, 5000-5500 units / ml, 5500-6000 units / ml, 6000-6500 units / ml, 6500-7000 units / ml, 7000-7500 units / ml, or 7500-8000 units / ml. In one embodiment, the IL-2 is at a concentration of 4000-8000 units / ml. In one embodiment, the IL-2 is at a concentration of 6000 units / ml.

[0075] In some embodiments, the cells in the small pieces become isolated as individual cells during this incubation. In some embodiments, the cells are removed from the first media after 1-2 days. In some embodiments, the cells are removed from the first media after 2 days.

[0076] In some embodiments, the cells are incubated in a second media comprising at least one antibody and at least one cytokine to the second media. In some embodiments, the at least one antibody is anti-CD3 and anti-CD28. In some embodiments, the cells are incubated in the second media for 2-4 days. In some embodiments, the cells are incubated in the second media for 4 days. In one embodiment, the anti-CD3 antibody is at a concentration of 0.5-1 gg / ml, 1- 1.5 gg / ml, 1.5-2 gg / ml, 2-2.5 gg / ml, 2.5-3 gg / ml, 3-3.5 gg / ml, 3.5-4 gg / ml, 4-4.5 gg / ml, or 4.5-5 gg / ml. In one embodiment, the anti-CD3 antibody is at a concentration of 2 gg / ml.

[0077] In one embodiment, the anti-CD28 antibody is at a concentration of 0.5-1 gg / ml, 1-1.5 gg / ml, 1.5-2 gg / ml, 2-2.5 gg / ml, 2.5-3 gg / ml, 3-3.5 gg / ml, 3.5-4 gg / ml, 4-4.5 gg / ml, or 4.5-5 gg / ml. In one embodiment, the anti-CD28 antibody is at a concentration of 2 gg / ml.

[0078] In some embodiments, at least one interleukin is added to the second media. In some embodiments, the at least one interleukin is IL-2. In some embodiments, the cells are cultured in the second media in the presence of IL-2 for an additional 2-4 days. In some embodiments, the cells are cultured in the second media in the presence of IL-2 for an additional 4 days.

[0079] In one embodiment, the IL-2 is at a concentration of 150-450 units / ml. In one embodiment, the IL-2 is at a concentration of 300 units / ml.

[0080] In some embodiments, programmed meTILs are isolated from the second media via any commonly used or applicable technique to isolate cells based upon affinity. In some embodiments, programmed meTILs are isolated by immunoprecipitation.

[0081] In some embodiments, the programmed meTILs are subsequently expanded, wherein the expanding comprises mixing feeder cells with the programmed meTILs in a 1 : 50- 1 : 100 ratio, wherein the feeder cells are obtained by irradiating human peripheral blood mononuclear cells. In some embodiments, the programmed meTILs are separated from the feeder cells and cultured in a gas permeable cell culture flask for 5-9 days in a third media wherein the third media comprises at least one interleukin and at least one antibody. In some embodiments, the gas permeable cell culture flask is the GREX-100™ flask. In some embodiments, the programmed meTILs are cultured in the third media for 7 days. In some embodiments, the at least one interleukin is IL-2. In some embodiments, the at least one antibody is anti-CD3. In some embodiments, the programmed meTILs are transferred to a second gas permeable cell culture flask and cultured for 5-9 days in a replenished third media. In some embodiments, the second gas permeable cell culture flask is the GREX-500™ flask. In some embodiments, the programmed meTILs are cultured for 7 days to obtain expanded programmed meTILs. In some embodiments, the expanded programmed meTILs are removed from the GREX-500™ flask to obtain clinical grade meTILs.

[0082] In one embodiment, the IL-2 is at a concentration of 150-450 units / ml. In one embodiment, the IL-2 is at a concentration of 300 units / ml.

[0083] In one embodiment, the anti-CD3 antibody is at a concentration of 1-5 ng / ml, 5- 10 ng / ml, 10-15 ng / ml, 15-20 ng / ml, 20-25 ng / ml, 25-30 ng / ml, 30-35 ng / ml, 35-40 ng / ml, 40-45 ng / ml, 45-50 ng / ml. In one embodiment, the anti-CD3 antibody is at a concentration of 30 ng / ml

[0084] In certain embodiments the at least one agent may be coupled to the same surface (i.e., in “cis” formation) or to separate surfaces (i.e., in “trans” formation). Alternatively, one agent may be coupled to a surface and the other agent in solution. In certain embodiments, both agents can be in solution. In another embodiment, the at least one agent may be in soluble form, and then cross-linked to a surface, such as a cell expressing Fc receptors or an antibody or other binding agent which will bind to the agents. In this regard, see for example, U.S. Patent Application Publication Nos. 20040101519 and 20060034810 for artificial antigen presenting cells (aAPCs) that are contemplated for use in activating and expanding the cells of the present invention.

[0085] In one embodiment, the at least one agent immobilized on beads, either on the same bead, i.e., “cis,” or to separate beads, i.e., “trans.” By way of example, the at least one agent is an anti-CD3 antibody or an antigen-binding fragment thereof, and an anti- CD28 antibody or antigen-binding fragment thereof; and both agents are co-immobilized to the same bead in equivalent molecular amounts. In one embodiment, a 1 : 1 ratio of each antibody bound to the beads for CD4+ T cell expansion and T cell growth is used. In certain aspects of the present invention, a ratio of anti CD3:CD28 antibodies bound to the beads is used such that an increase in T cell expansion is observed as compared to the expansion observed using a ratio of 1 : 1. In one particular embodiment an increase of from about 1 to about 3 fold is observed as compared to the expansion observed using a ratio of 1: 1. In one embodiment, the ratio of CD3:CD28 antibody bound to the beads ranges from 100: 1 to 1 : 100 and all integer values there between. In one aspect of the present invention, more anti-CD28 antibody is bound to the particles than anti-CD3 antibody, i.e., the ratio of CD3:CD28 is less than one. In certain embodiments of the invention, the ratio of anti CD28 antibody to anti CD3 antibody bound to the beads is greater than 2: 1. In one particular embodiment, a 1 : 100 CD3:CD28 ratio of antibody bound to beads is used. In another embodiment, a 1 :75 CD3:CD28 ratio of antibody bound to beads is used. In a further embodiment, a 1 :50 CD3:CD28 ratio of antibody bound to beads is used. In another embodiment, a 1 :30 CD3:CD28 ratio of antibody bound to beads is used. In one preferred embodiment, a 1 : 10 CD3:CD28 ratio of antibody bound to beads is used. In another embodiment, a 1:3 CD3:CD28 ratio of antibody bound to the beads is used. In yet another embodiment, a 3 : 1 CD3 :CD28 ratio of antibody bound to the beads is used.

[0086] In further embodiments of the present invention, the cells, such as T cells, are combined with agent-coated beads, the beads and the cells are subsequently separated, and then the cells are cultured. In an alternative embodiment, prior to culture, the agent- coated beads and cells are not separated but are cultured together. In a further embodiment, the beads and cells are first concentrated by application of a force, such as a magnetic force, resulting in increased ligation of cell surface markers, thereby inducing cell stimulation.

[0087] Generating CD 137+ TILs for cancer immunotherapy

[0088] In one embodiment, the present invention comprises methods for generating metabolically reprogrammed CD137+ tumor infiltrating lymphocytes (CD137+ TILs) from a subject with a tumor, wherein the method comprises obtaining TILs from the subject’s tumor, programming the TILs and isolating CD137+ TILs to obtain programmed CD 137+ TILs, and expanding the CD 137+ TILs to obtain clinical grade CD 137+ TILs.

[0089] Generally, the CD 137+ TILs of the invention are generated by contact with at least one agent, wherein the at least one agent is anti-CD3 antibody, anti-CD28 antibody, IL-2, or any combination thereof.

[0090] In one embodiment, the CD137+ TILs of the invention are generated by surgically removing a tumor from a subject and cutting the tumor into small pieces. In some embodiments, the small pieces are incubated at 37°C in a first media comprising at least one interleukin. In some embodiments, the at least one interleukin is IL -2.

[0091] In one embodiment, the IL-2 is at a concentration of 4000-4500 units / ml, 4500- 5000 units / ml, 5000-5500 units / ml, 5500-6000 units / ml, 6000-6500 units / ml, 6500-7000 units / ml, 7000-7500 units / ml, or 7500-8000 units / ml. In one embodiment, the IL-2 is at a concentration of 4000-8000 units / ml. In one embodiment, the IL-2 is at a concentration of 6000 units / ml.

[0092] In some embodiments, the cells in the small pieces become isolated as individual cells during this incubation. In some embodiments, the cells are removed from the first media when the number of cells reach approximately 30 million cells.

[0093] In some embodiments, the cells are incubated in a second media comprising at least one antibody and at least one conditioning cytokine to the second media. In some embodiments, the at least one antibody is anti-CD3 and anti-CD28. In some embodiments, the cells are incubated in the second media for 2-4 days. In some embodiments, the cells are incubated in the second media for 4 days. In some embodiments, incubation in the second media comprises activation and programing of the cells into TILs.

[0094] In one embodiment, the anti-CD3 antibody is at a concentration of 2.5-3 pg / ml, 3-3.5 pg / ml, 3.5-4 pg / ml, 4-4.5 pg / ml, 4.5-5 pg / ml, 5-5.5 pg / ml, 5.5-6.0 pg / ml, 6.0-6.5 pg / ml, 6.5-7 pg / ml, or 7-7.5 pg / ml . In one embodiment, the anti-CD3 antibody is at a concentration of 5 pg / ml.

[0095] In one embodiment, the anti-CD28 antibody is at a concentration of 2.5-3 pg / ml, 3-3.5 pg / ml, 3.5-4 pg / ml, 4-4.5 pg / ml, 4.5-5 pg / ml, 5-5.5 pg / ml, 5.5-6.0 pg / ml, 6.0-6.5 pg / ml, 6.5-7 pg / ml, or 7-7.5 pg / ml . In one embodiment, the anti-CD28 antibody is at a concentration of 5 pg / ml.

[0096] In some embodiments, at least one interleukin is added to the second media. In some embodiments, the at least one interleukin is IL-2. In some embodiments, the cells are cultured in the second media in the presence of IL-2 for an additional 2-4 days. In some embodiments, the cells are cultured in the second media in the presence of IL-2 for an additional 4 days. In one embodiment, the IL-2 is at a concentration of 150-450 units / ml. In one embodiment, the IL-2 is at a concentration of 300 units / ml.

[0097] In some embodiments, when the number of TILs growing in the second media reach approximately 50 million cells, the TILs are stained with anti-CD137 antibody and CD137+ TILs are isolated via sorting based upon the association of the anti-CD137 antibody with the TIL. In some embodiments, CD 137+ TILs the sorting of CD 137+ TILs from the second media is performed by any commonly used or applicable technique to sort and isolate cells. In some embodiments, programmed CD137+ TILs are sorted via flow cytometry. In some embodiments, the flow cytometry comprises Fluorescence- Activated Cell Sorting (FACS). In some embodiments, the anti-CD137 antibody is directly conjugated to a detectable marker such as a fluorescent marker which is detectable by FACS.

[0098] In some embodiments, the CD137+ TILs are mixed with feeder cells, wherein the feeder cells are prepared by irradiating human peripheral blood mononuclear cells (PBMCs) from three donors at 25 Gy. In some embodiments, the feeder cells are mixed with CD137+ TILs at a ratio of 1:50-1:100 cells.

[0099] In some embodiments, the CD137+ TILs are subsequently expanded. In some embodiments, the CD137+ TILs are separated from the feeder cells and cultured in a gas permeable cell culture flask for 8-12 days in a third media wherein the third media comprises at least one interleukin and at least one antibody. In some embodiments, the gas permeable cell culture flask is the GREX-100™ flask.

[0100] In some embodiments, the programmed meTILs are cultured in the third media for 10 days. In some embodiments, the at least one interleukin is IL-2. In some embodiments, the at least one antibody is anti-CD3. In some embodiments, the CD 137+ TILs are transferred to a second gas permeable cell culture flask and cultured for 5-9 days in a replenished third media. In some embodiments, the second gas permeable cell culture flask is the GREX-500™ flask. In some embodiments, the CD137+ TILs are cultured for 7 days to obtain expanded CD137+ TILs. In some embodiments, the expanded CD137+ TILs are removed from the GREX-500™ flask to obtain clinical grade meTILs.

[0101] In one embodiment, the IL-2 is at a concentration of 150-450 units / ml. In one embodiment, the IL-2 is at a concentration of 300 units / ml. In one embodiment, the anti-CD3 antibody is at a concentration of 1-5 ng / ml, 5- 10 ng / ml, 10-15 ng / ml, 15-20 ng / ml, 20-25 ng / ml, 25-30 ng / ml, 30-35 ng / ml, 35-40 ng / ml, 40-45 ng / ml, 45-50 ng / ml. In one embodiment, the anti-CD3 antibody is at a concentration of 30 ng / ml.

[0102] In one embodiment, the anti-CD3 antibody comprises OKT3® antibody. In one embodiment, the anti-CD3 antibody comprises Orthoclone OKT3®.

[0103] In some embodiments, the present invention comprises methods for generating metabolically reprogrammed CD 154+ TILs, CD25+ TILs, PD1+ TILs, or CD39+ TILs from a subject with a tumor, wherein the CD 154+ TILs, CD25+ TILs, PD1+ TILs, or CD39+ TILs are generated by the methods described above, wherein the TILs are isolated with anti-CD154, anti-CD25, anti-PDl, or anti-CD39 antibody, respectively, following growth in the second media.

[0104] In certain embodiments the at least one agent may be coupled to the same surface (i.e., in “cis” formation) or to separate surfaces (i.e., in “trans” formation). Alternatively, one agent may be coupled to a surface and the other agent in solution. In certain embodiments, both agents can be in solution. In another embodiment, the at least one agent may be in soluble form, and then cross-linked to a surface, such as a cell expressing Fc receptors or an antibody or other binding agent which will bind to the agents. In this regard, see for example, U.S. Patent Application Publication Nos. 20040101519 and 20060034810 for artificial antigen presenting cells (aAPCs) that are contemplated for use in activating and expanding the cells of the present invention.

[0105] In one embodiment, the at least one agent immobilized on beads, either on the same bead, i.e., “cis,” or to separate beads, i.e., “trans.” By way of example, the at least one agent is an anti-CD3 antibody or an antigen-binding fragment thereof, and an anti- CD28 antibody or antigen-binding fragment thereof; and both agents are co-immobilized to the same bead in equivalent molecular amounts. In one embodiment, a 1 : 1 ratio of each antibody bound to the beads for CD4+ T cell expansion and T cell growth is used. In certain aspects of the present invention, a ratio of anti CD3UD28 antibodies bound to the beads is used such that an increase in T cell expansion is observed as compared to the expansion observed using a ratio of 1 : 1. In one particular embodiment an increase of from about 1 to about 3 fold is observed as compared to the expansion observed using a ratio of 1 : 1 . In one embodiment, the ratio of CD3:CD28 antibody bound to the beads ranges from 100: 1 to 1 : 100 and all integer values there between. In one aspect of the present invention, more anti-CD28 antibody is bound to the particles than anti-CD3 antibody, i.e., the ratio of CD3:CD28 is less than one. In certain embodiments of the invention, the ratio of anti CD28 antibody to anti CD3 antibody bound to the beads is greater than 2: 1. In one particular embodiment, a 1 : 100 CD3:CD28 ratio of antibody bound to beads is used. In another embodiment, a 1 :75 CD3:CD28 ratio of antibody bound to beads is used. In a further embodiment, a 1 :50 CD3:CD28 ratio of antibody bound to beads is used. In another embodiment, a 1 :30 CD3:CD28 ratio of antibody bound to beads is used. In one preferred embodiment, a 1 : 10 CD3:CD28 ratio of antibody bound to beads is used. In another embodiment, a 1 :3 CD3:CD28 ratio of antibody bound to the beads is used. In yet another embodiment, a 3 : 1 CD3 :CD28 ratio of antibody bound to the beads is used.

[0106] In further embodiments of the present invention, the cells, such as T cells, are combined with agent-coated beads, the beads and the cells are subsequently separated, and then the cells are cultured. In an alternative embodiment, prior to culture, the agent- coated beads and cells are not separated but are cultured together. In a further embodiment, the beads and cells are first concentrated by application of a force, such as a magnetic force, resulting in increased ligation of cell surface markers, thereby inducing cell stimulation.

[0107] Genetic modification

[0108] In one embodiment, the TILs of the present invention are further modified to express a gene or protein of interest. For example, the TILs can be modified to prolong survival, resist suppression, enhance function, or target a tumor site. In certain aspects, the TIL is modified to express a chimeric antigen receptor (CAR). In one embodiment, the CAR comprises an antigen binding domain which is specific for at least one marker of at least one cancer cell. In some embodiments, once bound to the at least one cancer cell, the CAR expressing TIL cell facilitates the destruction of the at least one cancer cell (e.g., by phagocytosis, T cell-mediated cytotoxicity, etc.), thereby treating or preventing a disease or disorder (e.g., cancer, etc.) in the subject. The term “chimeric antigen receptor” or “CAR,” as used herein, refers to an artificial cell receptor that is engineered to be expressed on an immune effector cell (e.g., a TIL), and specifically bind an antigen on at least one cancer cell CARs may be used as a therapy with adoptive cell transfer. Generally, immune cells of interest, are removed from a patient and modified so that they express the receptors specific to a particular form of antigen. In some embodiments, the CARs have specificity to at least one cancer cell CARs may also comprise an intracellular activation domain, a transmembrane domain and an extracellular domain comprising an antigen binding region that specifically binds to at least one cancer cell.

[0109] In various embodiments, the CARs contemplated herein comprise an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain comprises a target-specific binding element otherwise referred to as an antigen binding domain. In some embodiments, the extracellular domain also comprises a hinge domain. In certain embodiments, the intracellular domain or otherwise the cytoplasmic domain comprises, a costimulatory signaling region and a zeta chain portion. The costimulatory signaling region refers to a portion of the CAR comprising the intracellular domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigens receptors or their ligands that are required for an efficient response of lymphocytes to antigen.

[0110] Vectors

[0111] The present invention also provides vectors in which a nucleic acid molecule of the invention is inserted. In some embodiments, the vector is a DNA, a RNA, a plasmid, a lentivirus vector, an adenoviral vector, or a retrovirus vector. Vectors derived from retroviruses, such as the lentivirus, are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells. Lentiviral vectors have the added advantage over vectors derived from onco-retroviruses, such as murine leukemia viruses, in that they can transduce nonproliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity.

[0112] In one embodiment, the vector further comprises a promoter. In one embodiment, the vector comprising the nucleic acid encoding the desired nucleic acid sequences of the invention is an adenoviral vector (A5 / 35). In one embodiment, the expression of nucleic acids encoding the desired nucleic acid sequences of the invention can be accomplished using of transposons, such as sleeping beauty, crisper, CAS9, and zinc finger nucleases. See below June et al. 2009 Nature Reviews Immunology 9.10: 704-716, is incorporated herein by reference.

[0113] The nucleic acid can be cloned into a number of types of vectors. For example, the nucleic acid can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0114] Further, the expression vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno- associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Pat. No. 6,326,193).

[0115] Additional promoter elements, e.g., enhancers, regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription. An example of a promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto. However, other constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the elongation factor- la promoter, the hemoglobin promoter, and the creatine kinase promoter. Further, the invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention. The use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

[0116] Another example of a promoter is the EFla promoter. The native EFla promoter drives expression of the alpha subunit of the elongation factor- 1 complex, which is responsible for the enzymatic delivery of aminoacyl tRNAs to the ribosome. The EFla promoter has been extensively used in mammalian expression plasmids and has been shown to be effective in driving CAR expression from transgenes cloned into a lentiviral vector. See, e.g., Milone et al., Mol. Ther. 17(8): 1453-1464 (2009).

[0117] In order to assess the expression of polypeptide or portions thereof, the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In various embodiments, the selectable marker may be carried on a separate piece of DNA and used in a co- transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers include, for example, antibiotic-resistance genes, such as neo and the like.

[0118] Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences. In general, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expression systems are well known and may be prepared using known techniques or obtained commercially. In general, the construct with the minimal 5' flanking region showing the highest level of expression of reporter gene is identified as the promoter. Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter- driven transcription.

[0119] Methods of introducing and expressing genes into a cell are known in the art. In the context of an expression vector, the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art. For example, the expression vector can be transferred into a host cell by physical, chemical, or biological means.

[0120] Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and / or exogenous nucleic acids are well-known in the art. See, for example, Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY).

[0121] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362.

[0122] Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g. , an artificial membrane vesicle). Other methods of state-of-the-art targeted delivery of nucleic acids are available, such as delivery of polynucleotides with targeted nanoparticles.

[0123] In the case where a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of the nucleic acids into a host cell (in vitro, ex vivo or in vivo). In one embodiment, the nucleic acid may be associated with a lipid. The nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid / DNA or lipid / expression vector associated compositions are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or with a “collapsed” structure. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances which may be naturally occurring or synthetic lipids. For example, lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0124] Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine (“DMPC”) can be obtained from Sigma, St. Louis, MO; dicetyl phosphate (“DCP”) can be obtained from K & K Laboratories (Plainview, NY); cholesterol (“Choi”) can be obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) and other lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL.). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at about -200C. Chloroform is used as the only solvent since it is more readily evaporated than methanol. “Liposome” is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). However, compositions that have different structures in solution than the normal vesicular structure are also encompassed. For example, the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules. Also contemplated are lipofectamine-nucleic acid complexes.

[0125] Methods of treating a subject with cancer

[0126] In one aspect the present invention comprises method of treating a subject with cancer. In one aspect, the method comprises generating clinical grade meTILs from a tumor in the subject with cancer by the methods of the present invention and administering to the subject a composition comprising the clinical grade meTILs. In one aspect, the method comprises generating clinical grade CD 137+ TILs from a tumor in a subject with cancer by the methods of the present invention and administering to the subject a composition comprising the clinical grade CD137+ TILs.

[0127] Cancers that may be treated include tumors that are not vascularized, or not yet substantially vascularized, as well as vascularized tumors. The cancers may comprise solid tumors. Types of cancers to be treated with the TILs of the invention include, but are not limited to, carcinoma, blastoma, and sarcoma, benign and malignant tumors, and malignancies e.g., sarcomas, carcinomas, and melanomas. Adult turn ors / cancers and pediatric tumors / cancers are also included. Exemplary types of cancers include, but are not limited to, adrenocortical carcinoma (ACC); bladder urothelial carcinoma (BLCA); B-cell acute lymphoblastic leukemia (B-ALL); breast invasive carcinoma (BRCA); cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC); cholangio carcinoma (CHOL); colon adenocarcinoma (COAD); esophageal carcinoma (ESCA); glioblastoma multiforme (GBM); head and neck squamous cell carcinoma (HNSC); kidney chromophobe (KICH); kidney renal clear cell carcinoma (KIRC); kidney renal papillary cell carcinoma (KIRP); brain lower grade glioma (LGG); liver hepatocellular carcinoma (LIHC); lung adenocarcinoma (LU AD); lung squamous cell carcinoma (LUSC); mesothelioma (MESO); ovarian serous cystadenocarcinoma (OV); pancreatic adenocarcinoma (PAAD); pheochromocytoma and paraganglioma (PCPG); pancreatic ductal adenocarcinoma (PDAC); prostate adenocarcinoma (PRAD); rectum adenocarcinoma (READ); sarcoma (SARC); skin cutaneous melanoma (SKCM); stomach adenocarcinoma (STAD); testicular germ cell tumors (TGCT); thyroid carcinoma (THCA); thymoma (THYM); uterine corpus endometrial carcinoma (UCEC); uterine carcinosarcoma (UCS); and uveal melanoma (UVM). In some embodiments, the cancer to be treated is lung cancer, melanoma, ovarian cancer, head and neck cancer, or prostate cancer.

[0128] In some embodiments, the TILs of the present invention may be administered either alone, or as a pharmaceutical composition in combination with diluents and / or with other components. Briefly, pharmaceutical compositions of the present invention may comprise TILs as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. Compositions of the present invention are preferably formulated for intravenous administration.

[0129] Pharmaceutical compositions of the present invention may be administered in a manner appropriate to the disease to be treated (or prevented). The quantity and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease, although appropriate dosages may be determined by clinical trials.

[0130] When “an immunologically effective amount”, “an anti-tumor effective amount”, “an tumor-inhibiting effective amount”, or “therapeutic amount” is indicated, the precise amount of the compositions of the present invention to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject). It can generally be stated that a pharmaceutical composition comprising the TILs cells described herein may be administered at a dosage of 104to 109cells / kg body weight, preferably 105to 106cells / kg body weight, including all integer values within those ranges. T cell compositions may also be administered multiple times at these dosages. The cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). The optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly.

[0131] Compositions

[0132] Compositions comprising meTILs.

[0133] In one aspect the present invention comprises compositions comprising meTILs as obtained by the methods disclosed elsewhere herein. In one aspect the present invention comprises compositions comprising a population of meTILs as obtained by the methods disclosed elsewhere herein. In one aspect the compositions comprise an antitumor effective amount of a population of meTILs.

[0134] In one embodiment, the composition further comprises a pharmaceutically acceptable carrier. In one embodiment, the compositions can be surgically implanted at a site of a tumor in a subject with a tumor. In one embodiment, the compositions are suitable for administration to a subject with a tumor via intravenous (IV) infusion.

[0135] Compositions comprising CD137+ TILS. In one aspect the present invention comprises compositions comprising CD137+ TILs as obtained by the methods disclosed elsewhere herein. In one aspect the present invention comprises compositions comprising a population of CD137+ TILs as obtained by the methods disclosed elsewhere herein. In one aspect the compositions comprise an anti-tumor effective amount of a population of CD137+ TILs.

[0136] In one embodiment, the composition further comprises a pharmaceutically acceptable carrier. In one embodiment, the compositions can be surgically implanted at a site of a tumor. In one embodiment, the compositions are suitable for administration to a subject with a tumor via intravenous (IV) infusion.

[0137] Therapeutic Application

[0138] In one embodiment, the present invention includes a type of cellular therapy using the one or more TILs described herein. In one embodiment, the method comprises administering one or more TILs described herein to a subject having cancer.

[0139] In one embodiment, the one or more TILs described herein can be infused to a recipient in need thereof. In one embodiment, the infused TIL is able to kill tumor cells in the recipient. Unlike antibody therapies, the one or more TILs of the invention are able to result in long-term persistence that can lead to sustained tumor control.

[0140] In another embodiment, the present invention includes a type of cellular therapy wherein the one or more TILs are additionally administered an agent that further alters the characteristics of the one or more TILs. In one embodiment, one or more TILs can then be infused to a recipient in need thereof. In one embodiment, the infused TIL is able to kill tumor cells in the recipient.

[0141] Pharmaceutical

[0142] In some embodiments, the present invention relates to pharmaceutical compositions comprising the TILs of the present invention.

[0143] Pharmaceutical compositions of the present invention may be administered in a manner appropriate to the disease to be treated (or prevented). The quantity and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient’s disease, although appropriate dosages may be determined by clinical trials.

[0144] When “an immunologically effective amount”, “an anti-tumor effective amount”, “a tumor-inhibiting effective amount”, or “therapeutic amount” is indicated, the precise amount of the compositions of the present invention to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject). It can generally be stated that a pharmaceutical composition comprising the T cells described herein may be administered at a dosage of 104to 109cells / kg body weight, preferably 105to 106cells / kg body weight, including all integer values within those ranges. T cell compositions may also be administered multiple times at these dosages. The cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). The optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly.

[0145] The administration of the subject compositions may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In one embodiment, the T cell compositions of the present invention are administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T cell compositions of the present invention are preferably administered by i.v. injection. The compositions of T cells may be injected directly into a tumor, lymph node, or site of infection.

[0146] Dosage / formulation

[0147] The present invention contemplates a variety of techniques and routes of administration of the compositions used in the practice of the invention. Non-limiting exemplary routes of administration suitable for use with the present invention include topical, nasal, oral, sublingual, rectal, transdermal, vaginal, transmucosal, or enteric. Parenteral delivery, including intramuscular, subcutaneous, intramedullary, intrathecal, intravenous, intraarterial, intraperitoneal, intranasal, intraocular, as well as direct intraventricular injections among others are contemplated. Indeed, it is not intended that the present invention be limited to any particular route of administration.

[0148] In one aspect of the invention, the amount of the compositions described herein administered to a subject is that of an effective amount to reduce or alleviate one or more symptoms associated with cancer.

[0149] The exact dose to be administered to a subject will depend on a number of factors unique to the individual subject to be treated, and is ultimately the responsibility of the treating physician (or veterinarian). It is contemplated that as part of the subject evaluation, it is well within the skill of the care provider to know how to and when to terminate, interrupt, or adjust administration due to toxicity, etc. Conversely, the care provider will also know how to adjust treatment to higher levels in circumstances where clinical response is inadequate, while precluding toxicity. The magnitude of an administered dose will vary with the particular agents administered, route of administration, the severity of the respiratory depression and the individual subject’s physiology, biochemistry, etc. The severity of the cancer can be evaluated in part by standard methodologies, and the dose, and frequency of administration will also depend in part on the age, body weight, sex, and response for each individual subject.

[0150] When the compositions of the invention are prepared for administration, they are preferably combined with a pharmaceutically acceptable carrier, diluent or excipient to form a pharmaceutical formulation, or unit dosage form. The total active ingredients in such formulations include from 0.1 to 99.9% by weight of the formulation. A “pharmaceutically acceptable” is a carrier, diluent, excipient, and / or salt that is compatible with the other ingredients of the formulation, and not deleterious to the recipient thereof. The active ingredient for administration may be present as a powder or as granules; as a solution, a suspension or an emulsion.

[0151] Pharmaceutical formulations containing the compositions of the invention can be prepared by procedures known in the art using well known and readily available ingredients. The compositions of the invention can also be formulated as solutions appropriate for parenteral administration, for instance by intramuscular, subcutaneous or intravenous routes.

[0152] The pharmaceutical formulations of the compositions of the invention can also take the form of an aqueous or anhydrous solution or dispersion, or alternatively the form of an emulsion or suspension.

[0153] Thus, the compositions may be formulated for parenteral administration (e.g., by injection, for example, bolus injection or continuous infusion) and may be presented in unit dose form in ampules, pre-fdled syringes, small volume infusion containers or in multi-dose containers with an added preservative. The active ingredients may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents. Alternatively, the active ingredients may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilization from solution, for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.

[0154] The pharmaceutical formulations of the present invention may include, as optional ingredients, pharmaceutically acceptable carriers, diluents, solubilizing or emulsifying agents, and salts of the type that are well-known in the art. Specific nonlimiting examples of the carriers and / or diluents that are useful in the pharmaceutical formulations of the present invention include water and physiologically acceptable buffered saline solutions, such as phosphate buffered saline solutions pH 7.0-8.0.

[0155] Additionally, standard pharmaceutical methods can be employed to control the duration of action. These are well known in the art and include control release preparations and can include appropriate macromolecules, for example polymers, polyesters, polyamino acids, polyvinyl, pyrolidone, ethylenevinylacetate, methyl cellulose, carboxymethyl cellulose or protamine sulfate. The concentration of macromolecules as well as the methods of incorporation can be adjusted in order to control release. Additionally, the agent can be incorporated into particles of polymeric materials such as polyesters, polyamino acids, hydrogels, poly (lactic acid) or ethylenevinylacetate copolymers. In addition to being incorporated, these agents can also be used to trap the compound in microcapsules. Accordingly, the compositions of the present invention may be delivered via various routes and to various sites in a mammal body to achieve a particular effect as is routinely performed in the art. One skilled in the art will recognize that although more than one route can be used for administration, a particular route can provide a more immediate and more effective reaction than another route. Local or systemic delivery can be accomplished by administration comprising application or instillation of the formulation into body cavities, inhalation or insufflation of an aerosol, or by parenteral introduction, comprising intramuscular, intravenous, peritoneal, subcutaneous, intradermal, as well as topical administration.

[0156] These methods described herein are by no means all-inclusive, and further methods to suit the specific application will be apparent to the ordinary skilled artisan. Moreover, the effective amount of the compositions can be further approximated through analogy to compounds known to exert the desired effect.

[0157] EMBODIMENTS

[0158] Embodiment l is a method for generating clinical grade metabolically enhanced Tumor Infiltrating Lymphocytes (TILs) from a subject with a tumor, wherein the method comprises: obtaining TILs from the subject’s tumor; and expanding the TILs.

[0159] Embodiment 2 is the method of embodiment 1, wherein the obtaining comprises: a. incubating small pieces of the tumor of the subject in a first media comprising at least one interleukin wherein cells in the small pieces become isolated as individual cells; b. removing the cells from the first media; c. placing the cells in a second media comprising at least one antibody; d. adding at least one conditioning cytokine to the second media; e. adding at least one interleukin to the second media; f. isolating TILs from the second media.

[0160] Embodiment 3 is the method of embodiment 1 or 2, wherein expanding the TILs comprises: a. mixing feeder cells with the TILs in a 1 : 50- 1 : 100 ratio; b. separating the TILs from the feeder cells and culturing the TILs in a gas permeable cell culture flask in a third media wherein the third media comprises at least one interleukin and at least one antibody; c. transferring the programmed meTILs to a second gas permeable cell culture flask and culturing the TILs in a replenished third media; d. removing the TILs to obtain clinical grade metabolically enhanced TILs.

[0161] Embodiment 4 is the method of any one of embodiments 1-3, wherein the at least one interleukin of is IL-2.

[0162] Embodiment 5 is the method of any one of embodiments 1-4, wherein the at least one antibody is selected from the group consisting of anti-CD3, anti-CD28, and a combination thereof.

[0163] Embodiment 6 is the method of any one of embodiments 1-5, wherein the at least one antibody is immobilized on a bead.

[0164] Embodiment 7 is the method of any one of embodiments 1-6, wherein isolating TILs from the second media comprises isolation via at least one technique selected from the group consisting of cell sorting, immunoprecipitation, antibody staining, and any combination thereof.

[0165] Embodiment 8 is the method of any one of embodiments 1-7, further comprising engineering the TILs to express a gene or protein of interest.

[0166] Embodiment 9 is the method of any one of embodiments 1-8, wherein the tumor comprises melanoma tumor, an ovarian tumor, a head and neck tumor, or a prostate tumor.

[0167] Embodiment 10 is a composition comprising the TILs generated by the method of any one of embodiments 1-9.

[0168] Embodiment 11 is the composition of embodiment 11, further comprising a pharmaceutically acceptable carrier.

[0169] Embodiment 12 is a method of treating a cancer in a subject in need thereof, the method comprising administering to the subject the composition of embodiments 10 or 11. Embodiment 13 is the method of embodiment 12, wherein the cancer comprises lung cancer, melanoma, ovarian cancer, head and neck cancer, or prostate cancer.

[0170] EXPERIMENTAL EXAMPLES

[0171] The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

[0172] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present invention and practice the claimed methods. The following working examples therefore are not to be construed as limiting in any way the remainder of the disclosure.

[0173] Example 1 : Optimizing Expansion of Metabolically Fit Tumor Infiltrating Lymphocytes for Multiple Cancers

[0174] Adoptive Cell Therapy (ACT) using Tumor-Infiltrating Lymphocyte (TIL)-based immunotherapy involves harvesting lymphocytes from a patient's tumor tissue and expanding these tumor-specific T cells manifold under ex-vivo conditioning. Effective expansion of TILs ex-vivo is critical for enhancing their therapeutic efficacy. Recent studies have successfully developed TIL cultures from various highly infiltrative solid tumors; however, this approach is not successful in ‘cold’ tumors, likely due to poor immunogenicity and low T cell infiltration. Furthermore, traditional manufacturing processes are time-consuming and require high doses of IL-2, which can compromise the quality and functionality of TILs. A 30-day ex-vivo protocol to expand TILs from a broad range of solid tumors, including lung, melanoma, ovarian, head and neck, and prostate was developed. After isolating TILs, they were cultured in a preformulated cytokine mixture for 6 days and then expanded in a G-Rex system using a rapid expansion protocol (REP). The process was monitored via flow cytometry, with TIL restimulation and cytokine analysis to assess quality and functionality. This approach expanded 0.4-1 .5 million TILs from human tumor tissue to 8-10 billion functional TILs, achieving over 2000-fold expansion. Expanded TILs maintained over 95% viability and produced key cytokines, such as IFNy, upon re-stimulation, confirming functional potency. This protocol effectively generates large quantities of functional TILs, even from tumors with limited TIL infiltration, like the prostate, offering significant potential for improving TIL-based immunotherapies and scalable clinical applications.

[0175] TIL Expansion Protocol allows for over 2000-fold expansion of TILs from melanoma, prostate, ovarian, and lung human patient tumor samples. Using the protocol, TILs can be metabolically reprogrammed providing a platform for optimizing T cell fitness before re-infusion. Expanded TILs are over 90% viable and metabolically active upon re-stimulation. This approach enhances the efficacy of TIL-based immunotherapies.

[0176] Benefits of strategy depicted in Figure 1 include 7-8L of media compared to 30- 40L with traditional TO 175 flask culture, 1 shelf of incubator space compared to 2-3 incubators, and 5-10 min media reduction of 4.5L utilizing Gather-X machine.

[0177] Example 2: Optimizing Expansion of Tumor Infiltrating Lymphocytes Utilizing the GREX platform

[0178] Limited capabilities of ACT in solid tumor models due to immunosuppressive tumor microenvironment. Transfused T cells experience terminal differentiation or exhaustion, limiting tumor killing effect. Tumor Infiltrating lymphocytes (TILs) from prostate and melanoma patients were generated and reprogrammed. Reprogrammed TILs are superior to conventional TILs in vivo (Figures 6-8).

[0179] Example 3 : Novel method to generate “metabolically-fit” TIL for Cancer Immunotherapy For this novel technology patent application, clinical grade metabolically enhanced Tumor Infiltrating Lymphocytes (meTILs) were generated. This approach targeting immunometabolism to program TILs is used to treat cancer patients. The compositions and method herein enable the therapeutic use of expanded anti-tumor T cells for treating different cancers.

[0180] Results Human T cells are programmed to hybrid phenotype.

[0181] It was determined that the ex vivo programming strategy has the potential to generate tumor-reactive human T cells for ACT. Using purified CD4+ T cells from regular healthy donors, human Thl / 17 hybrid cells were generated, as indicated by the IFNy and IL17 cytokine profile (Figure 9A). Further, data in Figure 9B show that ex vivo programming of TILs obtained from a metastatic melanoma patient resulted in increased ‘sternness’ features associated with the hybrid programming protocol. Importantly, the T 1 / 17 programmed TILs maintained the Tcm phenotype (CD62Lhi, CD28hi, CD127hi), exhibited less exhaustion (CD391o, PDllo), and increased metabolic fitness (PGClahi), along with enhanced effector function (IFNghi, Granzyme Bhi) as compared to the TILs that were expanded and underwent REP using the conventional IL2 method (Figure 9C).

[0182] Human melanoma TILs can be programmed to hybrid meTIL phenotype

[0183] After successfully establishing that human melanoma TILs can be programmed to hybrid T 1 / 17 phenotype (as in Figure 13), it was next determined that these meTIL preserve the phenotype after undergoing Rapid Expansion Protocol (REP) (67). Data in Figures 3 and 11 shows that hybrid programmed TILs have a higher fraction of CD8+ T cell subsets as compared to CD4+ fraction, whereas CD4+ fraction is higher in conventional IL2-TILs (Figure 10). Next, overnight TCR stimulation leads to increased secretion of effector cytokine interferon-gamma (IFNg) by hybrid TILs as compared to IL2-TILs (Figure 3B), whereas TNFa levels were unchanged (Figure 3C). Importantly, hybrid meTILs exhibited higher cytolytic ability against human melanoma 624-MEL cells as compared to conventional IL2 TILs (Figure 3D). Next, an evaluation of both CD8+ and CD4+ fraction in the TILs for cell surface markers showed increased CD 103, CD62L, and CD28 expression on CD8+ fraction of the hybrid TILs as compared to IL2 TILs (Figure 3E). While CD28 and CD62L are associated with Tcm or young TIL phenotype (22) (68), CD 103 has been associated with the efficient tumor-killing trait in TILs (69). It was also noted that hybrid meTILs were not overly activated as the expression of CD38 and CD25 was lower than that in IL2 TILs (Figure 3E). Reduced CD38 expression was also shown earlier to correlate with higher NAD+ levels and metabolically fit phenotype (31). Further, co-culture with human melanoma tumor 624- MEL of the hybrid meTILs showed increased CD137 expression, which is an established surrogate marker for antigen-specificity (70). Thus, it is likely that melanoma epitope reactive TILs are quantitatively and qualitatively enhanced upon hybrid programming. A similar phenotype was also observed in the CD4+ fraction of hybrid TILs (Figure 3F). This data establishes that hybrid meTILs can be obtained from human melanoma samples and retain their phenotype upon REP.

[0184] Distinct molecular signature in CD4 and CD8 fraction of hybrid meTILs from melanoma

[0185] To establish if hybrid programming imprinted a long-lived molecule signature that was distinct from the IL2 conventional TILs, a single cell sequencing on the TILs was performed. Data in Figure 11 shows a predominant CD4 and CD8 population that is expanded after REP (Figure 11 A) and more expression of CD8-related genes (Figure 1 IB). While the cluster differentiation between IL2 and hybrid TILs seemed to be identical (Figure 11C), a more significant number of genes were modulated in hybrid fraction (Figure 1 ID). The volcano plot highlights some of the genes in CD4 and CD8 fraction that is modulated after hybrid programming and expansion as compared to the 112 conventional TILs (Figure 1 IE). As an example, hybrid CD4+ TIL exhibited higher expression of Pyruvate Kinase M, an enzyme that converts phosphoenolpyruvate (PEP) to pyruvate during glycolysis. It has been shown that modulation of PKM activity affects the differentiation of TH17 cells (71). Additionally, it has been shown that activation of PKM2 interfered with TGF-pi signaling, which is necessary for the development of regulatory T cells. Similarly, Enolase was also higher in the CD4+ fraction of hybrid TILs. Enolase is the enzyme responsible for the reversible conversion D-2-phosphoglycerate (2PGA) and phosphoenolpyruvate (PEP) in glycolysis and gluconeogenesis, two metabolic pathways that are often vital for cellular function (72). It has been reported that a downregulation in the activity of ENOLASE 1, a critical enzyme in the glycolytic pathway, represses glycolytic activity in CD8+ TILs (73). DDIT, which was also high in the CD4+ fraction of hybrid TILs is a DNA damage-inducible transcript 4 (DDIT4), and its interactions with various protein molecules, immune and metabolic cell-related proteins, and participator in the oxygen sensing pathway are known (74). Overall, the signaling pathways enriched in hybrid TIL-derived CD4 and CD8 fractions over the IL2 TIL fraction shown in Figure 1 IF highlights that CD4+ and CD8+ fractions of hybrid meTILs had higher expression of metabolic genes and lower activation of several lymphocyte differentiation genes. This data comprehensively establishes that hybrid meTILs have unique imprinting of metabolic dependence and a less differentiated phenotype, which can correlate to better tumor control in vivo.

[0186] Human Prostate TILs can be programmed to hybrid meTIL phenotype

[0187] Next, it was determined that TILs from a poorly immunogenic prostate tumor can be reproducibly obtained and programmed with a hybrid phenotype. Data in Figure 12A shows that similar to melanoma TILs, prostate TILs programmed with hybrid protocol also exhibit increased CD8+ fraction than CD4+ ones. Further, IFNg secretion after TCR restimulation was higher in hybrid TILs as compared to the IL2 TILs (Figure 12B). Like observed in melanoma TILs, the secretion of TNFa was not different (Figure 12C), whereas CD25 expression without restimulation was lower (Figure 12D). The expression levels of cytolytic molecule GzmB (Figure 12E), antigen specificity determinant CD137 (Figure 12F), and surrogate marker for degranulation CD107a (Figure 12G) were identical to IL2 TILs after overnight co-culturing with prostate tumor cells 22-Rvl. Further, characterization of CD8+ (Figure 12H) and CD4+ (Figure 121) fractions in hybrid meTILs also showed upregulation of activation molecules CD25, CD44 and that of specificity determinant CD137 along with degranulation marker CD107a upon coculture with two different prostate tumor cells 22Rvl and C4-2, whereas these markers were not activated when prostate TILs were co-cultured with melanoma cells 624-MEL, thereby indicating the prevalence of prostate antigen reactive clones in the TIL population.

[0188] Human Melanoma and Prostate hybrid meTILs can be reproducibly generated After establishing that melanoma and prostate meTILs could be developed successfully, various combinations of G-Rex™ flasks to determine the optimal protocol for expanding meTILs within three weeks was tested. These studies were restricted to prostate TILs as they are hard to expand, and successfully generating them would also help boost the strategy for other poorly immunogenic tumors. Table 1 shows that the output of the prostate meTILs increased with the increased G-Rex™ flask capacity (where yield from G-Rex-500™ > G-Rex 10™0 > G-Rex- 10™). Figure 13 shows the finalized TIL Expansion protocol for optimally expanding TILs from Prostate and Melanoma tumor tissues. Given the hypothesis that meTILs could control the tumors effectively even when used at lower numbers, we performed three successful clinical validation runs following this TIL Expansion protocol to meet the criterion for the highest cohort in future clinical trials: 4 billion TILs; assuming 40 million cells / kg body weight and 100kg patient (Figure 4A). It is believed that the demonstration of successfully generating "metabolically fit" TILs, which could evade tumor suppression, can be adapted for improving TIL therapy against cancer. The traditional method for expanding human Tumor-Infiltrating Lymphocytes (TILs) involves a pre-Rapid Expansion Protocol (pre-REP) followed by a 14-day Rapid Expansion Protocol (REP). The pre-REP phase is crucial for the initial activation and expansion of TILs from tumor tissue before they proceed to the REP phase. Historically, this pre-REP phase has been the longest part of the TIL production process, taking two to five weeks to culture a sufficient number of cells for the REP phase (PMID: 22453018, PMID: 30509772). However, the current protocol has reduced this time to just 6-7 days, with two days in high-dose IL-2 and an additional 3-4 days of activation with costimulation in programming conditions. This streamlined procedure significantly shortens the timeline and results in TILs that are "younger and more metabolically fit." More importantly, this will be a significant advance since the TILs used for ACT comprise CD4+ and CD8+ T cells, which have been activated and expanded using a high dose of IL2.

[0189] However, distinct steps that have been modified / developed to use hybrid programming will render both CD4+ and CD8+ T cells with enhanced tumor lytic ability and reduced ability of CD4+ T cells to transdifferentiate to immunosuppressive regulatory T cells due to increased dependence on the glutamine pathway (31). The compositions and methods described herein enable novel clinical trials for treating melanoma, prostate cancer and patients with other solid tumors, and thus hold an immense market potential. Example 4: Metabolic Reprogramming of Ovarian CD137+ TILs for Treatment

[0190] Ovarian cancer is the 18th most common cancer and accounts for 1% of cancer cases in the US. The majority of cases are diagnosed at an advanced stage with metastatic disease (Figure 14). Previous ovarian cancer clinical trials include NCT04389229 (2020- ongoing): TILs frozen before receiving, sponsor Immetacyte Ltd withdrew support before 1st patient enrolled; and NCT03287674 (2018, completed): 6 patients, 1 partial response, 5 stable disease up to 3-5 months, high PD1 and Lag3 expression on TILs.

[0191] Available therapies: 1st line - Carboplatin / Paclitaxel; 2nd line - Cytotoxics, immunotherapy. Limitations to immunotherapy include limited capabilities of ACT in solid tumor models due to immunosuppressive tumor microenvironment with less than 30% efficacy, and transfused T cells experience terminal differentiation or exhaustion, limiting tumor-killing effect.

[0192] This invention overcomes the barriers of TIL immunotherapy by reprogramming anti-tumor T cells ex-vivo during expansion to improve T cell fitness. This invention reduces non-specificity of the final TIL product by enriching for anti-tumor specific TILs during expansion.

[0193] Patient Population: 8 patients with stage 3A or greater (high grade serous carcinoma or endometroid adenocarcinoma); both pre and post-chemo tumor samples for 4 patient samples.

[0194] Expansion Outcomes: 3-18xl09cells post-expansion with Hybrid (CD137+ sorted); > 90% viability.

[0195] This disclosure allows for rapid expansion of ovarian TILs in 30 days, demonstrates that hybrid metabolic programming and CD137+ sorting improves CD8+ T cell expansion and reduces exhaustion markers CD38 and PD-1, and that CD 137+ sorting allows for hyperexpansion of select clones, which correlates to improved tumor control. This invention offers a scalable solution for clinical applications.

[0196] The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

CLAIMSWhat is claimed is:

1. A method for generating clinical grade metabolically enhanced Tumor Infiltrating Lymphocytes (TILs) from a subject with a tumor, wherein the method comprises: obtaining TILs from the subject’s tumor; and expanding the TILs.

2. The method of claim 1, wherein the obtaining comprises: a. incubating small pieces of the tumor of the subject in a first media comprising at least one interleukin wherein cells in the small pieces become isolated as individual cells; b. removing the cells from the first media; c. placing the cells in a second media comprising at least one antibody; d. adding at least one conditioning cytokine to the second media; e. adding at least one interleukin to the second media; f. isolating TILs from the second media.

3. The method of claim 1, wherein expanding the TILs comprises: a. mixing feeder cells with the TILs in a 1 :50-1: 100 ratio,; b. separating the TILs from the feeder cells and culturing the TILs in a gas permeable cell culture flask in a third media wherein the third media comprises at least one interleukin and at least one antibody; c. transferring the programmed meTILs to a second gas permeable cell culture flask and culturing the TILs in a replenished third media; d. removing the TILs to obtain clinical grade metabolically enhanced TILs.

4. The method of any one of claims 2-3, wherein the at least one interleukin of is IL-2.

5. The method of any one of claims 2-3, wherein the at least one antibody is selected from the group consisting of anti-CD3, anti-CD28, and a combination thereof.

6. The method of any one of claims 2-3, wherein the at least one antibody is immobilized on a bead.

7. The method of claim 2, wherein isolating TILs from the second media comprises isolation via at least one technique selected from the group consisting of cell sorting, immunoprecipitation, antibody staining, and any combination thereof.

8. The method of claim 1, wherein the method further comprises engineering the TILs to express a gene or protein of interest.

9. The method of claim 1, wherein the tumor comprises melanoma tumor, an ovarian tumor, a head and neck tumor, or a prostate tumor.

10. A composition comprising the TILs generated by the method of claim 1.

11. The composition of claim 10, further comprising a pharmaceutically acceptable carrier.

12. A method of treating a cancer in a subject in need thereof, the method comprising administering to the subject the composition of claim 10.

13. The method of claim 12, wherein the cancer comprises lung cancer, melanoma, ovarian cancer, head and neck cancer, or prostate cancer.

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