Selective expansion of t cells and populations generated thereby

Abstract

Methods of generating expanded populations of T cells are disclosed. The method includes culturing a population of CD3+cells in a medium comprising a cell-stimulating amount of at least one cytokine, a T cell activator selected from the group consisting of a CD3 activator and a CD28 activator, an RAR antagonist and an AHR antagonist; and harvesting the expanded population of T cells. Cell populations obtainable using the method are also disclosed.

Description

[0001] SELECTIVE EXPANSION OF T CELLS AND POPULATIONS GENERATED THEREBY

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit of priority of U.S. Patent Application No. 63 / 747,020 filed January 19, 2025, the contents of which are incorporated herein by reference in their entirety.

[0004] FIELD AND BACKGROUND OF THE INVENTION

[0005] The present invention, in some embodiments thereof, relates to methods for selectively expanding T cells for use in adoptive cell therapy.

[0006] Adoptive cell therapy (ACT) is a cell therapy that attempts to enhance the anti-tumor activity of immune cells. ACT strategies have made a significant impact on cancer immunotherapy and is currently one of the most promising and groundbreaking strategies therefore. It is based on immune cells usually taken from the patient's own blood or tumor tissue, which are typically processed ex-vivo, extensively expanded, and then transferred back to the patient (autologous therapy) or to another individual (allogenic therapy). Various immune cells including T lymphocytes (T cells), Natural Killer (NK) cells and dendritic cells can be used in ACT. More often, the immune cells are genetically modified in the laboratory to enhance their ability to target and kill the patient's cancer cells.

[0007] One source of cells that can be used for producing ACT compositions is peripheral blood mononuclear cells (PBMC), from which lymphocyte populations (T cells, B cells and NK cells) may be isolated, expanded and / or otherwise manipulated by various procedures, to enhance their efficacy.

[0008] US2020138861 discloses an adoptive transfer procedure for treating inflammatory bowel disease comprising isolating CD3“ and CD56+NK cells from peripheral blood of a donor.

[0009] US 10149863 discloses a preparation method of isolated mammal NKT-like cells, comprising in vitro culturing isolated mononuclear cells, and adding culture cytokine(s) that stimulate the proliferation and activation of T cell, and sorting the NKT-like cells by using a cell sorting technique with NKT-like cell surface markers.

[0010] US2012258085 relates to a method of obtaining expanded and activated NK cells with the phenotype CD3 CD56+and NK-like T cells with the phenotype CD3+CD56+. The method comprises providing a cell sample of peripheral blood from a tumor bearing subject, incubating and expanding the cells until at least 35% of the expanded cell population comprises activated NK cells and NK-like T cells, and harvesting the expanded cells. In particular, US2012258085 discloses a method whereby PBMCs are initially cultured in the presence of monoclonal anti-CD3 antibody(OKT-3, 10 ng / ml) and 500 U / ml IL-2, wherein after five days, the cultures were replenished with fresh medium with 5% human serum and IL-2 but without OKT-3, every 2-3 days until the end of the culture.

[0011] Additional Background Art includes WO2022 / 153295; US Patent No. 10,653, 756; Anderson et al. Blood, American society of Hematology, US, Vol. 80, No. 7 1 January 1992, pages 1846-1853.

[0012] SUMMARY OF THE INVENTION

[0013] According to an aspect of the invention, there is provided a method of generating an expanded population of CD3+ immune cells useful for adoptive cell transfer (ACT), comprising culturing a population of CD3+cells in a medium comprising a cell- stimulating amount of at least one cytokine, a T cell activator selected from the group consisting of a CD3 activator and a CD28 activator, an RAR antagonist and an AHR antagonist; and harvesting the expanded population of T cells.

[0014] According to embodiments of the invention, the method further comprises supplementing, during, or on the same day as the harvesting a cell- stimulating amount of:

[0015] the at least one cytokine,

[0016] the T cell activator selected from the group consisting of a CD3 activator and a CD28 activator.

[0017] According to embodiments of the invention, the method further comprises supplementing the RAR antagonist and the AHR antagonist during, or on the same day as the harvesting.

[0018] According to embodiments of the invention, the at least one cytokine comprises IL-2 or a combination of IL- 15 and IL-7.

[0019] According to embodiments of the invention, the culturing is effected for a minimum of 3 days.

[0020] According to embodiments of the invention, the culturing is effected for no more than 6 days.

[0021] According to an aspect of the invention, there is provided a method of generating an expanded population of T cells useful for adoptive cell transfer (ACT), comprising:

[0022] culturing a population of CD3+ immune cells in a medium comprising a cell stimulating amount of IL-2 and a CD3 activator for at least nine days and no more than 16 days under conditions that increase the number of T cells of the population by at least 20 fold, wherein the medium is devoid of effective stimulating amounts of additional cytokines or antibodies;supplementing the medium with the IL-2 and the CD3 activator at least one time and no more than three times during the culturing, wherein the supplementing is not affected during the first four days of culturing; and

[0023] harvesting the expanded population of T cells, thereby generating the expanded cell composition.

[0024] According to embodiments of the invention, the medium further comprises a small molecule selected from the group consisting of a Cytochrome P450 Family 1 (Cypl) Inhibitor, an AHR antagonist and a RAR antagonist.

[0025] According to embodiments of the invention, the supplementing the medium further comprises supplementing with the small molecule during, or on the same day as the harvesting.

[0026] According to embodiments of the invention, the harvesting is affected following 10-12 days of the culturing.

[0027] According to embodiments of the invention, the supplementing is affected on the day of the harvesting.

[0028] According to embodiments of the invention, the supplementing is affected during the harvesting process.

[0029] According to embodiments of the invention, the supplementing is affected no more than two times during the method.

[0030] According to embodiments of the invention, the at least one time is a single time, on the day of the harvesting.

[0031] According to embodiments of the invention, the at least one time is no more than two times, when one of the times is affected on the day of the harvesting.

[0032] According to embodiments of the invention, the supplementing is affected more than six days following the start of the culturing.

[0033] According to embodiments of the invention, the supplementing is affected more than eight days following the start of the culturing.

[0034] According to an aspect of the invention, there is provided a method of generating an expanded population of T cells useful for adoptive cell transfer (ACT), comprising:

[0035] culturing a population of CD3+ immune cells in a medium comprising a cell-stimulating amount of at least one cytokine and a CD3 activator for at least nine days and no more than 16 days under conditions that increase the number of T cells of the population by at least 20 fold;

[0036] harvesting the expanded population of T cells;supplementing the medium with the at least one cytokine and the CD3 activator no more than two times during the culturing, wherein at least one of the two times is affected on the day of harvesting the expanded population of T cells.

[0037] According to embodiments of the invention, the medium further comprises a small molecule selected from the group consisting of a Cytochrome P450 Family 1 (Cypl) Inhibitor, an AHR antagonist and a RAR antagonist.

[0038] According to embodiments of the invention, the supplementing the medium further comprises supplementing with the small molecule during, or on the same day as the harvesting.

[0039] According to an aspect of the invention, there is provided a method of generating an expanded population of T cells comprising:

[0040] (a) culturing a population of CD3+ immune cells in a medium comprising least one cytokine, a CD3 activator, an RAR antagonist and an AHR antagonist under conditions that generate a population of activated T cells;

[0041] (b) culturing the population of activated T cells in a medium comprising the at least one cytokine, the RAR antagonist and the AHR antagonist, for at least 3 days and no more than 12 days to obtain a population of expanded T cells, wherein the medium of step (b) is substantially devoid of the CD3 activator; and subsequently

[0042] (c) contacting the population of expanded T cells with the at least one cytokine and the CD3 activator.

[0043] According to embodiments of the invention, the at least one cytokine is IL-2, and the medium is devoid of effective amounts of additional cytokines.

[0044] According to embodiments of the invention, the cell- stimulating amount of the IL-2 is between 100-1500 lU / ml.

[0045] According to embodiments of the invention, the at least one cytokine is IL- 15 and IL-7, and the medium is devoid of effective amounts of additional cytokines.

[0046] According to embodiments of the invention, the cell- stimulating amount of the IL- 15 is between 1-100 ng / ml and the cell- stimulating amount of the IL-7 is between 1-100 ng / ml.

[0047] According to embodiments of the invention, the harvesting is affected following 10-12 days of the culturing.

[0048] According to embodiments of the invention, the supplementing is affected at a single time, on the day of the harvesting.

[0049] According to embodiments of the invention, the second of the two times is affected more than six days following the start of the culturing.According to embodiments of the invention, the second of the two times is affected more than eight days following the start of the culturing.

[0050] According to embodiments of the invention, the amount of the CD3 activator is between 10-60 ng / ml.

[0051] According to embodiments of the invention, the cells are not subjected to additional enrichment, stimulation or expansion steps.

[0052] According to embodiments of the invention, the culturing is effected under conditions that increase the number of T cells of the population by at least 30-fold.

[0053] According to embodiments of the invention, the culturing is effected under conditions that increase the number of CD8+ cells of the population by at least 100-fold.

[0054] According to embodiments of the invention, the at least 70% of the cells of the expanded population are CD3+CD8+cells expressing NKG2D and granzyme B.

[0055] According to embodiments of the invention, the cells of the expanded population are capable of specifically eradicating hematopoietic tumor cells in vitro at a ratio of 0.25:1 within 24 hours.

[0056] According to embodiments of the invention, the cells of the expanded population are capable of specifically eradicating ovarian cancer cells in vitro at a ratio of 0.5:1 within 24 hours.

[0057] According to embodiments of the invention, the CD3+ immune cells are genetically modified during the culturing.

[0058] According to embodiments of the invention, the CD3+ immune cells are genetically modified to express at least one of a chimeric antigen receptor (CAR), T cell receptor (TCR), a cytokine, a chemokine, a receptor of a cytokine or chemokine, or any combination thereof.

[0059] According to embodiments of the invention, the CD3+ immune cells are genetically modified to express: (i) a CD19-specific CAR or a CD123-specific CAR; (ii) a modified CXCR4 receptor comprising a mutation or truncation at the C-terminal tail domain; and (iii) at least one cytokine selected from the group consisting of IL-2, IL- 15 and IL-21.

[0060] According to embodiments of the invention, the culturing is affected in a bioreactor.

[0061] According to embodiments of the invention, the medium is RPMI.

[0062] According to embodiments of the invention, the medium is a serum free medium (SFM). According to embodiments of the invention, the method further comprises cryopreserving the obtained cell composition following the harvesting.

[0063] According to embodiments of the invention, the CD3+ immune cells comprise CD3+ enriched T cells.According to embodiments of the invention, the CD3+ immune cells comprise peripheral blood mononuclear cells (PBMCs).

[0064] According to embodiments of the invention, the CD3+ immune cells are non-activated CD3+ immune cells.

[0065] According to an aspect of the invention, there is provided a cell composition obtainable according to the methods described herein.

[0066] According to an aspect of the invention, there is provided a cell composition, comprising in

[0067]

[0068] CD3+ immune cells, of which at least 39 % of the cells of the composition are CD8+CD38“, at least 80 % of the cells of the composition are CD8+CD62L+, at least 32 % of the cells of the composition are CD8+CCR7+and at least 9 % of the cells of the composition are CD3+CD4 CD8“ the composition exhibiting significant cytotoxic activity against tumor cells in vitro and in vivo.

[0069] According to embodiments of the invention, 70-85% of the cell composition are CD3+CD8+cells expressing NKG2D and granzyme B, at least 70% of the cells of the composition are CD56’ cells, and up to 5% of the cells of the composition are CD3“ cells.

[0070] According to embodiments of the invention, at least 39% of the cell composition are CD8+CD38“, at least 80 % of the cells of the composition are CD8+CD62L+, at least 32 % of the cells of the composition are CD8+CCR7+and at least 9% of the cells of the cell composition are CD3+CD4-CD8-.

[0071] According to embodiments of the invention, the cell composition comprises at least 70 % CD3+CD8+T cells, wherein at least 65 % of the T cells are CD62L+and at least 40 % of the cells are CCR7+.

[0072] According to embodiments of the invention, at least 40 % of the T cells are naive stem cell memory T cells characterized by expression of CD45RA+CD62L+.

[0073] According to embodiments of the invention, at least 20 % of the T cells are central memory T cells characterized by expression of CD45RA CD62L+.

[0074] According to embodiments of the invention, the cell composition comprises greater than 100 pmol of NAD per IxlO6cells.

[0075] According to embodiments of the invention, the cells secrete IFN-y, TNF-a, CCL1, CCL5, CCL3 and / or CXCL10.

[0076] According to embodiments of the invention, the cells are genetically modified.

[0077] According to embodiments of the invention, the cells are genetically modified to express at least one of a chimeric antigen receptor (CAR), T cell receptor (TCR), a cytokine, a chemokine, a receptor of a cytokine or chemokine, or any combination thereof.According to embodiments of the invention, the cells are genetically modified to express: (i) a CD19-specific CAR or a CD123-specific CAR; (ii) a modified CXCR4 receptor comprising a mutation or truncation at the C-terminal tail domain; and (iii) at least one cytokine selected from the group consisting of IL-2, IL- 15 and IL-21.

[0078] According to embodiments of the invention, the cells are for use in treating cancer in a subject in need thereof.

[0079] According to embodiments of the invention, the cancer is a hematopoietic cancer or a solid tumor.

[0080] According to embodiments of the invention, the cancer is selected from the group consisting of leukemia, multiple myeloma, a prostate cancer, ovarian cancer and mesothelioma.

[0081] According to embodiments of the invention, the tumor cells of the cancer are characterized by expression of at least one NKG2D ligand.

[0082] According to embodiments of the invention, the tumor cells express a plurality of NKG2D ligands selected from the group consisting of: MICA, MICB, HLAE, ULBP1, ULBP256, and ULBP3.

[0083] According to embodiments of the invention, the tumor cells express MICA, MICB, HLAE, ULBP1, ULBP256, and ULBP3.

[0084] According to embodiments of the invention, the tumor cells of the cancer are characterized by expression of at least one chemokine selected from the group consisting of CXCR6 ligands, CXCR4 ligands, CXCR2 ligands and CXCR3 ligands.

[0085] According to embodiments of the invention, the tumor cells of the cancer are characterized by expression of at least one ligand of an inhibitory immune checkpoint molecule selected from the group consisting of CTLA-4, PD-1, and TIGIT.

[0086] According to embodiments of the invention, the cancer is resistant to treatment by at least one checkpoint molecule inhibitor.

[0087] According to embodiments of the invention, the tumor is resistant to treatment by at least one CTLA-4- specific blocking antibody.

[0088] According to embodiments of the invention, the subject is not under a treatment regimen with checkpoint molecule inhibitors.

[0089] According to embodiments of the invention, the cell composition is autologous, allogeneic, or non-allogeneic to the subject.

[0090] According to embodiments of the invention, the cell composition is non-allogeneic to the subject.According to embodiments of the invention, the tumor is characterized by down-regulation of MHC I expression and / or activity.

[0091] According to an aspect of the invention, there is provided a method of treating a tumor in a subject in need thereof, comprising contacting the tumor cells with a therapeutically effective amount of the cell composition described herein, thereby treating the tumor in the subject.

[0092] According to embodiments of the invention, the cell composition is autologous, allogeneic, or non-allogeneic to the subject.

[0093] According to embodiments of the invention, the cell composition is non-allogeneic to the subject.

[0094] According to embodiments of the invention, the contacting is performed in vivo.

[0095] According to embodiments of the invention, the contacting is performed ex vivo.

[0096] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[0097] BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

[0098] Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

[0099] FIGs. 1A-B: On day 0, PBMCs were seeded into G-Rex 10M flasks containing 30 ml of RPMI complete medium with IL-2 and anti-CD3. Restimulation involved adding an additional 30 ml of fresh medium with IL-2 and anti-CD3 to each flask on days 4, 7, or 8 as indicated. For the group without restimulation, 30 ml of medium without activators was added on days 4 and 8.

[0100] On day 11, T cells were harvested and analyzed for cell numbers (fold expansion, Figure 1A). T cells were then incubated with CFSE-labeled MV4-11 leukemic cells at different target-to-effector (E: T) cell ratios. The percentage (%) of lysed cells after 24 hours of incubation was evaluated using FACS analysis (Figure IB). Data are mean± standard deviation from triplicate at each ratio.FIGs. 2A-B: PBMCs were seeded into G-Rex 10M flasks with 90 ml of RPMI complete medium with IL-2, and anti CD3. The second restimulation process occurred either on day 8 or on day 11, for 1.5 or 3 hours - during harvesting T cells. On day 11, T cells were harvested and analyzed for cytotoxic efficacy against the leukemic cells MV4-11 (Figure 2A) and the ovarian cancer cell SKOV3 (Figure 2B). Data are mean± standard deviation from triplicate.

[0101] FIGs. 3A-B: PBMCs were seeded into G-Rex 10M flasks with 90 ml of RPMI complete medium with IL-2, and anti CD3. The second restimulation process occurred either on day 11 or on day 14, for 1.5 hours during harvesting T cells. T cells were harvested and analyzed for fold expansion (Figure 3 A) and their cytotoxic efficacy against the leukemic cells MV4-11 (Figure 3B). Data are mean± standard deviation from triplicate.

[0102] FIGs. 4A-C. PBMCs were seeded into G-Rex 10M flasks with 90 ml of RPMI complete medium with IL-2 and anti CD3 either in the presence or the absence (control) of Rhapontigenin, (SP163 (0.5nM)), CYP1 selective inhibitor. The second restimulation process (IL-2 and anti CD3) occurred 1.5 hours prior to cell harvesting on day 11. T cells were harvested and analyzed for fold expansion (Figure 4A) and their cytotoxic efficacy against the leukemic cells MV4-11 (Figure 4B) and SKOV3 ovarian cancer (Figure 4C). Data are mean± standard deviation from triplicate.

[0103] FIGs. 5A-G. PBMCs were seeded into G-Rex 10M flasks with 90 ml of RPMI complete medium with IL-2 and anti CD3 either in the presence or the absence of RAR antagonist (AGN194310, 0.25pM). The second restimulation process (IL-2 and anti CD3) occurred during cell harvesting for 1.5 hours followed by final wash on day 11. T cells were harvested and analyzed for their cytotoxic efficacy against the leukemic cells MV4-11 (Figure 5A) and SKOV3 ovarian cancer (Figure 5B). Data are mean± standard deviation from triplicate. T-cells characteristics were analyzed by MITOSOX™ staining (Figure 5C) and phenotype by Flow cytometry analysis of CD8+ cells. Expression of CD57+(Figure 5D), CD38+(Figure 5E) and CD62L+, CCR7+(Figure 5F) on T cells analyzed by FACS. % of naive cells, central memory (CM), effector memory (EM) and effector cells were analyzed by FACS (Figure 5G).

[0104] FIG. 6: PBMCs were seeded into G-Rex 10M flasks with 90 ml of RPMI complete medium with IL-2 and anti CD3 along with RAR antagonist (AGN194310 0.2pM). The “RAR+ Restimulation” group underwent a second restimulation process (IL-2 and anti CD3) for 1.5 hours during cell harvesting on day 11, while the “RAR” group did not undergo restimulation. T cells were harvested and analyzed for their expression of CD62L+CCR7+.

[0105] FIGs. 7A-E: PBMCs were seeded into G-Rex 10M flasks with 90 ml of RPMI complete medium with IL-2, and anti CD3. The medium was supplemented with either 10% FBS or 5% HS. The second restimulation process occurred on day 11 for 1.5 hours during harvesting T cells. Onday 11, T cells were harvested and analyzed for fold expansion (Figure 7A) and their cytotoxic efficacy against the leukemic cells MV4-11 (Figure 7B). Data are mean± standard deviation from triplicate. Expression of CD57+, CD38+(Figure 7C) and CD62L+ (Figure 7D) on T cells was analyze by FACS. Figure 7E: The cells were cultured as described in Figures 7A-D in either RPMI complete medium supplemented with 10% FCS or in the serum-free medium NutriT. Following harvest, the cells were analyzed for cytotoxic efficacy against the ovarian cancer cell lines SKOV3 and OVCAR using a 3D tumor spheroid model.

[0106] FIGs. 8A-C. PBMCs were seeded into G-Rex 10M flasks with 90 ml of RPMI complete medium with IL-2 and anti CD3 either in the presence or the absence of AHR antagonist (CH223191 0.2pM). The second restimulation process (IL-2 and anti CD3) occurred 1.5 hours during harvesting on day 11. Following restimulation, T cells were harvested and analyzed for fold expansion (Figure 8 A) and their cytotoxic efficacy against the leukemic cells MV4-11 (Figure 8B) and SKOV3 ovarian cancer (Figure 8C). Data are mean± standard deviation from triplicate.

[0107] FIGs. 9A-B. PBMCs were seeded into G-Rex 10M flasks with 90 ml of RPMI complete medium with IL-2 and anti CD3 either in the presence or the absence of AHR antagonist (CH223191 0.05pM), RAR antagonist (AGN194310 O.OlpM or 0.05pM) or a combination of both. The second restimulation process (IL-2 and anti CD3) occurred 1.5 hours during harvesting on day 11. Following restimulation, T cells were analyzed for cytotoxic efficacy against the leukemic cells MV4-11 (Figure 9A). Data are mean± standard deviation from triplicate. T cells were analyzed for cytotoxic efficacy against ovarian cancer cells SKOV3 in 3D tumor spheroid model (Figure 9B).

[0108] FIG. 10. PBMCs were seeded into G-Rex 10M flasks with 90 mL of RPMI complete medium containing IL-2 and anti-CD3, in the presence and absence (control) of the AHR antagonist (CH223191, 0.05 pM) and the RAR antagonist (AGN194310, 0.01 pM). The "Restimulation" group underwent a second restimulation process (IL-2 and anti-CD3) 1.5 hours during harvesting on day 11, while the "w / o restimulation" group did not undergo restimulation. _ T cells were harvested and evaluated for cytotoxic efficacy against ovarian cancer SKOV3 cells in a 3D tumor spheroid model.

[0109] FIGs. 11A-G. PBMCs were seeded into G-Rex 10M flasks containing 90 ml of RPMI complete medium, supplemented with anti CD3 and either IL-2 or IL-15 and IL-7 (lOng / ml). The second restimulation process (IL-2 and anti CD3 or IL-15, IL-7 and anti CD3) occurred 1.5 hours during cell harvesting on day 11. T cells were harvested and analyzed for their fold change (Figure 11A) and cytotoxic efficacy against the leukemic cells MV4-11 (Figure 11B). Data are mean± standard deviation from triplicate. T phenotype was analyzed by Flow cytometry analysis of CD8+cells. Expression of CD57+(Figure 11C), CD38+(Figure 11D) and CD62L+and CCR7+(Figure1 IE) on T cells was analyzed by FACS. % of naive cells, CM, EM and effector cells were analyzed by FACS (Figure 11F). T phenotype was also analyzed by MITOSOX™ staining (Figure 11G).

[0110] FIG. 12A present results of FACS analysis of CD3 / CD4 / CD8 expression of an exemplary cell population obtained following expansion according to embodiments of the invention.

[0111] FIG. 12B presents results of RNA-Seq analysis which revealed distinct gene expression patterns between untreated T cells and T cells treated with RAR and AHR antagonists. Differentially expressed genes between untreated and RAR / AHR antagonist-treated T cells were subjected to canonical pathway enrichment analysis. The bar graph displays significantly enriched pathways ranked by -log(BH-adjusted p-value). Pathways shown represent those significantly upregulated in RAR and AHR antagonist-treated cells relative to Control (untreated cells), highlighting modulation of immune signaling, antigen presentation, and activation-related pathways.

[0112] FIGs. 13A-B are bar graphs illustrating an increased cellular NAD content following short stimulation during harvest with RAR. T cells generated as described in Method 2 of Example 2 were restimulated for 1.5 hours with either IE-2 and anti-CD3, or with IL-2, anti-CD3, and RAR antagonist. Following restimulation, cells were washed and cultured in fresh IL-2- supplemented medium alone for an additional 6 days. (A) Flow cytometric analysis of CD38Ahigh expression on CD8+T cells. (B) Intracellular NAD levels (pmol / lE6 cells).

[0113] FIGs. 14A-B. RAR antagonist effects: differential coupling to continuous TCR signaling. PBMCs were cultured in T-75 flasks in RPMI complete medium supplemented with IL-2, anti-CD3 antibody, RAR antagonist, and AHR antagonist for 48 h. On day 2, cells were washed and transferred to G-Rex culture vessels and expanded either in the presence or absence of anti-CD3 antibody. A final restimulation with IL-2 and anti-CD3 was performed 3 h prior to cell harvest on day 12. T-cell phenotype was analyzed by flow cytometry gating on CD8+cells. The percentage of naive, central memory (CM), effector memory (EM), and effector T cells is shown in Figure 14A, and CD38 expression is shown in Figure 14B.

[0114] FIGs. 15A-E are graphs which compare T-cell phenotypes of CAR-T cells in the presence and absence of small molecules. T-cell phenotype was assessed by flow cytometry with gating on CD8+T cells. CD38 expression is shown in Figure 15 A, and intracellular NAD levels are shown in Figure 15B. The distribution of naive, central memory (CM), effector memory (EM), and effector T-cell subsets is shown in Figure 15C. The frequency of CD62L+and CCR7+cells within the CD8+T-cell population is shown in Figure 15D. For cytotoxicity assessment, day-9 T cells were coincubated with CFSE-labeled THP-1 leukemic cells at varying effector-to-target (E:T) ratios. Targetcell lysis was quantified after 24 hours by flow cytometry (Figure 15E). Data are presented as mean ± standard deviation from triplicate samples at each E:T ratio.

[0115] FIGs. 16A-B illustrate the contribution of the small molecules when cells are cultured without continuous exposure to anti CD3. FIG. 16A. Short anti-CD3 restimulation at harvest enhances T-cell cytotoxic activity. T cells were expanded for 9 days in the presence of IL-7, IL-15, and RAR and AHR antagonists. On the day of harvest, T cells were restimulated for 1.5 hours with either IL-7 and IL- 15 alone or with IL-7, IL- 15, and anti-CD3 antibody. For cytotoxicity assessment, day-9 T cells were co-incubated with CFSE-labeled THP-1 or MV411 leukemic cells, as well as U266 multiple myeloma cells, at varying effector-to-target (E:T) ratios. Target cell lysis was quantified after 24 hours by flow cytometry. Data are presented as mean ± standard deviation from triplicate samples at each E:T ratio. FIG. 16B. CD38 downregulation is independent of continuous TCR activation during T-cell expansion. Standard T cells were expanded for 9 days in the presence or absence of the small-molecule RAR and AHR antagonists. CD38 expression was assessed by flow cytometry, and the percentage of CD38+cells within the CD8+T-cell population is shown.

[0116] FIGs. 17A-E (CD123-CAR). T cells were transduced with gammaretroviral vectors encoding a CD123-CAR. (A) The frequency of CD123-CAR-expressing cells within the CD8+T-cell population was determined by flow cytometry. (B) Cytotoxic activity was assessed by co-incubating day-9 CAR-T cells with CFSE-labeled MV411 leukemic target cells at the indicated effector-to-target (E:T) ratios. Target cell lysis was quantified after 24 h by flow cytometry. Data are presented as mean ± SD of triplicate samples. (C) CD38 expression on CD8+T cells. (D) Proportions of naive and central memory (CM) CD8+T cells. (E) Expression of the memory-associated markers CD62L and CCR7 on CD8+ T cells.

[0117] FIGs. 17F-J (CD19-CAR). T cells were transduced with gammaretroviral vectors encoding a CD19-CAR. (F) Distribution of naive, central memory (CM), effector memory (EM), and effector CD8+T-cell subsets. (G) Frequency of CD62L+and CCR7+cells within the CD8+T-cell population, as determined by flow cytometry. (H) CD38 expression on CD8+T cells. (I) Intracellular NAD levels (pmol / 10E6 cells). (J) Production of ROS (MITOSOX™ experiment)

[0118] FIGs. 17K-L (HER2-CAR). T cells were transduced with gammaretroviral vectors encoding a HER2-CAR. CD8+T-cell phenotype was analyzed by flow cytometry. (K) Proportions of naive and central memory (CM) CD8+T cells. (L) CD38 expression within the CD8+T-cell population.

[0119] FIGs. 17M-0 (BCMA-CAR). T cells were transduced with gammaretroviral vectors encoding a BCMA-CAR. CD8+T-cell phenotype was assessed by flow cytometry. (M) CD38 expression on CD8+T cells. (N) Distribution of naive, central memory (CM), effector memory (EM),and effector CD8+T-cell subsets. (O) Expression of CD62L and CCR7 within the CD8+T-cell population.

[0120] FIGs. 18A-B. Small molecules enhance cytokine secretion by both T-cells and CAR-T cells. T-cells (Figure 18A) and CAR-T cells (Figure 18B) were co-cultured with MV411 leukemic cells and CD14+monocytes (from the same donor) at a ratio of 2:1:0.25 for 24 hours. Culture supernatants were collected and analyzed using a human cytokine array. The detected levels of IFN-y and TNF-a are shown.

[0121] FIGs. 19A-H. Small molecules enhance chemokine secretion in T-cells (Figure 19A-D) and CAR-T cells (Figure 19 E-H). T-cells and CAR-T cells were co-cultured with MV411 leukemic cells and CD14+monocytes at a ratio of 2:1:0.25 for 24 hours. Culture supernatants were collected and analyzed using a human cytokine array. The detected levels of chemokines are shown.

[0122] FIGs. 20A-C. Short-term expansion of T cells in the presence of small molecules results in superior cytotoxic activity compared with standard T cells.

[0123] Rapid manufacturing of T cells was performed using either PBMCs as the starting material (Figure 20A) or purified CD3+T cells as the starting material (Figure 20B). Cytotoxic activity was evaluated by co-incubating day-6 T cells with CFSE-labeled RAJI target cells (derived from Eymphoma patient) at the indicated effector-to-target (E:T) ratios. Target cell lysis was quantified after 24 hours by flow cytometry. Data are presented as mean ± SD of triplicate samples (Figures 20A and 20B). CD38 expression on CD8+T cells generated from CD3+T cells (in the presence of small molecules) were analyzed by flow cytometry and is shown in Figure 20C.

[0124] FIGs. 21A-C. Short-term expansion of CD 19 CAR-T cells in the presence of small molecules compared with standard CD 19 CAR-T cells. (A) Expression levels of CD 19 CAR molecules on CAR-T cells in the presence and absence of small molecules. (B) CD38 expression on CD8+standard CAR-T cells and CAR-T cells generated from either CD3+T cells or PBMCs in the presence of small molecules, as analyzed by flow cytometry. (C) Cytotoxic activity of standard CAR-T cells and CAR-T cells in the presence of small molecules produced using PBMCs as the starting material.

[0125] FIGs. 22A-B: Ultra Short-term (4 days) manufacturing of T-cells and CD 19 CAR-T cells in the presence and absence of small molecules. Expression levels of CD 19 CAR molecules on CAR-T cells in the presence and absence of small molecules. (B) CD38 expression on CD8+T-cells and CAR-T cells generated from PBMCs in the presence and absence of small molecules, as analyzed by flow cytometry.DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

[0126] The present invention, in some embodiments thereof, relates to methods for selectively expanding T cells for use in adoptive cell therapy.

[0127] Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

[0128] Whilst conceiving embodiments of the present invention, the present inventors have uncovered novel PBMC culturing protocols that result in the expansion of particularly tumor-cytotoxic cell populations, including CD3+T cells and genetically modified T cells such as chimeric antigen receptor (CAR)-expressing T cells.

[0129] Such cell populations were significantly more effective at eradicating tumor cells compared to other PBMC-derived effector cell compositions such as those disclosed in WO2022 / 153295 and other cytokine-induced killer cells (CIK) and interleukin-2 (IL-2)-expanded T cell compositions, produced according to hitherto known ACT protocols.

[0130] As is illustrated hereinbelow and in the Examples section which follows, the present inventors have discovered that supplementation of particular cytokines in combination with a CD3 activator after at least four days from the start of culture enhances the tumor cytotoxicity of the cells (Figures 1A-B). In particular, restimulation with the cytokines and CD3 activator on the same day as (or during) cell harvesting resulted in the most anti-tumor cytotoxic cell populations (Figure 2A).

[0131] Whilst further reducing the present invention to practice, the present inventors uncovered particular advantageous small molecule agents that can be included in the culturing protocol. These small molecule agents, when added to the expansion cultures, significantly enhance T fitness by increasing resistance to reactive oxygen species (ROS) and raising the proportion of naive and central memory cells within the cell composition and optionally increase the cellular Nicotinamide adenine dinucleotide (NAD) content. Most significantly, the small molecules enhanced the eradication of solid tumor cells in a 3D, 6-day, spheroid model (Figure 9B and Figure 10). Of note, this effect was seen even under stress conditions, e.g., in the absence of IL-2.

[0132] Incorporation of the disclosed small molecule agents into the expansion protocol was shown to increase viral transduction efficiency (Figures 17A, Figure 21 A & Figure 22A) and to produce superior CAR-T cells as demonstrated in Figures 17A-O, 18B and 19E-H as compared to the same protocol in the absence of the small molecule agents.Using the disclosed small molecule agents further allowed for generation of CAR-T cells with enhanced cytotoxic activity using a shorter expansion protocol (Figures 21A-C and Figures 22A-B).

[0133] Thus, provided in embodiments of the invention are cell compositions for ACT, characterized by improved therapeutic properties. In other embodiments, the invention relates to improved processes for producing cell compositions for ACT. According to some embodiments, the cell compositions of the invention may advantageously be produced directly from PBMC samples by ex vivo expansion protocols as disclosed herein, without a preceding step of apheresis or other purification steps, and without the need to isolate tumor- specific cells from tumor biopsies. In some embodiments, the cells of the sample are expanded by incubation or culturing in the presence of IL-2 and a CD3 activator (e.g. CD3-specific stimulating antibody) for at least 3, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days and typically for 9-16 days, more typically 10-12 days. In other embodiments, the cells of the sample are expanded by incubation or culturing in the presence of IL-15 and IL-7 and a CD3 activator (e.g., CD3-specific stimulating antibody, or CD3 / CD28 activator beads such as those available from ThermoFisher) for at least 3 days, at least 4 days, at least 5 days at least 6 days, at least 7 days, at least 8 days, at least 9 days and typically for 9-16 days, more typically 10-12 days. It is to be understood, that the anti-CD3 antibody may conveniently be used in a free suspension form, and need not be plate bound or presented by allogeneic feeder cells. In particular, the cells are initially expanded in the presence of effective amounts of the IL-2 (or IL 15 and IL-7) and CD3 activator.

[0134] Thus, according to a first aspect of the invention, there is provided a method of generating an expanded population of T cells useful for adoptive cell transfer (ACT), comprising:

[0135] culturing a population of CD3+ immune cells in a medium comprising a cell stimulating amount of IL-2 and a CD3 activator for at least nine days and no more than 16 days under conditions that increase the number of T cells of the population by at least 20 fold, wherein the medium is devoid of effective stimulating amounts of additional cytokines or antibodies;

[0136] supplementing the medium with said IL-2 and said CD3 activator at least one time and no more than three times during the culturing, wherein said supplementing is not affected during the first four days of culturing; and

[0137] harvesting the expanded population of T cells, thereby generating the expanded cell composition.

[0138] As used herein, and unless otherwise specified, the term "adoptive transfer" refers to a form of passive immunotherapy where previously sensitized immunologic agents (e.g., cells or serum) are transferred to the recipients. The phrases “adoptive transfer immunotherapy”, “adoptive celltherapy” and “adoptive cell immunotherapy” are used interchangeably herein to denote a therapeutic or prophylactic regimen or modality, in which effector immunocompetent cells, such as the cell compositions of the invention, are administered (adoptively transferred) to a subject in need thereof, to alleviate or ameliorate the development or symptoms of cancer.

[0139] The term “CD3+ immune cells” as used throughout the specification, refers to a population of mature T cells and includes both CD4+ and CD8+ cells.

[0140] In one embodiment, the CD3+ immune cells are PBMCs.

[0141] In another embodiment, the CD3+ immune cells are CD3+ enriched cells.

[0142] The term "PBMCs" or "peripheral blood mononuclear cells sample" or "un-fractionated PBMC", as used herein, refers to PBMC, i.e., to a population of viable white blood cells having a round nucleus. In one embodiment, the PBMCs have not been substantially enriched in a given subpopulation. Typically, the PBMC sample has been obtained from peripheral blood and has not been subjected to a selection step so as to contain only adherent PBMC (which consist essentially of >90% monocytes) or non-adherent PBMC (which contain T cells, B cells, natural killer (NK) cells, NK T cells and DC precursors). Typically, PBMCs can be obtained (e.g. extracted or partially purified from peripheral blood) by centrifugation (e.g., from a buffy coat), by apheresis, by density gradient centrifugation (e.g., through a Ficoll-Hypaque) or by other methods known in the art. For example, PBMC may be extracted from whole blood using Ficoll, a hydrophilic polysaccharide that separates layers of blood, with the PBMC forming a cell ring under a layer of plasma. Additionally, PBMC can be extracted from whole blood using a hypotonic lysis which will preferentially lyse red blood cells. Such procedures are known to the expert in the art.

[0143] In one embodiment, the CD3+ immune cells (e.g. CD3+ enriched T cells) have been genetically modified, as further described below.

[0144] In another embodiment, the CD3+ immune cells (e.g. PBMCs) have been pre-activated as further described below.

[0145] In another embodiment, the CD3+ immune cells (e.g. PBMCs) have not been pre-activated as further described below.

[0146] In another embodiment, the CD3+ immune cells PBMCs have been pre-expanded, as further described below.

[0147] CD3+ enriched T cells may be purified from PBMCs or whole blood or apheresis using techniques known in the art including for example magnetic bead separation (e.g., with anti-CD3 antibodies conjugated to magnetic beads or by flow cytometry-based cell sorting (FACS). The phrase “CD3+ enriched T cells refers to a population of cells” refers to a population of T cells inwhich more than 70 % thereof, 80 % thereof, 90 % thereof, 95 % thereof express the surface marker CD3.

[0148] The PBMCs are cultured so as to increase the number of T cells (i.e. expanded) due to cell replication.

[0149] In particular, PBMC expansion as disclosed herein refers to an in vitro or ex vivo culturing process comprising polyclonal activation and multiplication of cell populations within the PBMC, so as to produce an ACT cell composition in accordance with the invention. Typically, e.g. for producing a clinical-grade ACT composition for treatment of a human subject, expansion is performed in a specific cGMP grade environment and cGMP grade medium. In some embodiments, expansion is performed for 9-16 days or 11-16 days, e.g. 9, 10, 11, 12, 13, 14, 15, or 16 days. In other embodiments, expansion is performed for 3-6 days.

[0150] In one embodiment, a stimulating amount of interleukin-2 (IL-2) and a CD3 activator are included in the culture medium at the start of the culturing process. In some embodiments, the cell composition is prepared by a process as described above, wherein additional effective amounts of IL-2 and CD3 activator are supplemented only after 4 days, 5 days, 6 days, seven days, eight days, nine days, or ten days.

[0151] The term “supplementing” as used throughout the specification, refers to the addition of factors to the culture medium other than at the start of the culturing protocol.

[0152] For any of the aspects described herein, in one embodiment, the IL-2 and CD3 activator are supplemented only one additional time during the course of the culturing, or only two additional times during the course of the culturing. Optionally, one of the times supplementation is affected is on the day of harvesting (e.g. day 10, 11, or 12). Optionally, the only time supplementation is affected is on the day of harvesting (e.g. day 10, 11, or 12). More specifically, the only time supplementation is affected is during the initiation of the harvesting process itself. For example, a portion of the medium has been removed from the cell culture, the IL-2 and CD3 are supplemented. The cells may be left in contact with the activators for a period of time that is sufficient to allow restimulation (e.g. 1.5-3 hours). Following optional rinsing, the cells may then be removed from the culture surface. It will be appreciated that supplementation does not occur more than three times during the course of the culturing.

[0153] As used herein, “interleukin 2” or “IL-2” refers to mammalian IL-2, preferably human IL-2 (e.g. recombinantly produced human IL-2). In some embodiments, polypeptides recognized in the art as biological equivalents of human IL-2, capable of selectively binding to IL-2 receptors and exerting comparable activity in supporting the expansion and proliferation of human lymphocytes (e.g. IL-2 fusion proteins, conjugates and variants) may be used. IL-2 may be producedrecombinantly (e.g. by methods as disclosed herein) and is commercially available from a variety of sources. For example, recombinant human IL-2 (rhIL-2) may be purchased from R&D Systems. Proleukin® (aldesleukin) is a highly purified rhIL-2 protein, approved for clinical use in cancer immunotherapy .

[0154] In the context of in vitro cell culture methods, a “stimulating” amount an agent is an amount sufficient to exert advantageous phenotypic modulations as disclosed herein, including, but not limited to cell expansion, up-regulation of activation markers (e.g. DNAM-1, NKG2D) and enhancement in cytotoxic capacity.

[0155] According to a particular embodiment, IL-2 is initially present in the culture medium at a concentration of about 50-1500 or 500-1500 lU / ml. Furthermore, supplementation with IL-2 is carried out at a concentration of about 50-1500 or 500-1500 lU / ml.

[0156] The CD3 complex is associated with T cell receptor (TCR) chains, forming the TCR-CD3 complex on the surface of T cells. Thus, CD3 is considered a T cell marker, although certain other cell populations such as NK-T cells, are also characterized by CD3 surface expression (CD3+cells), whereas other cell types, such as NK cells, B cells and myeloid cells are not generally characterized by lack of CD3 surface expression (CD3“ cells). In mammals, the TCR-CD3 complex is comprised of a CD3y chain, a CD35 chain, and two CD3s chains, complexed with the TCR chains and the CD3-zeta (^-chain). As used herein, a "CD3 activator" is an agent capable of inducing or mediating polyclonal stimulation and proliferation by binding to a CD3 molecule on the surface of a lymphocyte, typically, a human CD3 molecule. Examples of CD3 activators include stimulatory CD3-specific antibodies, antigen-binding portions thereof, and conjugates thereof.

[0157] Typically, the CD3 activator is a stimulatory anti-CD3 monoclonal antibody (mAb), typically directed to a CD3 signaling domain such as on CD3s. In a particular embodiment, an advantageous CD3 activator to be used in embodiments of the invention is OKT3, a murine monoclonal antibody directed against the human CD3s chain. Other exemplary antibodies that crosslink the T cell receptor / CD3 complex include the HIT3a and UCHT1 anti-CD3 mAbs. Additional examples of CD3 activators are CD3 ligands and CD3-binding mitogens. Such antibodies and agents are commercially available from a variety of sources. For example, OKT3 and other anti-CD3 mAbs are available from Biolegend and eBioscience.

[0158] According to a particular embodiment, CD3 activator is initially present in the culture medium at a concentration of about 1-60 ng / ml (e.g. about 30 ng / ml). Furthermore, supplementation with the CD3 activator is carried out at a concentration of about 1-60 ng / ml (e.g. about 30 ng / ml).For example, without limitation, effective amounts for IL-2 may include e.g. 500-1500, 600-1300, 750-1500 or 500-1200 lU / ml for recombinant human IL-2, and for anti-CD3 antibodies e.g.

[0159] 10-60, 20-40, 10-40 or 20-60 ng / ml for OKT3.

[0160] According to embodiments of the invention, the PBMC are expanded in the absence of other exogenously added cytokines or antibodies. In other words, cytokines and antibodies other than IL-2 and the CD3 activator are not supplemented (added exogenously) to the culture. Therefore, expansion processes in accordance with the invention are distinguishable from other expansion protocols, used for producing other types of cell compositions (for example CIK cells are produced by expansion of e.g. PBMC in the presence of inter alia IFN-y). It is to be understood, however, that during the expansion process, various factors, including cytokines, may be produced by the cultured cells and secreted to the culture media.

[0161] Other cell stimulators used to induce lymphocyte activation and proliferation during in vitro or ex-vivo expansion processes, such as antibodies directed to co- stimulatory molecules (e.g. CD28), antigens (e.g. MHC-antigen complexes) and the like, which are of an exogenous source to the cultured cells, are not artificially introduced (supplemented). It is to be understood, that certain cell stimulators may be produced endogenously by the cultured cells during the expansion process. It is further understood, that various factors included in standard tissue culture media (for example, in complete medium such as RPMI) are not considered to be exogenously-added cell stimulators according to these embodiments. For example, expansion may conveniently be performed in the presence of tissue culture serum, e.g. fetal calf serum (FCS) or human serum at a final concentration of 5-15%, typically 7-12% or 8-13% and more typically about 10%, or in serum-free mediums (such as LymphoONE, NutriT, CTS™ AIM V, X-Vivo etc.

[0162] As disclosed herein, expansion is advantageously performed so as to enhance the number of viable cells in the culture by at least 20-fold (or in some embodiments 30-60-fold), to thereby obtain a cell composition comprising 5xl06to 10xl08or in other embodiments up to 10xl09or even 10xl010viable cells. In one embodiment, expansion is advantageously performed so as to enhance the number of viable T cells in the culture by at least 20-fold (or in some embodiments 30-60-fold), to thereby obtain a cell composition comprising 5xl06to 10xl08viable T cells or in other embodiments up to 10xl09or even 10xl010viable T cells.

[0163] In one embodiment, expansion is advantageously performed so as to enhance the number of viable CD8 T cells in the culture by at least 100-fold (or in some embodiments 100-200-fold), to thereby obtain a cell composition comprising 5xl06to 10xl08viable T cells or in other embodiments up to 10xl09or even 10xl010viable CD8 T cellsThe term "viable cells", as used herein, refers to cells not undergoing necrosis or cells which are not in an early or late apoptotic state. Assays for determining cell viability are known in the art, such as using propidium iodide (PI) staining which may be detected by flow cytometry. Accordingly, in one embodiment, viable cells are cells which do not show propidium iodide intake and do not express phosphatidylserine. Necrosis can be further identified, by using light, fluorescence or electron microscopy techniques, or via uptake of the dye trypan blue.

[0164] In addition, the production processes of the invention comprise a step of collecting the cell composition resulting from the expansion step. For example, the cells may be harvested and subjected to centrifugation or other washing steps, to remove residual antibodies, cytokines and / or small molecules. In some embodiments, cells may be re-suspended in a suitable diluent or vehicle (e.g. PBS) prior to contacting with tumor cells or administration to a subject.

[0165] In some embodiments, the cells have not been subjected to additional steps of enrichment (e.g. apheresis, lymphocytoapheresis, or immunocapture of leukocytes prior to the culturing (i.e. expansion step), stimulation (e.g. antigen- specific) and / or expansion (e.g. a rapid expansion protocol in the presence of feeder cells or propagation in the presence of other / additional cytokines).

[0166] In another aspect, there is provided a method of generating an expanded population of T cells (e.g. CAR-T cells) useful for adoptive cell transfer (ACT), comprising:

[0167] culturing a population of T cells in a medium comprising a cell-stimulating amount of:

[0168] at least one cytokine,

[0169] a T cell activator selected from the group consisting of a CD3 activator and a CD28 activator,

[0170] an RAR antagonist and

[0171] an AHR antagonist; and

[0172] harvesting the expanded population of T cells.

[0173] The cultured T cells may be comprised in a PBMC sample. In another embodiment, the cultured T cells are isolated T cells.

[0174] In still another embodiment, the cultured T cells are genetically manipulated isolated T cells. In still another embodiment, the cultured T cells are genetically manipulated PBMCs.

[0175] Exemplary cytokines contemplated by the present invention include, but are not limited to IL-2 (as described herein above) or a combination of IL- 15 and IL-7.

[0176] As used herein, “interleukin 15” or “IL-15” refers to mammalian IL-15, preferably human IL-15 (e.g., recombinantly produced human IL-15). In some embodiments, polypeptides recognized in the art as biological equivalents of human IL- 15, capable of selectively binding to IL- 15 receptorcomponents and exerting comparable activity in promoting the survival, activation, or proliferation of human lymphocytes, particularly CD8+T cells and natural killer (NK) cells may be used. These include, for example, IL-15 fusion proteins, IL-15 / IL-15Ra complexes (e.g., heterodimeric forms), conjugates, and functional variants. IL- 15 may be produced recombinantly or obtained from commercial sources. Recombinant human IL-15 (rhIL-15) is available from R&D Systems and other suppliers, and may be formulated alone or in combination with IL- 15 receptor alpha (IL-15Ra) to enhance bioactivity and stability.

[0177] As used herein, “interleukin 7” or “IL-7” refers to mammalian IL-7, preferably human IL-7 (e.g., recombinantly produced human IL-7). In some embodiments, biologically active variants or analogs of IL-7 may be used, including those capable of binding to the IL-7 receptor complex and exerting a comparable activity in supporting the survival and homeostatic expansion of human T cells, particularly naive and memory T cells. Such variants include IL-7 fusion proteins and glycosylation variants. IL-7 may be recombinantly expressed or sourced commercially; recombinant human IL-7 (rhIL-7) is available from multiple vendors including PeproTech and R&D Systems, and is under clinical evaluation for use in immunotherapy and lymphocyte reconstitution.

[0178] RAR antagonists and AHR antagonists are described herein below.

[0179] In one embodiment, the T cells are cultured in a medium comprising said above disclosed components for at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days or at least 11 days. For example, in one embodiment, the T cells are cultured in a medium comprising said above disclosed components for 3-6 days, 3-12 days, 6-12 days.

[0180] The components may be added at any time to the medium. Typically, the components are added at the start of culture.

[0181] Optionally, at least one, two, three or all of the above disclosed components are supplemented during the culturing phase.

[0182] Supplementation of the components may be once every three, four, five, six, seven, eight, nine, ten or more days, depending on the length of the culturing period. In one embodiment, the components (e.g. IL-2 and CD3 activator; IL-7, IL-15 and CD3 activator) are also supplemented on the day of (or during) harvesting, as further described herein above.

[0183] Exemplary methods of expanding T cells are described in Arcangeli S, (2020) Front. Immunol. 11:1217 doi: 10.3389 / fimmu.2020.01217; Abraham-Miranda J, (2022); Front. Immunol.

[0184] 13:1007042 doi: 10.3389 / fimmu.2022.1007042; Schanda et al., Cells 2021, 10, 3208. www(dot)doi(dot)org / 10(dot)3390 / cellsl0113208; Schubert et al., Cells 2021, 10, 3208.www(dot)doi(dot)org / 10(dot)3390 / cellsl0113208:Ghassemi et al., Nature Biomedical Engineering, VOL 6 118; February 2022 pages 118-128; Watanabe N, (2022) Front. Immunol. 13:876339, doi: 10.3389 / fimmu.2022.876339; Hu et al., STAR Protocols 5, 103333, December 20, 2024; Schubert M-L, et al. BMJ Open 2019;9:e026644. doi: 10.1136 / bmjopen-2018-026644, all of which are incorporated herein by reference.

[0185] In another aspect, there is provided a method of generating an expanded population of T cells useful for adoptive cell transfer (ACT), comprising:

[0186] culturing a population of peripheral blood mononuclear cells (PBMCs) in a medium comprising a cell-stimulating amount of at least one cytokine and a CD3 activator for at least nine days and no more than 16 days under conditions that increase the number of T cells of the population by at least 20-fold;

[0187] harvesting the expanded population of T cells;

[0188] supplementing the medium with said at least one cytokine and said CD3 activator no more than two times during the culturing, wherein at least one of the two times is affected on the day of harvesting the expanded population of T cells.

[0189] In one exemplary protocol, the PBMCs are expanded in a medium comprising IL-2 and the CD3 activator (as described herein above). Supplementation of the medium with IL-2 and optionally CD3 activator is contemplated.

[0190] According to this aspect of the invention, additional effective amounts of IL-2 and CD3 activator are supplemented only after 4 days, 5 days, 6 days, seven days, eight days, nine days, or ten days.

[0191] In one embodiment, the IL-2 and CD3 activator are supplemented only one additional time during the course of the culturing, or only two additional times during the course of the culturing. Optionally, one of the times supplementation is affected is on the day of harvesting (e.g. day 10, 11, 12, 13, 14, 15 or 16). Optionally, the only time supplementation is affected is on the day of harvesting (e.g. day 10, 11, 12, 13, 14, 15 or 16).

[0192] More specifically, the IL-2 and CD3 activator are supplemented during the harvesting process itself. Preferably, when the medium is supplemented with the cytokine (e.g. IL-2) and CD3 activator on the day of (or during) harvesting, the contacting is affected for at least 30 minutes, at least one hour, at least two hours or even three hours. In one embodiment, the contacting is not affected for longer than 6 hours. For example, the contacting may be affected for 30 minutes to two hours.

[0193] Following optional rinsing, the cells may then be removed from the culture surface.It will be appreciated that supplementation does not occur more than three times during the course of the culturing.

[0194] In another embodiment, supplementation of the above described factors does not occur more than two times during the course of the culturing.

[0195] In another embodiment, supplementation of the above described factors occurs only once (at the time of harvesting).

[0196] When IL-2 is used as the stimulating cytokine, the culture medium of this aspect of the invention typically does not comprise any cytokine other than IL-2 (e.g. devoid of IFN-y).

[0197] In another exemplary protocol, the PBMCs are expanded in a medium comprising IL-15 and IL-7 and the CD3 activator. Supplementation of the medium with IL- 15 and IL-7 and the CD3 activator is contemplated.

[0198] When IL- 15 and IL-7 are used as the stimulating cytokine, the culture medium of this aspect of the invention typically does not comprise any cytokine other than IL- 15 and IL-7 (e.g. devoid of IFN-y).

[0199] Additional effective amounts of IL- 15 and IL-7 and the CD3 activator are supplemented only after 4 days, 5 days, 6 days, seven days, eight days, nine days, or ten days.

[0200] In one embodiment, the IL- 15, IL-7 and the CD3 activator are supplemented only one additional time during the course of the culturing, or only two additional times during the course of the culturing. Optionally, one of the times that supplementation is affected is on the day of harvesting (e.g. day 10, 11, or 12) (or during the harvesting process itself). Optionally, the only time supplementation is affected is on the day of harvesting (e.g. day 10, 11, or 12) or during the harvesting process itself. It will be appreciated that supplementation does not occur more than three times during the course of the culturing.

[0201] According to any of the culturing methods described herein, the culture medium may further comprise at least one small molecule agent.

[0202] Contemplated small molecule agents include, but are not limited to retinoic acid receptor (RAR) antagonists, Cytochrome P450 Family 1 (Cypl) inhibitors and aryl hydrocarbon receptor (AHR) antagonists.

[0203] According to a particular embodiment, the culturing is affected in the presence of a RAR antagonist. Examples of such are provided in Table 1, herein below:Table 1

[0204] &

[0205] & & & &

[0206] & &

[0207] & &

[0208] & &

[0209] & &

[0210] &

[0211]

[0212]

[0213] According to a particular embodiment, the culturing is affected in the presence of an AHR antagonist.

[0214] According to a particular embodiment, the culturing is affected in the presence of an AHR antagonist and a RAR antagonist.

[0215] Without being bound by theory, the AHR antagonist may bring about its effect by decreasing PD1 and Lymphocyte- Activation Gene 3 (LAG3) levels.

[0216] Exemplary AhR antagonists contemplated by the present inventors are summarized in Table 2, herein below.

[0217] Table 2

[0218]

[0219]

[0220] An exemplary CyplA inhibitor is Rhopontigenin (SP163, Selleckchem).

[0221] According to a specific embodiment, the initial culture medium comprises both an AHR antagonist and an RAR antagonist.

[0222] An exemplary rage of contemplated concentrations of AHR antagonists is between 0.005- 500pM, and more specifically between 0.05-5 pM

[0223] An exemplary rage of contemplated concentrations of RAR antagonists is between 0.005- 500pM, and more specifically between 0.05-5 pMIn one embodiment, the small molecule agent may be supplemented to the medium at the same time that the cytokine and CD3 activator are supplemented. Thus, for example, supplementation of the medium with the small molecule agent on the same day as harvesting (or during the harvesting process) is contemplated.

[0224] According to any of the culturing methods disclosed herein, the method may further comprise genetically modifying the cells to obtain a cell composition as disclosed herein. Advantageously, the cells are genetically modified (e.g., transduced or transfected) prior to or during culturing the cells in the expansion medium. In one embodiment, the cells are genetically modified following activation. Exemplary proteins that may be expressed in the cells include a recombinant T cell receptor (TCR) or a chimeric antigen receptor (CAR), as known in the art. Other contemplated proteins that may be expressed in the cells include those disclosed in WO2022 / 153295, the contents of which are incorporated herein by reference.

[0225] The CAR may be designed to recognize a tumor-associated antigen or surface marker. Exemplary CAR constructs include, but are not limited to, CD19-specific CARs, CD123-specific CARs, BCAM-specific CARs, and HER2-specific CARs. Each CAR may comprise an antigenbinding domain (e.g., a single-chain variable fragment or scFv), a transmembrane domain, and one or more intracellular signaling domains (e.g., CD3(^, CD28, 4-1BB). These CARs may be introduced into T cells via viral or non-viral gene transfer methods and may be used to direct the cytotoxic activity of the expanded cell population against specific cancer cell types.

[0226] As mentioned, the starting population of cells comprise CD3+ immune cells (e.g., PBMCs or CD3+ enriched T cells) may be pre-activated with anti-CD3 / CD28, IL15 and IL-7 (and optionally small molecules, as described herein). Following washing the cells are then subject to the culturing step in the presence of anti-CD3 / CD28, IL15 and IL-7 (and optionally small molecules), as further described herein. The culturing step may be carried out for at least 2, 3, 4, 5, or 6 days. In a particular embodiment, the culturing step is effected for at least 9 days and no more than 16 days.

[0227] In another embodiment, starting population of cells comprise CD3+ immune cells (e.g., PBMCs or CD3+ enriched T cells) may be pre-activated with anti-CD3 / CD28, IL-2 (and optionally small molecules, as described herein). Following washing the cells are then subject to the culturing step in the presence of anti-CD3 / CD28 and 11-2 (and optionally small molecules), as further described herein. The culturing step may be carried out for at least 2, 3, 4, 5 or 6 days. In a particular embodiment, the culturing step is effected for at least 9 days and no more than 16 days.

[0228] In some embodiments, the resulting cell composition is advantageously amenable for cryopreservation. Thus, the cell compositions of the invention may be frozen following expansionand thawed prior to administration to the subject, without substantial loss of potency. Thus, in another embodiment, the process further comprising freezing the resulting cell composition. For example, without limitation, a cryopreservation protocol may be performed using liquid nitrogen, wherein the cells are suspended in suitable media (e.g. containing 90% FCS) + 10% Dimethyl sulfoxide (DMSO). Optionally, commercially available systems or excipients (e.g. CryoSure® USP grade cryo-protective agents).

[0229] According to another aspect the cells are not cultured continuously in the presence of the CD3 / CD28 activator.

[0230] According to this aspect the T cells are initially cultured in a medium comprising a CD3 / CD28 activator (as further described herein above) at least one cytokine (as further described herein above), an RAR antagonist and an AHR antagonist under conditions that generate a population of activated T cells.

[0231] Once activated, the cells are expanded for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 days in the presence of the cytokine, RAR antagonist and AHR antagonist, but which is substantially free of the CD3 / CD28 activator (i.e., the amount of CD3 / 28 activator in the medium is not sufficient to activate or drive proliferation of CD3+cells). In one embodiment, the medium is devoid of the CD3 / CD28 activator.

[0232] The cells are then re-exposed to CD3 or CD28 or CD3 / CD28 activator in the presence of the at least one cytokine and optionally the RAR antagonist and the AHR antagonist during harvest, for 1.5 - 3 hours.

[0233] The resultant cell populations generated by any of the methods described herein may be enriched in CD3+CD8+cells. In exemplary embodiments, at least 50 %, 60 %, 70 %, 75 %, 80 %, 85 %, 90 % of the cells of the expanded population are CD3+CD8+cells (i.e., cytotoxic T cells). Typically, the CD3+CD8+cells further express NKG2D and granzyme B and low CD38 positive cells.

[0234] According to exemplary embodiments, at least 50 %, 60 % or 70% are CD56’ cells, and up to 5% are CD3“ cells, and at least 9% are CD3+CD4-CD8-.

[0235] As used herein, a cell is considered "positive" for a cell-surface marker if it expresses the marker on its cell-surface in amounts sufficient to be detected using methods known to those of skill in the art, such as contacting a cell with an antibody that binds specifically to that marker, and subsequently performing flow cytometric analysis of such a contacted cell to determine whether the antibody is specifically bound the cell. It is to be understood that while a cell may express messenger RNA for a cell-surface marker, in order to be considered positive for the compositions and methods described herein, the cell must express the marker of interest on its surface. Similarly,a cell is considered "negative" for a cell-surface marker if it does not express the marker on its surface in amounts sufficient to be detected using methods known to those of skill in the art, such as contacting a cell with an antibody that binds specifically to that marker and subsequently performing flow cytometric analysis of such a contacted cell to determine whether the antibody is bound the cell.

[0236] In various embodiments, the cell compositions of the invention are characterized by high relative CD8+cell levels. In one embodiment, less than 20 %, 30 %, 40% or 50% of the cells express CD4+. In some embodiments the compositions are characterized by an enhanced ratio of CD3+CD8+cells to CD3+CD4+cells (at least 6 : 1, e.g. 6.5-8.5 : 1, typically 7-8 : 1). In other words, the amount of CD3+CD8+cells in the composition is 6-8 times and typically 7-8 times the amount of the CD3+CD4+cells in the composition.

[0237] For example, in some embodiments, the cell compositions of the invention are characterized by the presence of at least 60% and typically at least 75% on average (e.g., 70-100%, 70-95%, 70-90% or 70-80%) cytotoxic CD3+CD8+cells. In another embodiment, the cell compositions are characterized by the presence of 80-95% are CD3+CD8+cells. In other embodiments, the cell compositions are characterized by the presence of up to 15% (e.g., 7-12%, 11-13%, 9-12% or 8-11%) CD3+CD4+cells, typically up to 10% or 20 % on average CD3+CD4+cells.

[0238] In one embodiment, at least 50 %, 60 %, 70 %, 80 % or 90% of the cells are CD56’ cells. For example, at least 50 %, 60 %, 70 % , 80 % or 90% of the cells are CD8+CD56“ cells.

[0239] In another embodiment, at least 30% and typically at least about 40% of the CD3+CD8+cells are characterized by surface expression of at least one marker selected from the group consisting of CXCR3, CXCR6 and DNAM-1. In one embodiment, at least 90% of the CD3+CD8+cells are characterized by surface expression of CXCR3. In another embodiment, at least 90% and typically substantially all of the CD3+CD8+cells are characterized by surface expression of DNAM-1. In another embodiment, at least 30% of the CD3+CD8+cells are characterized by surface expression of CXCR6. In another embodiment, about 90% of the CD3+CD8+cells are characterized by surface expression of CXCR3, substantially all of the CD3+CD8+cells are characterized by surface expression of DNAM-1, and about 30% of the CD3+CD8+cells are characterized by surface expression of CXCR6. In another embodiment, at least 70 % of the CD3+CD8+cells express NKG2D+and granzyme B.

[0240] Typically, at least 60 %, 45 %, 70 %, 75 % or even 80% of the CD3+CD8+cells of the cell composition are CD62L+.

[0241] Typically, at least 20 %, 30 % 40 %, 45 %, 50 %, 55 % or even 60 % of the CD3+CD8+cells of the cell composition are CCR7+.In further embodiments, at least 46% of the CD3+CD8+cells are N / SCM (Naive / Stem Cell Memory T cells), characterized by the expression of CD45RA+CD62L+. At least 25% of the cells are CM (Central Memory T cells), expressing CD45RACD62L+.

[0242] A maximum of 20% of the CD3+CD8+cells are EM (Effector Memory T cells), expressing CD45RACD62L’, and a maximum of 20% are EMRA (Effector Memory RA+T cells), expressing CD45RA+CD62L“.

[0243] According to a particular embodiment, the CD3+CD8+cells have low expression levels of CD38. At least 39% of the cell population are CD8+CD38“. Additionally, at least 40% of CD8+cells express low levels of CD38, while the percentage of CD8+cells expressing high levels of CD38 is below 14%.

[0244] According to still another embodiment, the CD3+CD8+cells are also characterized by lower expression levels of CD57, with the proportion of CD8+CD57+cells being below 36%.

[0245] The cells of the composition are also characterized by lower expression levels of PD-1+, with the proportion of CD8+PD-1+cells being below 8%.

[0246] The cells of the composition are also characterized by high content of NAD as shown in Figures 13A-B.

[0247] Typically, the level of NAD present in the cells described herein is equal or greater than 100 pmol of NAD per IxlO6cells, as measured by NAD / NADH Assay Kit ,Ab221821 kit II, Abeam In one embodiment, the level of NAD present in the cells described herein is between 90-700pmol / l*106cells.

[0248] As shown in Figures 18A-B, the cells described herein secrete high levels of interferon gamma (IFN-y) as measured by an immunoassay. Typically, the level of IFN-y is at least two time, at least three times, at least four times, at least five times greater T cells which have been expanded under the same protocol, but without restimulation at harvest and in the absence of small molecules, when measure using the same assay.

[0249] In addition, CAR-T cells described herein secrete high levels of tumor necrosis factor alpha (TNF-a) as measured by an immunoassay. Typically, the level of TNF-a is at least 1.5 times, at least two times, at least three times, at least four times, at least five times greater than T cells which have been expanded under the same protocol, but without restimulation at harvest and in the absence of small molecules, when measured using the same assay.

[0250] In addition, non-CAR-T cells described herein secrete high levels of C-C motif chemokine ligand 1 (CCL1) as measured by an immunoassay. Typically, the level of CCL1 is at least 10 times, 100 times or even 1000 times greater than T cells which have been expanded under the same protocol, but without restimulation at harvest and in the absence of small molecules, whenmeasured using the same assay. For CAR-T cells, typical levels of CCL1 are at least 1.2, 1.3, 1.4, 1.5, 2 times greater than CAR-T cells which have been expanded under the same protocol, but without restimulation at harvest and in the absence of small molecules, when measure using the same assay.

[0251] In addition, non-CAR-T cells described herein secrete high levels of C-C motif chemokine ligand 5 (CCL5) as measured by an immunoassay. Typically, the level of CCL5 is at least 10 times, 100 times or even 1000 times greater than T cells which have been expanded under the same protocol, but without restimulation at harvest and in the absence of small molecules, when measured using the same assay. For CAR-T cells, typical levels of CCL5 are at least 1.2, 1.3, 1.4, 1.5, 2 times greater than CAR-T cells which have been expanded under the same protocol, but without restimulation at harvest and in the absence of small molecules, when measure using the same assay.

[0252] In addition, non-CAR-T cells described herein secrete high levels of C-C motif chemokine ligand 3 (CCL3) as measured by an immunoassay. Typically, the level of CCL3 is at least 3, 6, 10, 12, 15 times greater than T cells which have been expanded under the same protocol, but without restimulation at harvest and in the absence of small molecules, when measured using the same assay. For CAR-T cells, typical levels of CCL3 are at least 1.5, 2, 2.5, 3, 5 times greater than CAR-T cells which have been expanded under the same protocol, but without restimulation at harvest and in the absence of small molecules, when measure using the same assay.

[0253] In addition, non-CAR-T cells described herein secrete high levels of C-C motif chemokine ligand 10 (CCL10) as measured by an immunoassay. Typically, the level of CCL10 is at least 1.2, 1.3, 1.5, 2, 2.5 times greater than T cells which have been expanded under the same protocol, but without restimulation at harvest and in the absence of small molecules, when measured using the same assay. For CAR-T cells, typical levels of CCL10 are at least 1.5, 2, 2.5, 3, 5 times greater than CAR-T cells which have been expanded under the same protocol, but without restimulation at harvest and in the absence of small molecules, when measure using the same assay.

[0254] Exemplary antibodies useful for characterization of the cell populations described herein are summarized in Table 3, herein below.

[0255] Table 3

[0256]

[0257]

[0258] In another embodiment, the cell compositions exert a cytotoxic activity against tumor cells. In some embodiments, the tumor cells are of hematopoietic origin. The phrase "tumor cells of a hematopoietic origin" refers to malignant cells derived from blood cells or precursors thereof. In another embodiment the tumor cells are of lymphoid origin. In another embodiment the tumor cells are of myeloid origin. In a particular embodiment the tumor cells are leukemia cells, e.g. acute myeloid leukemia (AML) cells. In another particular embodiment, the tumor cells are multiple myeloma cells. In still another embodiment, the tumor cells are ovarian cells.

[0259] According to exemplary embodiments, the cell compositions are capable of specifically eradicating (killing) hematopoietic tumor cells when incubated with said cells in vitro at a ratio of at least 0.25: 1 (respectively) for 24 hours.

[0260] In another embodiment, expansion is performed so as to produce a cell composition capable of specifically eradicating tumor cells (e.g. leukemic cells) when incubated with the tumor cells in vitro at a ratio of 0.25: 1 for 24 hours to a greater extent (e.g. at least 10 % more effective, at least 20 % more effective, at least 30 % more effective, at least 40 % more effective, at least 50 % more effective) than cell populations described in WO2022 / 153295.

[0261] According to additional exemplary embodiments, the cell compositions are capable of specifically eradicating at least 50% and typically 60-99%, more typically about 80-90% of said hematopoietic tumor cells when incubated with said cells in vitro at a 1:1 ratio for 24 hours. In other embodiments, the tumor cells express at least one and typically a plurality of NKG2D ligands, including, but not limited to MICA, MICB, HLAE, ULBP1, ULBP256, and ULBP3. In another embodiment said tumor cells express MICA, MICB, HLAE, ULBP1, ULBP256, and ULBP3. In other embodiments, as exemplified herein, the cell compositions are capable of specifically eradicating said hematopoietic tumor cells to a greater extent than CIK compositions or cellpopulations disclosed in WO2022 / 153295 under the same conditions (e.g. at least 3-fold higher at a 1:1 effector cell to tumor cell ratio or at least about 3 -fold higher at a 0.5:1 effector cell to tumor cell ratio or at least 6-fold higher at a 0.25: 1 effector cell to tumor cell ratio). In other embodiments, said tumor cells are solid tumor cells. In a particular embodiment, said cells are ovarian tumor cells, prostate tumor cells or mesothelioma cells. Each possibility represents a separate embodiment of the invention.

[0262] As used herein, the term "specific eradication" with respect to tumor cells indicates targeted tumor cell death, which is selective (or preferential) to the target tumor cells. Typically, eradication is exerted by cytotoxic activity that is induced by said tumor cells (e.g. upon contact of the cell composition with said tumor cells under conditions as disclosed herein).

[0263] As used herein, the terms "cytotoxicity" and "cytotoxic activity" refer in particular to cell-mediated cytotoxicity or cytolysis, i.e. to the cell killing activity of immune cells, in particular of effector immune cells. Among the prominent cytotoxic effector cells of the immune system are NK cells, cytotoxic T cells (e.g., CD8+T cells) and NK-T cells. A cytotoxic cell (e.g. CTL or NK cell) may kill a target cell via target cell apoptosis or lysis using one or more different mechanisms including, but not limited to, release of one or more cytotoxins stored in cytolytic granules, or expression of a Fas ligand (FasL). Exemplary cytotoxins include e.g. a perforin, a granzyme and a granulysin. Currently known granzymes are Granzymes A, B, H, K and M. Such cytotoxic or cytolytic activities can be measured and quantified using standard techniques, e.g., by assays employing selective labeling of viable cells. For example, without limitation, diacetate succinimidyl ester (CFSE)- and / or propidium iodide-based assays, or other suitable assays known in the art may be employed.

[0264] In another embodiment, the cytotoxic activity is mediated by Granzyme B and / or perforin. In another embodiment said cytotoxic activity is not substantially mediated by FAS-FasE interactions.

[0265] The cell compositions in accordance comprise both MHC-restricted and non-MHC restricted cytotoxic activities against various tumor cells. Thus, in one embodiment, the cytotoxic activity is MHC-dependent (i.e. requires the presence or activity of MHC molecules on the cells). In another embodiment, the cytotoxic activity is MHC-independent. In another embodiment, the cytotoxic activity is substantially MHC I-independent. In another embodiment, the cytotoxic activity is substantially MHC Il-independent.

[0266] In another embodiment, the cell compositions of the invention are capable of exerting tumor- specific cytotoxic activity without substantial cytotoxic activity against non-tumor cells. In other embodiments, the cell compositions of the invention do not substantially induce graft-versus-host (GVH) reaction or graft-versus-host disease (GVHD). Thus, in some embodiments, the cell compositions of the invention need not be histocompatible with the subject to be treated.

[0267] The cell compositions described herein may be used in the treatment of cancer.

[0268] Thus, according to another aspect, there is provided a method of treating cancer in a subject in need thereof, comprising contacting the cancer cells with a therapeutically effective amount of a cell composition of the invention, thereby treating the cancer in the subject.

[0269] In one embodiment, the cells are autologous to the subject.

[0270] As used herein, the terms "tumor" and "cancer" may be used synonymously and refer to both solid tumors and non-solid malignancies. In another embodiment, the tumor is a hematopoietic tumor. Non-limitative examples of hematopoietic tumors of lymphoid lineage include e.g. leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell-lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma and Burkitt's lymphoma. Non-limitative examples of hematopoietic tumors of myeloid lineage, include e.g. acute and chronic myelogenous leukemias, myelodysplastic syndrome and promyelocytic leukemia; tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma. In another embodiment the tumor is a solid tumor. Non-limitative examples of solid tumors include e.g. lung tumors, stomach tumors, ovarian tumors, breast tumors, colorectal tumors, pancreatic tumors, and renal tumors. In various embodiments, said tumor is selected from the group consisting of leukemia, multiple myeloma, prostate cancer mesothelioma, lung cancer and pancreatic cancer, wherein each possibility represents a separate embodiment of the invention. In a specific embodiment, said tumor is selected from the group consisting of leukemia, multiple myeloma, prostate cancer and mesothelioma. In a particular embodiment said tumor is AML.

[0271] In another embodiment said tumor is characterized by expression of at least one NKG2D ligand. In some embodiments, the NKG2D ligand is selected from the group consisting of MICA, MICB, HLAE, ULBP1, ULBP256, and ULBP3. In another embodiment said tumor expresses a plurality of NKG2D ligands selected from the group consisting of: MICA, MICB, HLAE, ULBP1, ULBP256, and ULBP3. In another embodiment said tumor expresses MICA, MICB, HLAE, ULBP1, ULBP256, and ULBP3. Each possibility represents a separate embodiment of the invention.

[0272] Conveniently, the expression of markers such as NKG2D ligands and checkpoint molecule ligands by a tumor of a subject may be determined by obtaining tumor cells (e.g. from a solid tumor biopsy or from bodily fluids such as blood or urine), and examining expression of said markers on the surface of said tumor cells by suitable assays including, but not limited to immunostaining and flow cytometry. Non-limitative examples of such assays are exemplified herein.In another embodiment, said tumor is characterized by expression of at least one inhibitory immune checkpoint molecule and / or ligand thereof. In some embodiments, said tumor may express at least one ligand of CTLA-4, PD-1, TIGIT, Lag-3, Tim-3 and combinations thereof, wherein each possibility represents a separate embodiment of the invention. In another embodiment, said tumor is characterized by expression of at least one ligand of an inhibitory immune checkpoint molecule selected from the group consisting of CTLA-4, PD-1, and TIGIT. In another embodiment, said tumor is characterized by expression of ligands of a plurality of inhibitory immune checkpoint molecule selected from the group consisting of CTLA-4, PD-1, and TIGIT. In another embodiment, said tumor is characterized by expression of at least one CTLA-4 ligand (e.g. CD80 and / or CD86).

[0273] In another embodiment, said tumor is resistant to treatment by at least one checkpoint molecule inhibitor. In another embodiment, said tumor is resistant to treatment by at least one inhibitor (e.g. blocking antibody) directed to at least one checkpoint molecule selected from the group consisting of CTLA-4, PD-1, TIGIT, Lag-3, Tim-3 and ligands and combinations thereof, wherein each possibility represents a separate embodiment of the invention.

[0274] According to exemplary embodiments, the tumor is resistant to treatment by at least one CTLA4 -specific inhibitor (e.g. blocking antibody) selected from the group consisting of ipilimumab (YERVOY®) and tremelimumab. In other embodiments, the tumor is resistant to treatment by at least one PD1 -specific inhibitor (e.g. blocking antibody directed to PD-1 or PD-L1) selected from the group consisting of atezolizumab (TECENTRIQ®), durvalumab (IMFINZI®), avelumab (BAVENCIO®), nivolumab (OPDIVO®) and pembrolizumab (KEYTRUDA®). In other embodiments, the tumor is resistant to treatment by at least one TIGIT- specific inhibitor selected from the group consisting of tiragolumab (MTIG7192A), ociperlimab (BGB-A1217), vibostolimab (MK-7684), domvanalimab (AB-154), BMS-986207, EOS-448, ASP-8374, COM-902, etigilimab (MPH-313), IB 1-939, AGEN-1307, CASC-674, Anti-PVR antibody (NB-6253), and PH-804. In other embodiments, the tumor is resistant to treatment by at least one LAG3 -specific inhibitor selected from the group consisting of PRS-332, P13B02-3, LBL-007, eftilagimod alpha (IMP321), LAG525 (IMP701), MK-4280, REGN3767, relatlimab (B MS-986016), BI 754111, FS118, tebotelimab (MGD013), TSR-033, INCAGN2385, Sym022 and XmAb22841. In other embodiments, the tumor is resistant to treatment by at least one TIM3 -specific inhibitor, e.g. ICAGN02390, Sym023, LY3321367, MGB453, TSR022, BGBA425, and BMS986258.

[0275] In another embodiment, the tumor is resistant to treatment by a plurality of checkpoint inhibitors or by inhibitors of a plurality of checkpoint molecules. For example, without limitation, the tumor may be resistant to treatment by a dual inhibitor targeting both TIM3 and PD1 such as R07121661 or to combination therapy with nivolumab and ipilimumab.In another embodiment, said subject is under treatment regimen with one or more checkpoint molecule inhibitors. In another embodiment, said subject is not under treatment regimen with checkpoint molecule inhibitors. For example, YERVOY™ (ipilimumab) is a human CTLA-4-blocking antibody for intravenous infusion, indicated for the treatment of unresectable or metastatic melanoma. OPDIVO® (nivolumab) is a PD-1 -blocking antibody indicated for the treatment of unresectable or metastatic melanoma. KEYTRUDA® (pembrolizumab) is a PD-1 -blocking antibody indicated for treatment of melanoma, NSCLC, head and neck squamous cell cancer, classical hodgkin lymphoma (cHL), primary mediastinal large B-cell lymphoma, urothelial carcinoma, micro satellite instability-high or mismatch repair deficient cancer, microsatellite instability-high or mismatch repair deficient colorectal cancer, gastric cancer, esophageal cancer, cervical cancer, hepatocellular carcinoma (HCC), merkel cell carcinoma (MCC), renal cell carcinoma (RCC), endometrial carcinoma, tumor mutational burden-high cancer, cutaneous squamous cell carcinoma, and triple-negative breast cancer. TECENTRIQ® (atezolizumab) is a programmed death-ligand 1 (PD-L1) blocking antibody indicated for the treatment of locally advanced or metastatic urothelial carcinoma, metastatic NSCLC, metastatic non-squamous NSCLC with no EGFR or ALK genomic tumor aberrations, Small Cell Lung Cancer (SCLC), extensive-stage small cell lung cancer (ES-SCLC), unresectable or metastatic HCC, and melanoma. IMFINZI® (durvalumab) is a PD-L1 blocking antibody indicated for the treatment of adult patients with unresectable, Stage III NSCLC whose disease has not progressed following concurrent platinum-based chemotherapy and radiation therapy; in combination with etoposide and either carboplatin or cisplatin, as first-line treatment of adult patients with ES-SCLC. BAVENCIO® (avelumab) is a PD-L1 blocking antibody indicated for the treatment of MCC, UC, and RCC.

[0276] In another embodiment, the method or use further comprises administration of at least one checkpoint molecule inhibitor as disclosed herein (in concurrent or sequential combination with the cell composition of the invention). For example (e.g. wherein the tumor is characterized by surface expression of at least one ligand of Lag-3, Tim-3 or TIGIT), the method of use further comprises administration of at least one checkpoint molecule inhibitor directed to Lag-3, Tim-3 or TIGIT, respectively. In another embodiment, the method or use further comprises administration of at least one checkpoint molecule inhibitor directed to Lag-3, Tim-3, or combinations thereof. Yet in other embodiments, cell compositions of the invention may be used as the sole therapeutic agent. For example, without limitation, compositions of the invention may be used as monotherapy in the treatment of tumors characterized by surface expression of at least one ligand of CTLA-4 or PD-1. Each possibility represents a separate embodiment of the invention.In another embodiment, said tumor is characterized by expression of at least one chemokine selected from the group consisting of CXCR6 ligands, CXCR4 ligands, CXCR2 ligands and CXCR3 ligands. In another embodiment, said tumor is characterized by expression of at least one chemokine selected from the group consisting of CXCR6 ligands, CXCR4 ligands, and CXCR3 ligands. In another embodiment, said tumor is characterized by expression of a plurality of chemokines selected from the group consisting of CXCR6 ligands, CXCR4 ligands, CXCR2 ligands and CXCR3 ligands. In another embodiment, said tumor is characterized by expression of a plurality of chemokines selected from the group consisting of CXCR6 ligands, CXCR4 ligands, and CXCR3 ligands. In a particular embodiment said tumor is characterized by expression of a CXCR4 ligand, e.g. CXCL12. In another particular embodiment said tumor is characterized by expression of a CXCR6 ligand, e.g. CXCL16. For example, without limitation, CXCL16-expressing tumors may include non-small cell lung cancer (NSCLC) tumors, stomach tumors, ovarian tumors, breast tumors, colorectal tumors, pancreatic tumors, and renal tumors, wherein each possibility represents a separate embodiment of the invention.

[0277] In another embodiment the contacting is performed in vivo. Thus, the methods of the invention may comprise administering to said subject a therapeutically effective amount of a cell composition of the invention, e.g. an ACT cell composition, comprising at least 5xl06-10xl08viable in vilro-c^ivadcA PBMCs (as described herein). In another embodiment, the cells are administered to said subject with concomitant IL-2 or IL- 15 administration. Yet in other embodiments, the cells may advantageously be used in the absence of concomitant IL-2 or IL- 15 administration.

[0278] An “effective amount” or “therapeutically effective amount” refers to an amount sufficient to exert a beneficial outcome in a method of the invention. More specifically, a therapeutically effective amount means an amount of active ingredients (e.g., effector cells) effective to prevent, alleviate, or ameliorate symptoms of a disorder (e.g., cancer) or prolong the survival of the subject being treated. For example, the cell composition of the invention for administration to a subject comprises an effective amount of at least 5xl06-10xl08and typically up to about lOxlO9viable cells expanded from PBMC as disclosed herein. An effective amount of a cell composition for ex vivo contacting with tumor cells may comprise for example at least about 0.25-1 effector cells for every tumor cell in culture.

[0279] In another embodiment, said tumor is characterized by down-regulation of MHC I expression and / or activity. The cell compositions may advantageously be used even in the treatment of treatment-resistant tumors (e.g. refractory to immunotherapy or checkpoint molecule inhibitors) or tumors that exhibit an immune-evasive phenotype associated with impaired MHC I antigenpresentation due to e.g. reduced expression of MHC I molecules, loss of function mutations or other impairments in the MHC I pathway characteristic of tumors.

[0280] In another embodiment, said cell composition had undergone cryopreservation and a subsequent thawing protocol prior to contacting with said tumor cells. Thus, in another embodiment of the methods of the invention, the thawing protocol is performed within 1-3 days, e.g. within 36, 24, 12 , 6 or 1 hours of contacting said cell composition with said tumor cells. Each possibility represents a separate embodiment of the invention. In other embodiments, the cell composition may be autologous, histocompatible allogeneic, or non-histocompatible allogeneic to said subject, wherein each possibility represents a separate embodiment of the invention.

[0281] The cells may be provided per se or in a pharmaceutical composition.

[0282] As used herein a "pharmaceutical composition" refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

[0283] Herein the term "active ingredient" refers to cell composition accountable for the biological effect.

[0284] Hereinafter, the phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the cells. An adjuvant is included under these phrases.

[0285] Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intracardiac, e.g., into the right or left ventricular cavity, into the common coronary artery, intravenous, intraperitoneal, intranasal, or intraocular injections.

[0286] According to a particular embodiment, the cells are administered intravenously or locally. Pharmaceutical compositions suitable for use in context of some embodiments of the invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients (cell composition) effective to prevent, alleviate or ameliorate symptoms of a disorder (e.g., cancer) or prolong the survival of the subject being treated.

[0287] Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays. For example, a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.

[0288] Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p.l).

[0289] Dosage amount and interval may be adjusted individually to provide tissue levels of the active ingredient are sufficient to induce or suppress the biological effect (minimal effective concentration, MEC). The MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.

[0290] Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is affected or diminution of the disease state is achieved.

[0291] The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.

[0292] Compositions of some embodiments of the invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert. Compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as is further detailed above.Drug combinations are also contemplated with the multispecific antibody of the present teachings, such as with immune checkpoint modulators such as anti-CTLA4, anti-CD40, anti-41BB, anti-OX40, anti-PDl or anti-PDLl. According to a specific embodiment, the immune checkpoint modulator is anti-PDl or anti-PDLl.

[0293] It is expected that during the life of a patent maturing from this application many relevant agonistic CD40 antibodies will be developed and the scope of the term anti CD40 antibody is intended to include all such new technologies a priori.

[0294] As used herein the term “about” refers to ± 10 %.

[0295] The terms "comprises", "comprising", "includes", "including", “having” and their conjugates mean "including but not limited to".

[0296] The term “consisting of’ means “including and limited to”.

[0297] The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and / or parts, but only if the additional ingredients, steps and / or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

[0298] As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

[0299] Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the 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, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0300] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging / ranges between” a first indicate number and a second indicate number and “ranging / ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

[0301] As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques andprocedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

[0302] As used herein, the term “treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.

[0303] When reference is made to particular sequence listings, such reference is to be understood to also encompass sequences that substantially correspond to its complementary sequence as including minor sequence variations, resulting from, e.g., sequencing errors, cloning errors, or other alterations resulting in base substitution, base deletion or base addition, provided that the frequency of such variations is less than 1 in 50 nucleotides, alternatively, less than 1 in 100 nucleotides, alternatively, less than 1 in 200 nucleotides, alternatively, less than 1 in 500 nucleotides, alternatively, less than 1 in 1000 nucleotides, alternatively, less than 1 in 5,000 nucleotides, alternatively, less than 1 in 10,000 nucleotides.

[0304] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0305] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

[0306] EXAMPLES

[0307] Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion.

[0308] Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature.EXAMPLE 1

[0309] Materials and methods

[0310] T cell culture: Peripheral blood mononuclear cells (PBMCs) were isolated from human blood by Ficoll-Hypaque density centrifugation (Cytiva). PBMCs were resuspended in RPMI medium (Complete medium+ MEM NEAA xl and O.lmM 2-mercaptoethanol all from Gibco) and incubated at a concentration of 5xl06cells / Grex (G-Rex 10M).

[0311] PBMCs were stimulated on days 0 with 500 lU / ml recombinant human IL-2 (R and D system) and 30 ng / ml anti human CD3 (OKT-3, eBioscience). The cultures were then incubated at 37°C with 5% CO2 for a total of 11-14 days.

[0312] Stimulation Method (1): PBMCs were seeded into G-Rex 10M flasks with 30 ml of RPMI complete medium containing IL-2 and anti-CD3 for 11-14 days. On days 4 and 8, an additional 30 ml of complete medium with IL-2 and OKT3 was added to each flask.

[0313] Stimulation Method (2): PBMCs were seeded into G-Rex 10M flasks with 90 ml of RPMI complete medium containing IL-2 and anti-CD3 for 11-14 days, without any further addition of activation agents. During harvest procedure, the T cells were re-stimulated with IL-2 and anti CD3 for 1.5-3h (80 ml of medium was discarded and cells were restimulated in a total volume of 10 ml), washed twice with PBS. Following final wash the cells were analyzed by flow cytometry and subjected for cytotoxic experiments.

[0314] Small molecules were added to the G-Rex on day 0: AHR antagonist- CH223191 (Cayman). RAR antagonist- AGN194310 (MedChemExpress). Cytochrome P450 Family 1 Inhibitor (CYP1A) inhibitor- Rhopontigenin (SP163, Selleckchem), alone or in combination.

[0315] Alternative method for culturing T cells in the presence of IL-15 and IL-7: Briefly, PBMCs were seeded into G-Rex 10M flasks with 90 ml of RPMI complete medium containing lOng / ml IL-15, 10 ng / ml IL-7 (Peprotech) and anti-CD3 for 11 days, without any further addition of activation agents. On harvest day, during the harvest procedure, the expanded T cells were restimulated with IL-15, IL-7 and anti CD3 for 1.5 hours, analyzed by flow cytometry and subjected for cytotoxic experiments.

[0316] Small molecules were added to the G-Rex on day 0: AHR antagonist- CH223191 (Cayman). RAR antagonist- AGN194310 (MedChemExpress). CYP1A inhibitor- Rhopontigenin (SP163, Selleckchem), alone or in combination.

[0317] Cytotoxic assay: Cytotoxicity was measured using CFSE-based assay. Target cells were labeled with CFSE (eBioscience) according to Manufacturer’s instructions and plated at a concentration of 2X104cells / well in 96 well plate. Expanded T cells (generated according tomethod 1 or 2) were harvested on day 11-14 of culture and added to target cells at effector: target (E: T) ratios of 0.25:1, 0.5:1, 1:1, 2:1, 5:1 and 10:1.

[0318] After 24 hours, CFSE+PI- labeled target cells were analyzed by flow cytometry. The percentage of dead cells was calculated using the formula: { 100 - [(Number of CFSE+PI- cells with effector cells / Number of CFSE+PI- cells without effector cells) x 100}.

[0319] Measurement of mitochondrial ROS: Mitochondrial ROS production was detected using MITOSOX™ ™ Red Mitochondrial Superoxide Indicator for live-cell imaging (Thermo Fisher Scientific). Cells were incubated in warm PBS, pH 7.4, followed by incubation with MITOSOX™ Red (5 pM) at 37°C for 20 min. The fluorescence of MITOSOX™ Red in T cells was measured using a FACS CytoFEEX™.

[0320] FACS analysis: Expanded T cells population can be identified by expression of CD45RA, CD45RO, CCR7, and CD62L (L-selectin).

[0321] These subsets include:

[0322] naive (CD45RO-CD62E+CD45RA+ CCR7+)

[0323] central memory = Tc> CD45RO+CD62E+ CD45RA- CCR7+)

[0324] effector memory (TEM; CD45RO+CD62E-CD45RA- CCR7-)

[0325] effector cells (CD45RO-CD62E-CD45RA+ CCR7-).

[0326] Expression of CD57, CD62E, CCR7 and CD38 on CD8+ cells was detected using antibody (all from eBioscience).

[0327] 3D tumor spheroid modeling: To generate tumor microspheroids, 1 x 104SKOV-3-GFP cells were cultured in clear, round-bottom 96-well ultra-low attachment plates (Coming) in RPMI complete media. Following centrifugation at 1,400 x rpm for 10 min, the plates were placed in the IncuCyte™ for monitoring. After 5 days of culture, spheroids were used as targets for cytotoxicity assays. Effector cells (expanded T cells generated according to the methods described above), with and without small molecules, were added to the plates with spheroids at defined E:T ratios. Images were taken every 4 hours to monitor immune-mediated cytotoxicity, measuring total green intensity.

[0328] RESULTS

[0329] Enhancing T cell activity by stimulating T cells at different times during the expansion protocol

[0330] The present inventors compared the effect of stimulation of T cells by IL-2 and anti-CD3 on days 0, 4, and 8 to two stimulations on days 0 and 4, days 0 and 7, and days 0 and 8. It was found that two stimulations were sufficient to maintain T cell expansion and activation. Moreover, it was discovered that a single re-stimulation on a late day within the cells’ expansion processyielded better results than an earlier day within the expansion process (Figures 1A-B). Specifically, re- stimulation on day 8 improved T cell expansion (Figure 1A) and enhanced cytotoxic killing (Figure IB) compared to re- stimulation on day 4 or 7.

[0331] Thereafter, the present inventors compared the activity of T cells stimulated with IL-2 and anti-CD3 only on day 0 (control) to those re-stimulated either on day 8 or on the last day of the production process, day 11. The re-stimulation on day 11 was performed for 3 and 1.5 hours during the harvest process. It was found that a short restimulation on the last day of the production process (day 11) for 1.5 hours improved T cell activity compared to re-stimulation on day 8 or without restimulation (Figure 2A). The enhancement in T cell activity was also observed against solid tumors (Figure 2B). Restimulation on the day of cell harvesting (day 11) is advantageous as it enhances T cell activity.

[0332] Next, the present inventors investigated whether harvesting could be extended to day 14. In these experiments, T cells harvested on day 11 were compared to those harvested on day 14. Harvesting T cells on day 14 resulted in the same fold expansion (Figure 3A) and cytotoxic activity (Figure 3B) as on day 11.

[0333] Enhancing T cell efficacy and fitness by culturing in the presence of small molecules To further improve the functionality and fitness of the T cells, the present inventors sought a small molecule that would epigenetically improve T cell functionality: boosting metabolism, delaying differentiation, and consequently increase naive and memory T cell populations. Several small molecules were tested. Out of those tested, six of them were found to not affect the CD8 / CD4 ratio and only slightly influenced T fold expansion (data not shown). One of these candidates is CYP1A inhibitor (SP163) which slightly improved T cell expansion and cytotoxic activity against both AML cell and ovarian cancer (Figure 4A-C).

[0334] The RAR antagonist (AGN194310) had the most significant effect on both T cell cytotoxic activity and cell fitness (Figures 5A-G). Analysis of the RAR antagonist demonstrated that it enhanced T cell cytotoxic activity against AML cells and dramatically improved T cell killing of ovarian cancer cells (Figure 5A and 5B). Additionally, the RAR antagonist improved T cell fitness by increasing resistance to ROS (as evidenced by decreased MITOSOX™ ™ staining, Figure 5C), decreasing CD57 and CD38 expression (Figure 5D and 5E) and elevating the number of CD62L+and CCR7+positive cells (Figure 5F). Treatment with the RAR antagonist resulted in a higher percentage of naive and central memory cells while reducing the number of effector and effector memory cells (Figure 5G).The combination of the RAR antagonist with restimulation on day 11 was more effective than the RAR antagonist alone (Figure 6). This combined treatment increased the number of CD62L+ CCR7+ cells, which have a high potential to target the lymph nodes (Figure 6).

[0335] The present inventors also demonstrated that the improvement in T phenotype due to the addition of the RAR antagonist was maintained when culturing cells with human serum, similar to culturing with FBS or with a serum-free medium, NutriT (Figure 7E) As shown in Figures 7A-D, there were no significant differences in cT fold expansion (Figure 7A), cytotoxic activity (Figure 7B), or the percentage of CD38hlgh, CD57+(Figure 7C), and CD62L+cells (Figure 7D) between T cells expanded with FBS or human serum. In addition, the cytotoxic efficacy of the cells against the ovarian cancer cell lines SKOV3 and OVCAR, as assessed using a 3D tumor spheroid model, was comparable between cells cultured in RPMI supplemented with FCS and cells cultured in the serum-free medium NutriT (Figure 7E).

[0336] In addition to the RAR antagonist, the AHR antagonist (CH223191) was also found to have significant beneficial effects on T cell characteristics. AHR antagonist leads to increased T cell expansion (Figure 8A) and improved cytotoxic activity against both AML and ovarian cancer (Figures 8B and 8C).

[0337] When combining the AHR antagonist with the RAR antagonist, an enhancement in T cell cytotoxic activity was observed compared to using either the RAR antagonist or AHR antagonist alone (Figure 9A). Surprisingly, the combined treatment of the RAR antagonist and AHR antagonist have a synergistic effect when cytotoxicity was measured in an ovarian Spheroid 3D model (Figure 9B).

[0338] Moreover, the present inventors assessed the effect of restimulation with the combination of both the AHR antagonist and RAR antagonist in the 3D spheroid model of ovarian SKOV3 cells a significant effect of restimulation was observed. In this model, the cells cultured in the presence of both the AHR antagonist and RAR antagonist and restimulated prior to harvest were significantly more effective compared to the cells that did not undergo restimulation (Figure 10).

[0339] Expansion ofT cells using IL-15 and IL-7

[0340] In this example, the expansion of T cells using IL- 15 and IL-7 was compared to those expanded with IL-2. As shown in Figure 11 A, there were no changes in the fold expansion of T cells expanded with either IL-2 or IL- 15 and IL-7. The addition of IL- 15 and IL-7 also did not alter the T cell phenotype following incubation with a RAR antagonist.

[0341] When the RAR antagonist was added to the T cell cultures along with IL-15 and IL-7, it improved the cytotoxic activity against AML cells (Figure 11B), decreased CD57 and CD38 expression (Figures 11C and 11D), increased the number of CD62L+CCR7+cells (Figure HE),enhanced the percentage of naive cells (Figure 11F), and increased T cell fitness (Figure 11G). These effects were consistent with those observed in T cells produced with the presence of either IL-2 or IL- 15 and IL-7.

[0342] EXAMPLE 2

[0343] Effect of RAR & AHR antagonism on expansion of PBMCs Materials and methods

[0344] Experiment 1 (corresponds to results shown in Figure 12): Peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood by Ficoll-Hypaque density gradient centrifugation (Cytiva). PBMCs were resuspended in RPMI medium and seeded in G-Rex culture vessels at a density of 5 x 106cells per G-Rex. On day 0, cells were stimulated with 500 lU / mL recombinant human IL-2 and 30 ng / mL anti-human CD3 antibody, either in the presence or absence of 0.01 pM RAR antagonist and 0.05 pM AHR antagonist. Cultures were maintained at 37°C with 5% CO2 for 11 days without further addition of activation agents.

[0345] On the day of harvest, T cells of both groups were restimulated with IL-2 and anti-CD3 antibody for 1.5h, after which total RNA was isolated. Differentially expressed genes between control and RAR / AHR antagonist-treated T cells were identified from bulk RNA- sequencing data using standard statistical criteria. Genes meeting the significance thresholds for adjusted p-value and fold change were subjected to functional enrichment analysis. Canonical pathway enrichment was performed using a right-tailed Fisher’s exact test, with p-values corrected for multiple testing using the Benjamini-Hochberg method. Significantly enriched pathways were ranked by -log(BH-adjusted p-value) and visualized as a bar graph; only pathways meeting significance criteria were analyzed.

[0346] Gene Set Enrichment Analysis (GSEA). Bulk RNA-sequencing data were used to compare gene expression profiles between control and RAR / AHR antagonist-treated T cells. Genes were ranked based on their differential expression metric derived from the RNA-seq analysis. Gene Set Enrichment Analysis (GSEA) was performed using the Molecular Signatures Database (MSigDB) Hallmark gene sets.

[0347] Experiment 2 (corresponds to results shown in Figures 13A-B): PBMCs were isolated from human peripheral blood by Ficoll-Hypaque density gradient centrifugation (Cytiva). PBMCs were resuspended in complete RPMI medium (RPMI supplemented with MEM non-essential amino acids [lx] and 0.1 mM 2-mercaptoethanol; Gibco) and seeded at a density of 5 * 106cells per G-Rex®6M well. On day 0, cells were stimulated with recombinant human IL-2 (500 lU / mL; R&D Systems), anti-human CD3 antibody (30 ng / mL; OKT-3, eBioscience), a RAR antagonist (0.01pM), and an AHR antagonist (0.05 pM). Cultures were maintained at 37 °C in a humidified incubator with 5% CO2 for 11 days.

[0348] At harvest, cells were restimulated for 1.5 hours with either IL-2 and anti-CD3, or with IL-2, anti-CD3, and RAR antagonist (0.01 pM). Following restimulation, cells were washed and cultured in fresh IL-2-supplemented medium for an additional 6 days in 24-well plates. CD38 expression and intracellular NAD levels were assessed on days 2 and 6 post-restimulation using flow cytometry and the NAD / NADH Assay Kit II (Abeam), respectively.

[0349] Experiment 3 (corresponds to results shown in Figures 14A-B): PBMCs were isolated from human peripheral blood by Ficoll-Hypaque density gradient centrifugation (Cytiva). PBMCs were resuspended in RPMI complete medium supplemented with MEM non-essential amino acids (lx) and 0.1 mM 2-mercaptoethanol (all from Gibco) and seeded at a density of 1 x 106cells / mL in T-75 flasks. Cells were stimulated with 500 lU / mL recombinant human IL-2, 30 ng / mL anti-human CD3 antibody, 0.01 pM RAR antagonist, and 0.05 pM AHR antagonist, and incubated at 37°C with 5% CO2 for 48 hours.

[0350] On day 2, cells were washed and resuspended in fresh medium containing either: (i) IL-2, anti-CD3, RAR antagonist, and AHR antagonist (continuous anti-CD3 condition), or (ii) IL-2, RAR antagonist, and AHR antagonist (anti-CD3 withdrawal condition). Cells were then transferred to G- Rex culture vessels and expanded for an additional 10 days.

[0351] On day 12, cells were harvested and restimulated with IL-2 and anti-CD3 for 3 hours, followed by flow cytometric analysis and use in cytotoxicity assays.

[0352] The experiment is summarized in Table 4 herein below.

[0353] Table 4

[0354]

[0355] Experiment 4 (corresponds to results shown in Figures 15A-E): PBMCs were isolated from human peripheral blood by Ficoll-Hypaque density gradient centrifugation (Cytiva). PBMCswere resuspended in complete RPMI medium and seeded at a density of 1 x 106cells / mL in T-75 culture flasks. Cells were activated under one of the following conditions:

[0356] (a) Standard T-cell expansion: CD3 / CD28 stimulation using ImmunoCult™ Human CD3 / CD28 T Cell Activator (STEMCELL Technologies) in the presence of recombinant human IL-7 (10 ng / mL) and IL- 15 (10 ng / mL) (PeproTech).

[0357] (b) Standard T-cell expansion supplemented with RAR and AHR antagonists: CD3 / CD28 stimulation as above, supplemented with IL-7 (10 ng / mL), IL-15 (10 ng / mL), a RAR antagonist (0.01 pM), and an AHR antagonist (0.05 pM).

[0358] Cells were incubated at 37°C in a humidified atmosphere containing 5% CO2 for 48 hours. On day 2, cells were washed, resuspended in fresh complete RPMI medium containing IL-7 and IL- 15, and transferred to 6-well plates for an additional 24 hours.

[0359] On day 3, cells were transferred to G-Rex culture vessels for further expansion. Group (a) cells were cultured in RPMI medium supplemented with IL-7 and IL- 15, whereas group (b) cells were cultured in RPMI medium containing IL-7, IL-15, anti-CD3 antibody, and the RAR and AHR antagonists. Cells were expanded for an additional 6 days.

[0360] On day 9, cells were harvested. Group (b) cells were restimulated with IL-7, IL-15, and anti- CD3 antibody for 1.5 hours prior to downstream analyses. Cells were subsequently analyzed by flow cytometry, used in cytotoxicity assays, and assessed for intracellular NAD levels according to the manufacturer’s instructions (NAD / NADH Assay Kit II, Abeam).

[0361] The experiment is summarized in Table 5 herein below.

[0362] Table 5

[0363]

[0364] Experiment 5 (corresponds to results shown in Figures 16A-B):

[0365] Experiment 5A - Cell Expansion (in the absence of continuous exposure to anti CD3) with or without Anti-CD3 Restimulation at harvest

[0366] PBMCs were isolated from human peripheral blood by Ficoll-Hypaque density gradient centrifugation (Cytiva). PBMCs were resuspended in complete RPMI medium and seeded at a density of lx 106cells / mL in T-75 culture flasks. Cells were activated using CD3 / CD28 stimulationwith ImmunoCult™ Human CD3 / CD28 T Cell Activator (STEMCELL Technologies), supplemented with recombinant human IL-7 (10 ng / mL), IL-15 (10 ng / mL), a RAR antagonist (0.01 pM), and an AHR antagonist (0.05 pM). Cultures were incubated at 37 °C in a humidified atmosphere containing 5% CO2 for 48 hours.

[0367] On day 2, cells were washed, resuspended in fresh complete RPMI medium containing IL-7 and IL-15, and transferred to 6-well plates for an additional 24 hours. On day 3, cells were transferred to G-Rex culture vessels for further expansion and maintained in RPMI medium supplemented with IL-7, IL- 15 RAR and AHR antagonists. Cells were expanded for an additional 6 days.

[0368] On day 9, cells were harvested and subjected to one of the following conditions for 1.5 hours: (a) control, with IL-7 and IL- 15 only; or (b) restimulation at harvest, with IL-7, IL- 15, and anti-CD3 antibody. Following treatment, cells were used in cytotoxicity assays against leukemic and multiple myeloma target cells and analyzed as indicated.

[0369] Experiment 5A, which corresponds to Figure 16A is summarized in Table 6 herein below.

[0370] Table 6

[0371]

[0372] Experiment 5B. Standard T cells expansion (in the absence of continuous exposure to anti CD3) with and without small molecules

[0373] PBMCs were isolated from human peripheral blood by Ficoll-Hypaque density gradient centrifugation (Cytiva). PBMCs were resuspended in complete RPMI medium and seeded at a density of 1x106cells / mL in T-75 culture flasks. Cells were activated under one of the following conditions:

[0374] (a) Standard T-cell expansion: CD3 / CD28 stimulation using ImmunoCult™ Human CD3 / CD28 T Cell Activator (STEMCELL Technologies) in the presence of recombinant human IL-7 (10 ng / mL) and IL-15 (10 ng / mL) (PeproTech).(b) T-cell expansion supplemented with small molecules: CD3 / CD28 stimulation as above, supplemented with IL-7 (10 ng / mL), IL-15 (10 ng / mL), a RAR antagonist (0.01 pM), and an AHR antagonist (0.05 pM).

[0375] Cells were incubated at 37°C in a humidified atmosphere containing 5% CO2 for 48 hours. On day 2, cells were washed, resuspended in fresh complete RPMI medium containing IL-7 and IL-15, and transferred to 6-well plates for an additional 24 hours.

[0376] On day 3, cells were transferred to G-Rex culture vessels for further expansion. Group (a) cells were cultured in RPMI medium supplemented with IL-7 and IL- 15, whereas group (b) cells were cultured in RPMI medium containing IL-7, IL- 15, and the RAR and AHR antagonists. Cells were expanded for an additional 6 days.

[0377] On day 9, cells were harvested. Group (b) cells were restimulated with IL-7, IL- 15, and anti-CD3 antibody for 1.5 hours prior to downstream analyses. Cells were subsequently analyzed by flow cytometry.

[0378] Experiment 5B which corresponds to Figure 16B is summarized in Table 7 herein below.

[0379] Table 7

[0380]

[0381] Experiment 6 (corresponds to Figures 17A-O): PBMCs were isolated from human peripheral blood by Ficoll-Hypaque density gradient centrifugation (Cytiva). PBMCs were resuspended in complete RPMI medium and seeded at a density of 1 x 106cells / mL in T-75 culture flasks. Cells were activated under one of the following conditions:

[0382] (a) T-cell expansion: Cells were stimulated with CD3 / CD28 using ImmunoCult™ Human CD3 / CD28 T Cell Activator (STEMCELL Technologies) in the presence of recombinant human IL-7 (10 ng / mL) and IL- 15 (10 ng / mL) (PeproTech).

[0383] (b) T-cell expansion supplemented with small molecules: Cells were stimulated with recombinant human IL-2 (500 lU / mL), anti-human CD3 antibody (30 ng / mL), together with a RAR antagonist (0.01 pM) and an AHR antagonist (0.05 pM).

[0384] Cells were incubated at 37°C in a humidified atmosphere containing 5% CO2 for 48 hours. On day 2, cells were washed and resuspended in fresh complete RPMI medium supplemented withIL-7 and IL- 15 for group (a) or IL-2 for group (b). Cells were then transduced with gammaretroviral vectors encoding CD 19 CAR, CD 123 CAR, BCAM CAR, or HER2 CAR and incubated in 6-well plates for an additional 24 hours.

[0385] On day 3, cells were transferred to G-Rex culture vessels for further expansion. Group (a) cells were cultured in RPMI medium supplemented with IL-7 and IL- 15, whereas group (b) cells were cultured in RPMI medium containing IL-2, anti-CD3 antibody, and the RAR and AHR antagonists. Cells were expanded for an additional 6 days.

[0386] On day 9, cells were harvested. Group (b) cells were restimulated with IL-2 and anti-CD3 antibody for 1.5 hours prior to downstream analyses. Cells were subsequently analyzed by flow cytometry, used in cytotoxicity assays, and assessed for intracellular NAD levels according to the manufacturer’s instructions (NAD / NADH Assay Kit II, Abeam).

[0387] Experiment 6 is summarized in Table 8 herein below.

[0388] Table 8

[0389]

[0390] Experiment 7 (corresponds to Figures 18A-B) and Experiment 8 (corresponds to Figures 19A-H):

[0391] In vitro-co-culture assay to analyze cytokine and chemokine secretion: CD14+monocytes, MV411 leukemia cells, and either T-cells or CAR-T cells were co-incubated for 24 hours. Monocytes, T-cells and CAR-T cells were generated from the same whole-blood donor. Standard T cells and T cells produced in the presence of small molecules, as well as CAR-T cells and CAR-T cells produced in the presence of small molecules as described in Experiment 6 and summarized in Table 8. CD14+monocytes were purified from whole blood using the RosetteSep™ Human Monocyte Enrichment Cocktail (STEMCELL Technologies). After 24 hours, culture supernatants were collected, and cytokine and chemokine levels were quantified using a human cytokine array (R&D Systems).

[0392] Experiment 9 (corresponds to Figures 20A-C) and Experiment 10 (corresponds to Figures 21A-C):Short-term expansion of standard T cells and Sakura Bio T cells with or without CD 19 CAR.

[0393] On day 1, PBMCs were isolated from human peripheral blood by Ficoll-Hypaque density gradient centrifugation (Cytiva). CD3+T cells were purified from PBMCs using a Pan T Cell Isolation Kit (Miltenyi Biotec). T-cell expansion, with or without CD 19 CAR expression, was performed starting from either PBMCs or purified CD3+T cells. Cells were resuspended in complete RPMI medium and seeded at a density of 1 x 106cells / mL in T-75 culture flasks. Cells were activated under one of the following conditions:

[0394] (a) Standard T-cell expansion: PBMCs or CD3+T cells were stimulated with CD3 / CD28 using ImmunoCult™ Human CD3 / CD28 T Cell Activator (STEMCELL Technologies) in the presence of recombinant human IL-7 (10 ng / mL) and IL-15 (10 ng / mL) (PeproTech).

[0395] (b) Experimental conditions for expansion: PBMCs or CD3+T cells were stimulated with recombinant human IL-2 (500 lU / mL), anti-human CD3 antibody (30 ng / mL), a RAR antagonist (0.01 pM), and an AHR antagonist (0.05 pM).

[0396] Cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2. On day 3, cells were washed and resuspended in fresh complete RPMI medium supplemented with IL-7 and IL- 15 for group (a), or IL-2, anti-CD3 antibody, and the RAR and AHR antagonists for group (b). Cells were then transduced with gammaretroviral vectors encoding a CD 19 CAR or left non-transduced and incubated in 6-well plates for an additional 24 hours.

[0397] On day 4, cells were washed and cultured in fresh medium in 6-well plates. Group (a) cells were maintained in RPMI supplemented with IL-7 and IL- 15, whereas group (b) cells were maintained in RPMI containing IL-2, anti-CD3 antibody, and the RAR and AHR antagonists. Cells were expanded for an additional 2 days.

[0398] On day 6, cells were harvested and subsequently analyzed by flow cytometry and used in cytotoxicity assays.

[0399] Experiment 9 is summarized in Table 9 herein below.

[0400] Table 9

[0401]

[0402]

[0403] Experiment 11 (corresponds to Figures 22A-B)

[0404] Ultra- short-term (3-4 days) production of standard T cells and Sakura Bio T cells with or without CD 19 CAR.

[0405] On day 1, PBMCs were isolated from human peripheral blood by Ficoll-Hypaque density gradient centrifugation (Cytiva). Cells were resuspended in complete RPMI medium and seeded at a density of 1 x 106cells / mL in T-75 culture flasks. Cells were activated under one of the following conditions:

[0406] (a) Standard T-cell expansion: PBMCs were stimulated with CD3 / CD28 using ImmunoCult™ Human CD3 / CD28 T Cell Activator (STEMCELL Technologies) in the presence of recombinant human IL-7 (10 ng / mL) and IL- 15 (10 ng / mL) (PeproTech). (b) Experimental conditions for expansion: PBMCs were stimulated with recombinant human IL-2 (500 lU / mL), anti-human CD3 antibody (30 ng / mL), a RAR antagonist (0.01 pM), and an AHR antagonist (0.05 pM).

[0407] Cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2. On day 2, cells were washed and resuspended in fresh complete RPMI medium supplemented with IL-7 and IL- 15 for group (a), or IL-2, anti-CD3 antibody, and the RAR and AHR antagonists for group (b). Cells were then transduced with gammaretroviral vectors encoding a CD 19 CAR or left untransduced and incubated in 6- well plates for an additional 24 hours.

[0408] On day 3, cells were washed and cultured in fresh medium in 6-well plates. Group (a) cells were maintained in RPMI supplemented with IL-7 and IL- 15, whereas group (b) cells were maintained in RPMI containing IL-2, anti-CD3 antibody, and the RAR and AHR antagonists. Cells were incubated for 24 hours.

[0409] On day 4, cells were harvested and subsequently analyzed by flow cytometry and used in cytotoxicity assays.

[0410] Experiment 11 is summarized in Table 10 herein below.

[0411] Table 10

[0412]

[0413]

[0414] Cytotoxicity assay: Cytotoxicity was measured using CFSE-based assay. Target cells labeled with CFSE (eBioscience) according to manufacture instruction and plate at a concentration of 2X104cells / well in 96 well plate. T cells harvested on day 11-14 of culture and added to target cells at effector: target (E:T) ratios of 0.25:1, 0.5:1, 1:1, 2:1, and 5:1. After 24h, the CFSE+PI- labeled target cells were analyzed by flow cytometry. % of dead cells were calculated as:

[0415] { 100-(# of CFSE+PI- cells with effector cells / #of CFSE+PI- cells without effector cells) xlOO}.

[0416] FACS analysis: The cell population was identified by expression of CD45RA, CD45RO, CCR7. and CD62L (L-selectin). These subsets include naive (CD45RO-CD62L+CD45RA+ CCR7+), central memory (TCM; CD45RO+CD62E+ CD45RA- CCR7+), effector memory ('FEM; CD45RO+CD62E, CD45RA- CCR7-) and effector cells (CD45RO-CD62E-CD45RA+ CCR7-). Expression of CD57, CD62L, CCR7 and CD38 on CD8+ cells was detected using antibody (all from eBio science).

[0417] Small molecules: AHR antagonist- CH223191 (Cayman). RAR antagonist- AGN194310 (MedChemExpres s) .

[0418] Results

[0419] To demonstrate the significant effect of RAR and AHR antagonism on cell reprograming, RNA-seq analysis was performed to compare the gene expression profiles of cells expanded in the presence or absence of RAR and AHR as detailed in Experiment 1. An analysis of the composition of an exemplary cell population obtained following the culturing protocol is described in Figure 12A. As shown in Figure 12B, the addition of RAR and AHR during the expansion process resulted in a markedly different gene expression profile, indicative of cellular reprogramming. Treatment with RAR and AHR upregulates glycolytic, mTOR signaling pathway which plays a central role in promoting glycolysis and metabolic pathways that are known to support T cell proliferation, differentiation, and effector function. In addition, RNA-seq analysis revealed that RAR and AHR enhances the expression of genes involved in key immune pathways, including I Ny signaling, T cell activation, and lymphocyte-mediated immunity. Thus, the results support the conclusion that addition of RAR and AHR during the cell expansion process reprograms the cells to enhance their potency, metabolic fitness, and resilience.Next, the present inventors investigated whether the addition of RAR to cells restimulation process during the harvest phase could prolong the beneficial effects of RAR and sustain enhanced metabolic fitness over time. To this end, RAR was added during restimulation step at harvest, and cellular NAD levels and CD38 expression were assessed two and six days post-harvest. As shown in Figure 13B, the addition of RAR during harvest resulted in increased cellular NAD levels at both time points and maintained lower CD38 expression levels two and six days after harvest, indicating sustained metabolic fitness overtime.

[0420] Next, the present inventors investigated whether the effects brought about by RAR required continuous presence of CD3 activation. PBMCs were cultured as described in Experiment 3. The data show that some of the effects of the small molecules on cellular reprogramming are coupled to continuous TCR activation, whereas other appear to be independent of sustained TCR stimulation. As illustrated in Figures 14A-B, sustained stimulation throughout cell expansion is essential for the small molecules-dependent enrichment of less differentiated cell subsets, including naive and central memory cells expressing CD62L+(Figure 14A). In contrast, continuous TCR stimulation is not required for RAR-dependent downregulation of CD38 expression, as shown in Figure 14B.

[0421] To evaluate whether the addition of small molecules during the expansion phase and restimulation during harvest, enhances T-cell characteristics for CAR-T manufacturing, PBMCs were expanded under standard CAR-T manufacturing conditions in the presence or absence of the small molecules (Experiment 4). Briefly, on Day 0, cells were seeded in T75 flasks, activated with anti-CD3 / CD28 beads, and cultured in the presence of IL-7 and IL-15, cytokines commonly used for T-cell expansion for CAR-T manufacturing (group A). In the experimental arm (group B), small molecules were added at this stage. To mimic CAR-T transduction conditions, on Day 2 cells were transferred to 6-well plates and incubated for 24 hours with IL-7 and IL- 15. On Day 3, cells were washed and transferred to G-RexlOM flasks (100 mL) and maintained in the presence of IL-7 and IL-15 until harvest. Small molecules were also added to the experiment group (group B) at this stage. Cells were harvested on Day 9, at which point the experimental group was restimulated during harvest.

[0422] As demonstrated in Figures 15A-E, Group B cells exhibited significantly enhanced phenotypic and functional characteristics as compared to Group A. Specifically, these cells showed reduced CD38 expression (Figure 15 A), elevated intracellular NAD+levels indicative of improved metabolic fitness (Figure 15B), and a higher proportion of less differentiated naive and central memory T cells (Figure 15C). In addition, an increased frequency of CD62L+CCR7+T-cell subpopulations, previously associated with improved clinical outcomes in CAR-T therapies, was observed (Figure 15D). Importantly, these phenotypic improvements translated into enhancedfunctional potency, as demonstrated by significantly increased cytotoxic activity against cancer cells (Figure 15E).

[0423] Collectively, these results demonstrate that under CAR-T-compatible manufacturing conditions, inclusion of the small molecules leads to the generation of T cells with improved metabolic fitness, favorable differentiation state, and superior functional potency.

[0424] To assess the effect of restimulation during harvest in the absence of continuous CD3 stimulation, the experimental group were restimulated with anti CD3 at harvest, whereas the control group did not (Experiment 5). T cells were expanded in the presence of IL-7, IL-15, and the small molecules in the absence of anti CD3 during the expansion process (days 3-8).

[0425] Figure 16A demonstrates that restimulation at harvest significantly improved T-cell functionality, including enhanced tumor cell killing capacity, even when cells were expanded without continuous anti- CD3 stimulation. The effect of the addition of the small molecules during expansion and restimulation during harvest on cells expanded in the absence of continuous exposure to anti CD3 was evaluated. Cells expanded in the presence of small molecules (as summarized in Experiment 6), in the absence of continuous exposure to anti CD3, showed marked downregulation of CD38 expression (Figure 16B). These findings confirm that some of the beneficial effects of using the small molecules are preserved even in the absence of sustained CD3 stimulation during expansion. However, enhanced killing strictly depended on anti CD3 stimulation (for 1.5- 3.0 hours) during product harvest procedure.

[0426] To demonstrate that the use of small molecules supports the generation of superior CAR-T cells with increased viral transduction efficiency, enhanced potency, resilience, and metabolic fitness, thereby increasing therapeutic potential, a head-to-head comparison was made between CAR-T cells manufactured using a standard protocol (including IL-5 and IL-7 supplementation and activation using ImmunoCult™ Human CD3 / CD28 T Cell Activator) and CAR-T cells expanded in the presence of IL-2, anti CD3 and the small-molecule combination and restimulated during harvest (Experiment 6).

[0427] This comparison was conducted across multiple CAR constructs, including CD 123 (Figure 17A-E), CD 19 (Figure 17F-J), HER2 (Figure 17K-L), and BCMA (Figure 17M-O).

[0428] In a direct comparison of CD123-specific CAR-T cells, CAR-T cells generated by incorporating small-molecule supplementation and restimulation during harvest exhibited enhanced CAR expression on CD8+T cells (Figure 17A) and significantly improved killing of AML target cells (Figure 17B), indicating increased functional potency and therapeutic potential. Similarly, in head-to-head comparisons of CD19-specific CAR-T cells, CAR-T cells generated by incorporating small-molecule supplementation and restimulation during harvest exhibited demonstrated increasedresilience, as evidenced by reduced mitochondrial ROS production measured by MITOSOX™ assay (Figure 17J). In addition, these cells exhibited superior metabolic fitness, reflected by increased cellular energy levels (Figure 171).

[0429] Importantly, the beneficial effects of incorporating small-molecule supplementation and restimulation during harvest were observed consistently across all CAR specificities tested. CAR-T cells generated using this approach displayed significantly higher proportions of young, less-differentiated naive and central memory T-cell subsets expressing CD62L+(Figure 17D [CD123 CAR], Figure 17F [CD19 CAR], Figure 17K [HER2 CAR], and Figure 17N [BCMA CAR]). Moreover, enrichment of the CD62L+CCR7+population, previously associated with improved CAR-T therapeutic outcomes, was observed across multiple CAR constructs (Figure 17E [CD 123 CAR], Figure 17G [CD19 CAR], and Figure 170 [BCMA CAR]). These cells are advantageous for CAR-T applications due to their improved functional fitness, durability, and reduced exhaustion profile.

[0430] In addition, CAR-T cells produced using the protocol which includes small molecules consistently exhibited reduced CD38 expression across all CAR types tested (Figure 17C [CD123 CAR], Figure 17H [CD19 CAR], Figure 17L [HER2 CAR], and Figure 17M [BCMA CAR]).

[0431] To compare the functional activity of standard T-cells and CAR-T (CD123-specific) cells to T-cells and CAR-T (CD123-specific) cells manufactured using the method described in Experiment 7, the present inventors evaluated their ability to induce an effective anti-tumor immune response by measuring cytokine and chemokine secretion following cell stimulation. Specifically, the different cell types were co-cultured overnight with the AML cell line MV4-11 in the presence of CD14+monocytes. Supernatants were collected the following day, and secreted cytokine and chemokine levels were quantified.

[0432] The inclusion of monocytes is critical, as they act as accessory antigen-presenting cells and provide costimulatory signals that enhance T-cell and CAR-T-cell activation. In addition, monocytes shape the cytokine and chemokine milieu, promoting immune cell recruitment, effector function, and pro-inflammatory responses during T-cells-tumor interactions. Their presence therefore creates a more physiologically relevant system to assess the balance of immune signals regulating T-cells and CAR-T efficacy.

[0433] As shown in Figures 18 A, addition of small molecules in the expansion protocol produced T-cells significantly increased IFN-y secretion by T-cells, while having no significant effect on TNF-a secretion. Moreover, as shown in Figure 18B, addition of small molecules in the expansion protocol produced CAR-T cells resulted in significantly higher secretion levels of IFN-y and TNF-a compared with standard CAR-T cells. IFN-y plays a central role in antitumor immunity bypromoting Thl polarization, driving naive CD4+T-cell differentiation, and supporting cell-mediated immune responses. TNF-a induces the expression of adhesion molecules and enhances leukocyte migration into tissues, further strengthening antitumor activity.

[0434] In addition, as demonstrated in Figures 19A-H, T-cells (Figure 19A-D) and CAR-T (Figure 19 E-H) cells manufactured using small molecules exhibited increased secretion of key chemokines involved in antitumor immune activation, including CCL1 (immune cell recruitment and activation), CCL5 (T-cell trafficking into the tumor microenvironment), CCL3 (enhanced immune cell recruitment and activation), and CXCL10 (immune cell recruitment and antitumor immunity).

[0435] Next, the present inventors investigated whether the beneficial effects of small molecules which led to the generation of more potent, resilient, and metabolically fit T cells, are preserved when cells are manufactured using a rapid 4-6-day process. T cells generated using a standard protocol were compared with T cells produced in the presence of RAR and AHR. In parallel, the present inventors evaluated two starting materials: PBMCs and purified CD3+cells. All cell types were manufactured using the same rapid 4-6-day expansion protocol.

[0436] As shown in Figures 20A and 20B, T cells produced as described in Experiment 9, section b of the methods section, during rapid 6-day manufacturing exhibited significantly higher potency and cytotoxic activity against the lymphoma cell line Raji compared with standard T cells. These enhanced functional properties were observed regardless of whether PBMCs (Figure 20A) or purified CD3+cells (Figure 20B) were used as the starting material.

[0437] Importantly, when CD3+ cells were used as the starting material, low CD38 expression was maintained following rapid 6-day manufacturing (Figure 20C). Collectively, these results demonstrate that the beneficial effects of the protocol in generating potent, resilient, and metabolically fit T cells are preserved under rapid 4-6-day manufacturing conditions.

[0438] Next, the present inventors evaluated whether the beneficial effects of adding small molecules resulted in generating potent, resilient, and metabolically fit T cells are preserved under rapid 4-6-day CAR-T manufacturing conditions. CD19-specific CAR-T cells generated using a standard manufacturing protocol were compared with CAR-T cells produced using the use of small molecules, including the addition of RAR and AHR during cells’ growth and restimulation during harvest. As described in Figures 20A-C, two starting materials were assessed: PBMCs and purified CD3+cells. All CAR-T cell types were manufactured using the same rapid 6-day expansion protocol.

[0439] As shown in Figure 21 A, CAR-T cell production using small molecules resulted in a more efficient transduction process and higher CAR expression levels on CD8+T cells. In addition, as demonstrated in Figure 2 IB, the distinctive phenotypic features of CAR-T cells, including lowerCD38 expression, were maintained following rapid 6-day manufacturing, when PBMCs (left) or purified CD3+cells (right) were used as the starting material.

[0440] Furthermore, CAR-T cells produced using the small molecules through rapid 6-day manufacturing from PBMCs exhibited enhanced potency and cytotoxic activity against the lymphoma cell line Raji (Figure 21C).

[0441] Next, the present inventors evaluated whether the beneficial effects of incorporating small molecules into T-cell and CAR-T manufacturing processes are preserved under ultra-rapid 4-day CAR-T manufacturing conditions. T cells and CD19-specific CAR-T cells generated using a standard manufacturing protocol were compared with T cells and CAR-T cells produced in the presence of small molecules. PBMCs were used as the starting material in this experiment.

[0442] As shown in Figure 22A, CAR-T cell production in the presence of small molecules resulted in increased transduction efficiency and higher CAR expression levels on CD8+T cells. In addition, as demonstrated in Figure 22B, the distinctive phenotypic features of T cells and CAR-T cells cultured with small molecules, specifically reduced CD38 expression, were maintained following the ultra-rapid 4-day manufacturing process.

[0443] These results indicate that the small molecules reprogram immune cells to generate a unique population of T cells and CAR-T cells characterized by enhanced transduction efficiency and reduced CD38 expression, even under ultra-rapid 4-days manufacturing conditions.

[0444] Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

[0445] All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting. In addition, any priority document(s) of this application is / are hereby incorporated herein by reference in its / their entirety.

Claims

WHAT IS CLAIMED IS:

1. A method of generating an expanded population of CD3+ immune cells useful for adoptive cell transfer (ACT), comprising:culturing a population of CD3+cells in a medium comprising a cell-stimulating amount of:at least one cytokine,a T cell activator selected from the group consisting of a CD3 activator and a CD28 activator,an RAR antagonist andan AHR antagonist; andharvesting the expanded population of T cells.

2. The method of claim 1 further comprising supplementing, during, or on the same day as the harvesting a cell-stimulating amount of:said at least one cytokine,said T cell activator selected from the group consisting of a CD3 activator and a CD28 activator.

3. The method of claim 2 further comprising supplementing said RAR antagonist and said AHR antagonist during, or on the same day as the harvesting.

4. The method of any one of claims 1-3, wherein said at least one cytokine comprises IL-2 or a combination of IL- 15 and IL-7.

5. The method of any one of claims 1-4, wherein said culturing is effected for a minimum of 3 days.

6. The method of any one of claims 1-5, wherein said culturing is effected for no more than 6 days.

7. A method of generating an expanded population of T cells useful for adoptive cell transfer (ACT), comprising:culturing a population of CD3+ immune cells in a medium comprising a cell stimulating amount of IL-2 and a CD3 activator for at least nine days and no more than 16 days under conditions that increase the number of T cells of the population by at least 20 fold, wherein the medium is devoid of effective stimulating amounts of additional cytokines or antibodies;supplementing the medium with said IL-2 and said CD3 activator at least one time and no more than three times during the culturing, wherein said supplementing is not affected during the first four days of culturing; andharvesting the expanded population of T cells, thereby generating the expanded cell composition.

8. The method of claim 7, wherein the medium further comprises a small molecule selected from the group consisting of a Cytochrome P450 Family 1 (Cypl) Inhibitor, an AHR antagonist and a RAR antagonist.

9. The method of claim 8, wherein the supplementing the medium further comprises supplementing with said small molecule during, or on the same day as the harvesting.

10. The method of any one of claims 7-9, wherein said harvesting is affected following 10-12 days of the culturing.

11. The method of any one of claims 7-10, wherein said supplementing is affected on the day of said harvesting.

12. The method of claim 11, wherein said supplementing is affected during the harvesting process.

13. The method of any one of claims 7-12, wherein said supplementing is affected no more than two times during the method.

14. The method of any one of claims 7-13, wherein said at least one time is a single time, on the day of said harvesting.

15. The method of any one of claims 7-14, wherein said at least one time is no more than two times, when one of said times is affected on the day of said harvesting.

16. The method of any one of claims 7-13 and 15, wherein said supplementing is affected more than six days following the start of said culturing.

17. The method of any one of claims 7-13 and 15, wherein said supplementing is affected more than eight days following the start of said culturing.

18. A method of generating an expanded population of T cells useful for adoptive cell transfer (ACT), comprising:culturing a population of CD3+ immune cells in a medium comprising a cell-stimulating amount of at least one cytokine and a CD3 activator for at least nine days and no more than 16 days under conditions that increase the number of T cells of the population by at least 20 fold;harvesting the expanded population of T cells;supplementing the medium with said at least one cytokine and said CD3 activator no more than two times during the culturing, wherein at least one of the two times is affected on the day of harvesting the expanded population of T cells.

19. The method of claim 18, wherein the medium further comprises a small molecule selected from the group consisting of a Cytochrome P450 Family 1 (Cypl) Inhibitor, an AHR antagonist and a RAR antagonist.

20. The method of claim 19, wherein the supplementing the medium further comprises supplementing with said small molecule during, or on the same day as the harvesting.

21. A method of generating an expanded population of T cells comprising:(a) culturing a population of CD3+ immune cells in a medium comprising least one cytokine, a CD3 activator, an RAR antagonist and an AHR antagonist under conditions that generate a population of activated T cells;(b) culturing the population of activated T cells in a medium comprising said at least one cytokine, said RAR antagonist and said AHR antagonist, for at least 3 days and no more than 12 days to obtain a population of expanded T cells, wherein the medium of step (b) is substantially devoid of said CD3 activator; and subsequently(c) contacting said population of expanded T cells with said at least one cytokine and said CD3 activator.

22. The method of any one of claims 1-21, wherein said at least one cytokine is IL-2, and the medium is devoid of effective amounts of additional cytokines.

23. The method of claim 22, wherein said cell- stimulating amount of said IL-2 is between 100-1500 lU / ml.

24. The method of any one of claims 1-6 and 18-21, wherein said at least one cytokine is IL- 15 and IL-7, and the medium is devoid of effective amounts of additional cytokines.

25. The method of claim 24, wherein said cell- stimulating amount of said IL-15 is between 1-100 ng / ml and said cell-stimulating amount of said IL-7 is between 1-100 ng / ml.

26. The method of any one of claims 18-25, wherein said harvesting is affected following 10-12 days of the culturing.

27. The method of any one of claims 18-25, wherein said supplementing is affected at a single time, on the day of said harvesting.

28. The method of any one of claims 18-25, wherein a second of said two times is affected more than six days following the start of said culturing.

29. The method of any one of claims 18-25, wherein a second of said two times is affected more than eight days following the start of said culturing.

30. The method of any one of claims 1-29, wherein an amount of said CD3 activator is between 10-60 ng / ml.

31. The method of any one of claims 1-30, wherein the cells are not subjected to additional enrichment, stimulation or expansion steps.

32. The method of any one of claims 1-31, wherein culturing is affected under conditions that increase the number of T cells of the population by at least 30-fold.

33. The method of any one of claims 7-20, wherein said culturing is affected under conditions that increase the number of CD8+ cells of the population by at least 100-fold.

34. The method of any one of claims 1-33, wherein at least 70% of the cells of the expanded population are CD3+CD8+cells expressing NKG2D and granzyme B.

35. The method of any one of claims 1-34, wherein the cells of the expanded population are capable of specifically eradicating hematopoietic tumor cells in vitro at a ratio of 0.25:1 within 24 hours.

36. The method of any one of claims 1-34, wherein the cells of the expanded population are capable of specifically eradicating ovarian cancer cells in vitro at a ratio of 0.5:1 within 24 hours.

37. The method of any one of claims 1-36, wherein the CD3+ immune cells are genetically modified during the culturing.

38. The method of claim 37, wherein the CD3+ immune cells are genetically modified to express at least one of a chimeric antigen receptor (CAR), T cell receptor (TCR), a cytokine, a chemokine, a receptor of a cytokine or chemokine, or any combination thereof.

39. The method of claim 38, wherein the CD3+ immune cells are genetically modified to express: (i) a CD19-specific CAR or a CD 123 -specific CAR; (ii) a modified CXCR4 receptor comprising a mutation or truncation at the C-terminal tail domain; and (iii) at least one cytokine selected from the group consisting of IL-2, IL- 15 and IL-21.

40. The method of any one of claims 1-39, wherein the culturing is affected in a bioreactor.

41. The method of any one of claims 1-40, wherein said medium is RPMI.

42. The method of any one of claims 1-41, wherein said medium is a serum free medium (SFM).

43. The method of any one of claim 1-41, further comprising cry opreserving the obtained cell composition following the harvesting.

44. The method of any one of claims 1-43, wherein said CD3+ immune cells comprise CD3+ enriched T cells.

45. The method of any one of claims 1-43, wherein said CD3+ immune cells comprise peripheral blood mononuclear cells (PBMCs).

46. The method of any one of claims 1-45, wherein said CD3+ immune cells are nonactivated CD3+ immune cells.

47. A cell composition obtainable according to the method of any one of claims 1-46.

48. A cell composition, comprising in vitro-Qx^nA&A CD3+ immune cells, of which at least 39 % of the cells of the composition are CD8+CD38“, at least 80 % of the cells of the composition are CD8+CD62L+, at least 32 % of the cells of the composition are CD8+CCR7+and at least 9 % of the cells of the composition are CD3+CD4 CD8“ said composition exhibiting significant cytotoxic activity against tumor cells in vitro and in vivo.

49. The cell composition of claim 47 or 48, of which 70-85% of the cell composition are CD3+CD8+cells expressing NKG2D and granzyme B, at least 70% of the cells of the composition are CD56’ cells, and up to 5% of the cells of the composition are CD3“ cells.

50. The cell composition of claim 47, of which at least 39% of the cell composition are CD8+CD38“, at least 80 % of the cells of the composition are CD8+CD62L+, at least 32 % of the cells of the composition are CD8+CCR7+and at least 9% of the cells of the cell composition are CD3+CD4-CD8-.

51. The cell composition of any one of claims 47 or 48, comprising at least 70 % CD3+CD8+T cells, wherein at least 65 % of said T cells are CD62L+and at least 40 % of said cells are CCR7+.

52. The cell composition of claims 47-51, wherein at least 40 % of said T cells are naive stem cell memory T cells characterized by expression of CD45RA+CD62L+.

53. The cell composition of any one of claims 47-52, wherein at least 20 % of said T cells are central memory T cells characterized by expression of CD45RA CD62L+.

54. The cell composition of any one of claims 47-53, comprising greater than 100 pmol of NAD per IxlO6cells.

55. The cell composition of any one of claims 47-54, wherein the cells secrete IFN-y, TNF-a, CCL1, CCL5, CCL3 and / or CXCL10.

56. The cell composition of any one of claims 47-55, being genetically modified.

57. The cell composition of claim 56, wherein the cells are genetically modified to express at least one of a chimeric antigen receptor (CAR), T cell receptor (TCR), a cytokine, a chemokine, a receptor of a cytokine or chemokine, or any combination thereof.

58. The cell composition of claim 57, wherein the cells are genetically modified to express: (i) a CD19-specific CAR or a CD 123 -specific CAR; (ii) a modified CXCR4 receptor comprising a mutation or truncation at the C-terminal tail domain; and (iii) at least one cytokine selected from the group consisting of IL-2, IL- 15 and IL-21.

59. The cell composition of any one of claims 47-58, for use in treating cancer in a subject in need thereof.

60. The cell composition for use of claim 59, wherein the cancer is a hematopoietic cancer or a solid tumor.

61. The cell composition for use of claim 59, wherein said cancer is selected from the group consisting of leukemia, multiple myeloma, a prostate cancer, ovarian cancer and mesothelioma.

62. The cell composition for use of any one of claims 59-61, wherein tumor cells of said cancer are characterized by expression of at least one NKG2D ligand.

63. The cell composition for use of claim 62, wherein said tumor cells express a plurality of NKG2D ligands selected from the group consisting of: MICA, MICB, HLAE, ULBP1, ULBP256, and ULBP3.

64. The cell composition for use of claim 63, wherein said tumor cells express MICA, MICB, HLAE, ULBP1, ULBP256, and ULBP3.

65. The cell composition for use of any one of claims 59-64, wherein tumor cells of said cancer are characterized by expression of at least one chemokine selected from the group consisting of CXCR6 ligands, CXCR4 ligands, CXCR2 ligands and CXCR3 ligands.

66. The cell composition for use of any one of claims 59-64, wherein tumor cells of said cancer are characterized by expression of at least one ligand of an inhibitory immune checkpoint molecule selected from the group consisting of CTLA-4, PD-1, and TIGIT.

67. The cell composition for use of any one of claims 59-66, wherein said cancer is resistant to treatment by at least one checkpoint molecule inhibitor.

68. The cell composition for use of claim 60, wherein said tumor is resistant to treatment by at least one CTLA-4-specific blocking antibody.

69. The cell composition for use of any one of claims 59-68, wherein said subject is not under a treatment regimen with checkpoint molecule inhibitors.

70. The cell composition for use of any one of claims 59-69, wherein the cell composition is autologous, allogeneic, or non- allogeneic to said subject.

71. The cell composition for use of claim 70, wherein said cell composition is non-allogeneic to said subject.

72. The cell composition for use of any one of claims 60-71, wherein said tumor is characterized by down-regulation of MHC I expression and / or activity.

73. A method of treating a tumor in a subject in need thereof, comprising contacting the tumor cells with a therapeutically effective amount of the cell composition of any one of claims 47-58, thereby treating the tumor in the subject.

74. The method of claim 73, wherein the cell composition is autologous, allogeneic, or non-allogeneic to said subject.

75. The method of claim 74, wherein said cell composition is non-allogeneic to said subject.

76. The method of any one of claims 73-75, wherein the contacting is performed in vivo.

77. The method of any one of claims 73-75, wherein the contacting is performed ex vivo.

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