Compositions and methods for culturing t cells

Abstract

Cell culture medium for expanding a cell population (e.g., T cell population), cell culture compositions, and methods for culturing a cell population (e.g., T cell population) are provided. The medium can comprise a basal growth medium, a serum or serum replacement, and one or more cytokines and / or cytokine inhibitors (e.g., IL-2, IL-4, TGFβ, and / or an anti-IFNγ antibody). The method can comprising contacting the T cell population with the medium to expand the T cell population. The T cell population can comprise CD4+ and / or CD8+ primary human T cells or tumor infiltrating lymphocytes (TILs), which can be transformed to express a chimeric antigen receptor (CAR) or T cell receptor (TCR) and / or mutated by gene editing. The expanded T cell population using the medium and / or method provided herein can secrete IL-9, have increased viability, persistence, downregulation of exhaustion, mitogenesis, activation response, early memory T cell phenotype, or tumor control, and / or have an increased ratio of CD4+ / CD8+ T cells, relative to a control T cell population.

Description

[0001] COMPOSITIONS AND METHODS FOR CULTURING T CELLS

[0002] RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 63 / 512,006, filed on July 5, 2023, and U.S. Provisional Application No. 63 / 643,819, filed on May 7, 2024. The entire contents of each of the foregoing applications are incorporated herein by reference.

[0004] FIELD OF THE DISCLOSURE

[0005] The disclosure relates to culturing and expansion of T cells, e.g., primary human T cells.

[0006] BACKGROUND OF THE DISCLOSURE

[0007] Cell therapies, such as T cell receptor (TCR) therapy, adoptive cell therapy (ACT), chimeric antigen receptor (CAR)-T cell therapy, and tumor-infiltrating lymphocytes (TIL) therapy, is effective for treatment of malignancies. However, considerable numbers of patients relapse after the treatment, partially due to poor expansion or viability, antigen loss, and limited persistence of infused T cells. Limited persistence of T cells can be due to programmed cell death and exhaustion. Methods for efficiently culturing and expanding cells (e.g., T cells) and culturing and expanding cells (e.g., T cells) that persist in cancer therapy are therefore needed.

[0008] BRIEF SUMMARY OF THE DISCLOSURE

[0009] A cell culture medium for expanding a cell population (e.g., a T cell population), a cell composition comprising the cell culture medium and a population of cells cultured therein, and a method for culturing a cell population (e.g., a T cell population) is provided. The medium can comprise a basal growth medium, a serum or serum replacement, and one or more cytokines and / or cytokine inhibitors (e.g., IL-2, IL-4, TGFP, and / or an anti-IFNy antibody). The method can comprise contacting the T cell population with the medium to expand the T cell population. The T cell population can comprise CD4+and / or CD8+primary human T cells, or tumor infiltrating lymphocytes (TILs), which can be transformed to express any target-specific receptors, such as a chimeric antigen receptor (CAR) or T cell receptor (TCR), and / or mutated by gene editing. The expanded T cell population using the medium and / or method provided herein can secrete IL-9; express CCR4, CCR7, CD62L, and / or CD45RA; have increased viability, persistence, downregulation of exhaustion, and / or tumor control; have increased mitogenesis in response to a target antigen; have an early memory T cell phenotype or prolonged survival; have less differentiation into the effector phenotype (i.e., have a younger phenotype) or less T cell exhaustion; have enhanced proliferation or activation regardless of suppression by TGFP; have decreased potentiation of the TGFP signaling pathway in response to stimuli; do not require IL-2 for proliferation; and / or have an increased ratio of CD4+ / CD8+T cells, relative to a control T cell population (e.g., cultured using conventional T medium).

[0010] In one aspect, the present disclosure provides a method for culturing a T cell population. The method includes contacting the T cell population with expansion medium containing a basal growth medium, a serum or serum replacement, and one or more cytokines and / or cytokine inhibitors, such that the T cell population is expanded.

[0011] In some embodiments, the one or more cytokines and / or cytokine inhibitors in the expansion medium comprise IL-2, IL-4, TGFP, and / or an anti-IFNy antibody. In some embodiments, the expansion medium comprise IL-2 at a concentration of 10 lU / ml or more (e.g., 10-400 lU / ml); IL-4 at a concentration of 50 lU / ml or more (e.g., 250-500 lU / ml); TGFP at a concentration of 3 lU / ml or more (e.g., 20-150 lU / ml); and / or the anti-IFNy antibody at a concentration of 0 pg / ml or more (e.g., 0-20 pg / ml). In specific embodiments, the expansion medium comprises TGFP at a concentration of 4.5 lU / ml or more. In some embodiments, the expansion medium does not comprise the anti-IFNy antibody or IL-4. In some embodiments, the T cell population is not contacted with a medium or medium containing IL-2 and no other cytokines or cytokine inhibitors. In some embodiments, the basal growth medium comprises a Roswell Park Memorial Institute (RPMI) 1640 medium, X-VIVO™-15 medium, CTS™ OPTMIZER™ Serum Free Medium, CTS™ OPTMIZER™ Pro Serum Free Medium, and / or IMMLJNOCLrLT™-XF T Cell Expansion Medium. In some embodiments, the serum or serum replacement comprises fetal bovine serum (FBS), human AB serum, CTS™ Immune Cell Serum Replacement, and / or PHYSIOLOGIX™ Xeno-Free Serum Replacement.

[0012] In some embodiments, the T cell population comprise one or more primary T cells, such as one or more primary human T cells. In some embodiments, the T cell population consists essentially of one or more CD4+and / or CD8+T cells. In some embodiments, the T cell population comprises one or more tumor infiltrating lymphocytes (TILs).

[0013] In some embodiments, the expansion medium is replaced with fresh expansion medium every 2-3 days and / or the T cell population is cultured in the expansion medium for up to about 14 days. In some embodiments, the method includes culturing the T cell population in the expansion medium for at least 3 days optionally followed by culturing the T cell population in a control medium. In some embodiments, the method includes culturing the T cell population in the expansion medium for at least 7 days optionally after culturing the T cell population in a control medium.

[0014] In some embodiments, the method further comprises, after culturing the T cell population in the expansion medium, freezing the T cell population and / or administering the T cell population to a subject. In some embodiments, the T cell population is cultured in a system (e.g., bioreactor) comprising a gas permeable membrane culture surface, e.g., a G-REX® gas permeable flask.

[0015] In some embodiments, the method further comprises, prior to contacting the T cell population with the expansion medium: thawing a plurality of cells comprising the T cell population from a frozen stock; selecting from the plurality of cells one or more CD4+and / or CD8+T cells as the T cell population; contacting the T cell population with an activating reagent such that the T cell population is activated; introducing a polynucleotide encoding a chimeric antigen receptor (CAR) and / or a T cell receptor (TCR) into the T cell population such that one or more cells of the T cell population expresses the CAR or the TCR; and / or contacting the T cell population with one or more gene editing reagents to introduce a mutation at a target site in a genome of one or more cells of the T cell population and alter the level or activity of a gene of interest.

[0016] In some embodiments, said step of selecting one or more CD4+and / or CD8+T cells is performed within one day after the step of thawing a plurality of cells; said step of contacting the T cell population with an activating reagent is performed within one day after the step of selecting one or more CD4+and / or CD8+T cells; said step of introducing a polynucleotide encoding a CAR and / or a TCR into the T cell population is performed about 1-2 days after the step of contacting the T cell population with an activating reagent; said step of contacting the T cell population with one or more gene editing reagents is performed about 3 days after the step of contacting the T cell population with an activating reagent and / or about 1-2 days after the step of introducing a polynucleotide encoding a CAR and / or a TCR into the T cell population; and / or said step of contacting the T cell population with expansion medium is performed within one day after the step of introducing a polynucleotide encoding a CAR and / or a TCR into the T cell population or the step of contacting the T cell population with one or more gene editing reagents.

[0017] In some embodiments, said step of selecting one or more CD4+and / or CD8+T cells comprises contacting the plurality of cells with magnetic beads coupled with an anti- CD4+antibody and an anti-CD8+antibody, and selecting CD4+and / or CD8+T cells that are bound to the magnetic beads. In some embodiments, one or more cells of the T cell population are CD3+ and CD28+, and the activating reagent binds to and crosslinks CD3 and CD28 on surface of the one or more cells of the T cell population.

[0018] In some embodiments, introducing the polynucleotide encoding a CAR and / or a TCR into the T cell population comprises introducing the polynucleotide into the T cell population. In some embodiments, the polynucleotide is introduced via a viral vector (e.g. lentiviral vector or retroviruses or adenoviruses), and in other embodiments a non-viral delivery method may be used (e.g. transposition or mRNA transfection).

[0019] In some embodiments, contacting the T cell population with the one or more gene editing reagents comprises electroporating the one or more gene editing reagents into one or more cells of the T cell population.

[0020] In some embodiments, the cultured T cell population secretes IL-9; expresses CCR4, CCR7, CD62L, and / or CD45RA; has increased viability, persistence, downregulation of exhaustion, killing activity, and / or tumor control in vivo and / or in vitro., has increased mitogenesis in response to a target antigen; has an early memory T cell phenotype or prolonged survival; has less differentiation into the effector phenotype (i.e., have a younger phenotype) or less T cell exhaustion; have enhanced proliferation or activation regardless of suppression by TGFP; have decreased potentiation of the TGFP signaling pathway in response to stimuli; have decreased levels of phosphorylated SMAD3; do not require IL-2 for proliferation (have increased proliferation or activation in the absence of IL-2 in medium); and / or have an increased ratio of CD4+T cells over CD8+T cells, relative to a control T cell population, e.g., cultured in a control medium not comprising the expansion medium. In specific embodiments, in the cultured T cell population: the viability is statistically significantly increased by up to about 15%; the persistence is statistically significantly increased by up to about 100%; the exhaustion is statistically significantly downregulated by up to about 80%; a volume of a tumor contacted by the cultured T cell population is statistically significantly decreased, or eradicated; the mitogenesis is statistically significantly increased by up to about 50% in response to a target antigen; the differentiation into an effector phenotype is statistically significantly decreased down to 0%; the proliferation is statistically significantly increased by up to about 50% in the presence of TGFP; the proliferation is statistically significantly increased by up to about 50% in the absence of IL-2; the activation response is statistically significantly increased by up to 200% in the presence of TGFP; the activation response is statistically significantly increased by up to 500% in the absence of TGFP; and / or the ratio of CD4+T cells over CD8+T cells is statistically significantly increased by up to about 80%, as compared to the control T cell population. In some embodiments, expression of one or more genes associated with an early memory T cell phenotype or prolonged survival is increased, and / or expression of one of more genes associated with a T cell effector phenotype, T cell exhaustion, and / or suppression of T cell responses to tumors is decreased in the cultured T cell population relative to a control T cell population cultured in a control medium not comprising the expansion medium. In some embodiments, expression of PTGER2 is decreased in the cultured T cell population relative to a control T cell population cultured in a control medium not comprising the expansion medium. In some embodiments, the one or more genes associated with an early memory T cell phenotype or prolonged survival are one or more of RUNX2, CCR7, CTSL, MYB, and SELL (encoding CD62L), and / or the one or more genes associated with a T cell effector phenotype and / or a T cell exhaustion phenotype is one or more of LAG3, LAIR1, FASLG, IFNG, KLRG1, TBX21 (encoding Tbet), SLAMF7, GZMA, and GZMB. In some embodiments, expression of one or more of RUNX2, CCR7, CTSL, MYB, SELL, IL-9, IKZF2, CCR4, BATF3, SMAD2, , JUN, IRF4, USP18, MX1, IFNGR2, CD4, and TCF7 is increased, and / or expression of one or more of LAG3, LAIR1, FASLG, IFNG, KLRG1, TBX21, SLAMF7, GZMA, GZMB, PRF1, PTGER2, , AD0RA2A, BID, TGFBR1, ID2, and CD8a is decreased in the cultured T cell population relative to a control T cell population cultured in a control medium not comprising the expansion medium. In some embodiments, the cultured T cell population is a population of unmodified T cells or engineered CAR-T cells in the presence or absence of antigen challenge (e.g., repeated antigen stimulation).

[0021] In some embodiments, the CAR introduced into the T cells is bispecific for two antigens. In some embodiments, the CAR introduced into the T cells is specific for an antigen on a cancer cell. In some embodiments, the cancer cell is a cell of B cell lymphoma, T cell lymphoma, myeloma, leukemia, hematopoietic neoplasia, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, nonHodgkin’s lymphoma, Hodgkin’s lymphoma, uterine cancer, cervical cancer, endometrial cancer, adenocarcinoma, breast cancer, pancreatic cancer, colon cancer, anal cancer, renal cancer, bladder cancer, prostate cancer, ovarian cancer, primary or metastatic melanoma, squamous cell carcinoma, basal cell carcinoma, brain cancer, angiosarcoma, hemangiosarcoma, head and neck carcinoma, thyroid carcinoma, soft tissue sarcoma, bone sarcoma, testicular cancer, gastrointestinal cancer, stomach cancer, glioblastoma, small cell lung cancer, non-small cell lung cancer, or any combination thereof.

[0022] In one aspect, provided herein is a cell culture medium for expanding a cell population, such as a T cell population. The cell culture medium comprise a basal growth medium, a serum or serum replacement, and one or more cytokines and / or cytokine inhibitors. In some embodiments, one or more cytokines and / or cytokine inhibitors of the cell culture medium comprise IL-2, IL-4, TGFP, and / or an anti-fFNy antibody. In some embodiments, the cell culture medium comprises IL-2 at a concentration of 10 lU / ml or more (e.g., 10-400 lU / ml), IL-4 at a concentration of 50 lU / ml or more (e.g., 250-500 lU / ml), TGFP at a concentration of 3 lU / ml or more (e.g., 20-150 lU / ml), and / or the anti-IFNy antibody at a concentration of 0 pg / ml or more (e.g., 0-20 pg / ml). In specific embodiments, the expansion medium comprises TGFP at a concentration of 4.5 lU / ml or more. In some embodiments, the cell culture medium does not contain the anti-fFNy antibody or IL-4. In some embodiments, the cell culture medium does not contain IL-2 as the only cytokine and / or cytokine inhibitors. “Does not contain” a composition as used herein refers to not containing a substantive amount of the composition, including containing a negligible amount of the composition that does not impact the physiology of cells or the body in a significant manner. In some embodiments, the basal growth medium of the cell culture medium comprises a Roswell Park Memorial Institute (RPMI) 1640 medium, X-VIVO™-15 medium, CTS™ OPTMIZER™ Serum Free Medium, CTS™ OPTMIZER™ Pro Serum Free Medium, and / or IMMUNOCULT™-XF T Cell Expansion Medium. In some embodiments, the serum or serum replacement of the cell culture medium comprises fetal bovine serum (FBS), human AB serum, CTS™ Immune Cell Serum Replacement, and / or PHYSIOLOGIX™ Xeno-Free Serum Replacement.

[0023] In a certain aspect, provided herein is a cell culture composition comprising the cell culture medium provided herein and a cell population cultured therein.

[0024] In some embodiments, the cell population comprises a T cell population. In some embodiments, the T cell population comprises one or more primary human T cells, one or more tumor infiltrating lymphocytes (TILs), and / or one or more CD4+and / or CD8+T cells.

[0025] In some embodiments, the cell population comprises hematopoietic stem cells.

[0026] In some embodiments, the cell population has been modified by introducing an exogenous polynucleotide of interest or one or more gene editing reagents. In some embodiments, a polynucleotide encoding a chimeric antigen receptor (CAR) and / or a T cell receptor (TCR) has been introduced into the cell population such that one or more cells of the cell population express the CAR and / or the TCR.

[0027] In some embodiments, the cell population has not been modified with an exogenous polynucleotide of interest or one or more gene editing reagents.

[0028] In some embodiments, the T cell population has not been stimulated by an antigen. In other embodiments, the T cell population has been stimulated by an antigen. In some embodiments, the T cell population of the cell culture composition secretes IL-9; expresses CCR4, CCR7, CD62L, and / or CD45RA; comprises increased viability, increased persistence, increased downregulation of exhaustion, increased killing activity, increased tumor control, and / or increased mitogenesis; comprises an early memory T cell phenotype or prolonged survival; comprises less differentiation into an effector phenotype or less T cell exhaustion; comprises increased proliferation or activation in the presence or absence of TGFP or in the absence of IL-2 in medium; comprises decreased levels of phosphorylated SMAD3; comprises decreased potentiation of the TGFP signaling pathway in response to stimuli; and / or comprises an increased ratio of CD4+T cells over CD8+T cells, relative to a control T cell population cultured in a control medium not comprising the cell culture medium.

[0029] In some embodiments, in the T cell population of the cell culture composition, the viability is statistically significantly increased by up to about 15%; the persistence is statistically significantly increased by up to about 100%; the exhaustion is statistically significantly downregulated by up to about 80%; a volume of a tumor contacted by the cultured T cell population is statistically significantly decreased, or eradicated; the mitogenesis is statistically significantly increased by up to about 50% in response to a target antigen; the differentiation into an effector phenotype is statistically significantly decreased down to 0%; the proliferation is statistically significantly increased by up to about 50% in the presence of TGFP; the proliferation is statistically significantly increased by up to about 50% in the absence of IL-2; the activation response is statistically significantly increased by up to 200% in the presence of TGFP; the activation response is statistically significantly increased by up to 500% in the absence of TGFP; and / or the ratio of CD4+T cells over CD8+T cells is statistically significantly increased by up to about 80%, as compared to the control T cell population.

[0030] In some embodiments, expression of one or more genes associated with an early memory T cell phenotype or prolonged survival is increased, and / or expression of one of more genes associated with a T cell effector phenotype, T cell exhaustion, or suppression of T cell responses to tumors is decreased in the T cell population of the cell culture composition relative to a control T cell population cultured in a control medium not comprising the cell culture medium.

[0031] In some embodiments, expression of PTGER2 is decreased in the T cell population of the cell culture composition relative to a control T cell population cultured in a control medium not comprising the cell culture medium. In some embodiments, expression of one or more of RUNX2, CCR7, CTSL, MYB, SELL, IL-9, IKZF2, CCR4, BATF3, SMAD2, , JUN, IRF4, USP18, MX1, IFNGR2, CD4, and TCF7 is increased, and / or expression of one or more of LAG3, LAIR1, FASLG, IFNG, KLRG1, TBX21, SLAMF7, GZMA, GZMB, PRF1, PTGER2, , ADORA2A, BID, TGFBR1, ID2, and CD8a is decreased in the T cell population of the cell culture composition relative to a control T cell population cultured in a control medium not comprising the cell culture medium.

[0032] BRIEF DESCRIPTION OF THE DRAWINGS

[0033] FIG. l is a flowchart of a method of culturing a T cell population according to embodiments of the present disclosure.

[0034] FIGs. 2A and 2B depict proliferation of CAR-T cells cultured with the cell culture medium containing a combination of cytokines of the present disclosure (“OTX”) or IL-2 as the only cytokine (“Tconv”), illustrating that OTX cells are not reliant on IL-2 for proliferation. FIG. 2A depicts proliferation of OTX or Tconv CAR-T cells when cultured for 3 days without IL-2. FIG. 2B depicts CELL TRACE™ dye mean fluorescence intensity (“MFI”) for the OTX or Tconv CAR-T cells when cultured for 3 days without or with IL-2. Statistics represent paired T tests. ** p<0.01. n = 4, using 2 independent primary T cell donors; error bars represent SEM.

[0035] FIGs. 3 A and 3B depict proliferation of OTX CART-T cells (FIG. 3 A; expanded in the medium containing a combination of cytokines of the present disclosure) and conventional CAR-T cells (FIG. 3B; expanded in the medium containing IL-2 as the only cytokine) with or without TGFp, demonstrating proliferation of OTX CAR-T cells even in the presence of TGFp. Statistics represent paired 2 way ANOVA tests with a Sidak Post-hoc multiple comparison test. * p<0.05. n = 4, using 4 independent primary T cell donors; error bars represent SEM.

[0036] FIG. 3C depicts p-SMAD3 response to stimulus of primary T cells cultured in the OTX medium (medium containing a combination of cytokines of the present disclosure; “OTX”) and T cells cultured in conventional medium containing IL-2 as the only cytokine (“Tconv”).

[0037] FIGs. 4A and 4B depict activation responses of OTX cells (expanded in the medium containing a combination of cytokines of the present disclosure) and Tconv cells (expanded in the medium containing IL-2 as the only cytokine) without TGFP (FIG. 4A) or with TGFP (FIG. 4B) as measured by CD25 MFI, demonstrating enhanced activation responses of OTX CAR-T cells to low antigen levels, regardless of TGFp suppression. Statistics represent paired 2 way ANOVA tests with a Sidak Post-hoc multiple comparison test. **** p < 0.0001. n = 4 independent primary T cell donors; error bars represent SEM.

[0038] FIGs. 5A and 5B depict activation responses of OTX cells (expanded in the medium containing a combination of cytokines of the present disclosure) and Tconv cells (expanded in the medium containing IL-2 as the only cytokine) without TGFP (FIG. 5A) or with TGFP (FIG. 5B) as measured by CD71 MFI, demonstrating enhanced activation responses of OTX CAR-T cells to low antigen levels, regardless of TGFp suppression. Statistics represent paired 2 way ANOVA tests with a Sidak Post-hoc multiple comparison test. **** p<0.0001. n=4 independent primary T cell donors; error bars represent SEM.

[0039] FIGs. 6A and 6B depict mitogenesis in response to target antigen of OTX cells (expanded in the medium containing a combination of cytokines of the present disclosure) and Tconv cells (expanded in the medium containing IL-2 as the only cytokine), demonstrating increased mitogenesis in OTX cells in response to target antigen. FIG. 6A depicts MITOTRACKER™ Green total mitochondrial dye MFI. FIG. 6B depicts MITOTRACKER™ Green total mitochondrial dye MFI normalized to the MFI in Tconv cells, n = 2 independent primary T cell donors; error bars represent SEM.

[0040] FIG. 7 depicts a heatmap of genes differentially expressed in healthy human unmodified T cells conditioned in the OTX medium (medium containing a combination of cytokines of the present disclosure; “OTX”) or conventional medium (medium containing IL-2 as the only cytokine; “conv.”).

[0041] FIG. 8 depicts a heatmap of genes differentially expressed in gene edited CAR-T cells conditioned with OTX medium or conventional medium.

[0042] FIG. 9 depicts a heatmap of genes differentially expressed in gene edited CAR-T cells conditioned with OTX medium or conventional medium and stimulated by three rounds of antigen challenge.

[0043] FIG. 10A depicts the IL-9 secretion from T cells cultured in the OTX medium and / or conventional medium for the duration indicated.

[0044] FIG. 10B depicts the CCR4 expression in CD4+and CD8+T cells cultured in the OTX medium and / or conventional medium for the duration indicated.

[0045] FIG. IOC depicts the CCR7 expression in CD4+and CD8+T cells cultured in the OTX medium and / or conventional medium for the duration indicated.

[0046] FIG. 10D depicts the CD62L expression in CD4+and CD8+T cells cultured in the OTX medium and / or conventional medium for the duration indicated.

[0047] FIG. 11 depicts tumor volume over time in mice administered with OTX CD70 CAR-T cells (“OTX CAR-T”; expanded in the medium containing a combination of cytokines of the present disclosure), conventional CD70 CAR-T cells (“Conv CAR-T”; expanded in the medium containing IL-2 as the only cytokine), control T cells, or HBSS control on day 0, demonstrating superior tumor control of OTX CD70 CAR-T cells on tumor rechallenge in vivo. FIG. 12A depicts flow cytometry of excised / homogenized secondary tumor samples from FIG. 8 at the end of the study. FIG. 12B depicts percentage of human CD3+cells in the tumor from mice having received OTX CAR-T cells (“OTX”) or conventional CAR-T cells (“convT”).

[0048] FIG. 13 depicts activation response of control T cells (“control”), conventional CAR-T cells (“convT”; expanded in the medium containing IL-2 as the only cytokine), and OTX CAR-T cells (“OTX”; expanded in the medium containing a combination of cytokines of the present disclosure) obtained from the blood of subjects as measured by MFI of the activation marker CD25 on day 34, prior to tumor rechallenge, demonstrating superior activation response of OTX CAR-T cells.

[0049] FIG. 14A depicts exhaustion of control T cells (“control”), conventional CAR-T cells (“convT”), and OTX CAR-T cells (“OTX”) obtained from the spleen of subject as assessed by the presence of three independent exhaustion markers PD-1, LAG3, and KLRG1 at the end of the study described with respect to FIG. 13. FIG. 14B depicts percentages of an activation marker CD25- positive control, convT, and OTX cells at the end the same study.

[0050] FIGs. 15A-15D depict basal medium screening results for the cell culture medium (“OTX medium”) according to the embodiments of the present disclosure, demonstrating that OPTMIZER™ and OPTMIZER™ Plus medium yield high quality OTX cells with comparable results. FIG. 15A depicts process growth and recovery of total viable cells, as measured by the VICELL™ cell viability analyzer. FIG. 15B depicts transduction and transfection efficiency, and CD4:CD8 ratio. FIGs. 15C and 15D depict cell phenotyping (Naive, Central Memory, Effector Memory, and Effector Cells) in the CD4+T cells (FIG. 15C) and CD8+T cells (FIG. 15D).

[0051] FIGs. 16A and 16B depict CD4:CD8 ratios (FIG. 16A) and cell phenotypes (FIG. 16B) of OTX cells (expanded under OTX conditions) and conventional T cells (expanded in the conventional T medium containing IL-2 as the only cytokine), demonstrating that the OTX medium preferentially support the retention of naive and central memory CD4+and CD8+T cells and overall expansion of CD4+cells as compared to conventional T medium.

[0052] DETAILED DESCRIPTION

[0053] The present disclosure is explained in greater detail below. This description is not intended to be a detailed catalog of all the different ways in which the disclosure may be implemented, or all the features that may be added to the instant disclosure. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be deleted from that embodiment. In addition, numerous variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of the instant disclosure which do not depart from the instant disclosure. Hence, the following specification is intended to illustrate some particular embodiments of the disclosure, and not to exhaustively specify all permutations, combinations and variations thereof.

[0054] Unless the context indicates otherwise, it is specifically intended that the various features of the disclosure described herein can be used in any combination. Moreover, the present disclosure also contemplates that in some embodiments of the disclosure, any feature or combination of features set forth herein can be excluded or omitted.

[0055] In the following description, certain details are set forth such as specific quantities, sizes, etc. so as to provide a thorough understanding of the present embodiments disclosed herein. However, it will be obvious to those skilled in the art that the present disclosure may be practiced without such specific details. In many cases, details concerning such considerations and the like have been omitted inasmuch as such details are not necessary to obtain a complete understanding of the present disclosure and are within the skills of persons of ordinary skill in the relevant art.

[0056] The present disclosure is based on the discovery of media and methods for culturing and / or expanding a T cell population. The T cell population can for instance comprise primary human T cells, CD4+and / or CD8+T cells, CAR-T cells (including bispecific CAR-T cells), and / or T cells expressing an engineered TCR. The expanded T cell population can be used for cancer treatment.

[0057] I. Expansion Medium

[0058] In one aspect, provided herein is a cell culture medium for expanding a cell population, e.g., a T cell population, also referred to herein as “expansion medium.” The “cell culture medium” or the “expansion medium” can be used for culturing proliferating (or expanding) cells or a population of cells provided herein. The expansion medium provided herein comprise a basal growth medium, a serum or serum replacement, and one or more cytokines and / or cytokine inhibitors.

[0059] Any cytokines or cytokine inhibitors at any concentrations can be used in the expansion medium to support culturing and expansion of the T cell population. For example, the expansion medium can comprise IL-2, IL-4, TGFP, and / or anti-IFNy antibody.

[0060] The concentration of IL-2 in the medium can be about 10 lU / ml or more, such as about 10- 400, 50-400, 100-400, 200-400, 300-400, 10-50, 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400 lU / ml, about 50 lU / ml or more, 100 lU / ml or more, 150 lU / ml or more, 200 lU / ml or more, 250 lU / ml or more, 300 lU / ml or more, 350 lU / ml or more, or about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 250, 300, 350, or 400 lU / ml.

[0061] The concentration of IL-4 in the medium can be about 0-400 lU / ml, such as about 0-50, 50- 200, or 200-400 lU / ml. The concentration of IL-4 in the medium can be about 50 lU / ml or more, such as about 50-500, 100-500, 150-500, 200-500, 250-500, 300-500, 350-500, 400-500, 450-500, 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-450, 350-400, 400-450, 450-500 lU / ml, about 100 lU / ml or more, 150 lU / ml or more, 200 lU / ml or more, 250 lU / ml or more, 300 lU / ml or more, 350 lU / ml or more, 400 lU / ml or more, 450 lU / ml or more, 500 lU / ml or more, or about 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 lU / ml.

[0062] The concentration of TGFP in the medium can be about 3 lU / ml or more, or 4.5 lU / ml or more, such as about 4.5-10, 4.5-20, 4.5-30, 4.5-40, 4.5-50, 4.5-60, 4.5-70. 4.5-80, 4.5-90, 4.5-100, 4.5-150, 3-150, 10-150, 20-150, 30-150, 40-150, 50-150, 60-150, 70-150, 80-150, 90-150, 100-150, 110-150, 120-150, 130-150, 140-150, 3-10, 10-20, 20-25, 25-30, 30-40, 40-50, 50-60, 60-70, 70- 80, 80-90, 90-100, 100-110, 110-120, 120-130, 130-140, or 140-150 lU / ml, about 4.5 lU / ml or more, about 10 lU / ml or more, 50 lU / ml or more, 100 lU / ml or more, 150 lU / ml or more, or about 3, 4.5, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 lU / ml. In specific embodiments, the concentration of TGFP in the medium is 4.5 U / ml or more.

[0063] The concentration of the anti-IFNy antibody in the medium can be 0 pg / ml or more, such as about 0-20, 0.1-20. 1-20, 5-20, 10-20, 15-20, 0-1, 0-5, 5-10, 10-15, 15-20 pg / ml, about 20 pg / ml or less, 15 pg / ml or less, 10 pg / ml or less, 5 pg / ml or less, 1 pg / ml or less, 0.1 pg / ml or less, or about 0, 0.1, 1, 5, 10, 15, or 20 pg / ml, or more.

[0064] In some embodiments, the expansion medium does not contain the anti-IFNy antibody or IL-4. In some embodiments, the expansion medium does not contain IL-2 as the only cytokine and / or cytokine inhibitors. “Does not contain” a composition as used herein refers to not containing a substantive amount of the composition, including containing a negligible amount of the composition that does not impact the physiology of cells or the body in a significant manner. In specific embodiments, the expansion medium contains 100 HJ / mL IL-2, 290 HJ / mL IL-4, 25 lU / mL TGFP, and 10 pg / mL anti-IFNy.

[0065] “OTX cytokines” or “OUTAST cytokines” as used herein refer to a combination of cytokines contained in the expansion medium provided herein. For example, OTX cytokines can be a combination of IL-2 at a concentration of 10 lU / ml or more, IL-4 at a concentration of 50 lU / ml or more, TGFP at a concentration of 3 lU / ml or more or 4.5 lU / ml or more, and / or the anti-IFNy antibody at a concentration of 0-20 pg / ml. In specific embodiments, OTX cytokines can be a combination of 100 lU / mL IL-2, 290 lU / mL IL-4, 25 lU / mL TGFP, and 10 pg / mL anti-IFNy.

[0066] “OTX medium,” “OUTLAST medium,” “OTX media,” or “OUTLAST media” as used herein refers to any expansion medium provided herein containing a certain combination or concentrations of cytokines or cytokine inhibitors (“OTX cytokines”). The basal growth medium of the OTX medium can be any medium that supports growth of T cells as provided herein, including but not limited to Roswell Park Memorial Institute (RPMI) 1640 medium, CTS™ OPTMIZER™ Serum Free Medium (“OPTMIZER™ medium”), and CTS™ OPTMIZER™ Pro Serum Free Medium (“OPTMIZER™ Pro medium”). In certain embodiments, IL-2 is not the only cytokine or cytokine inhibitor in the OTX medium. In some embodiments, the OTX medium does not contain an anti-IFNy antibody or IL-4. In specific embodiments, the OTX medium contains 4.5 lU / ml or more TGFp. In specific embodiments, the OTX medium contains RPMI 1640 medium, 10% FBS, and OTX cytokines (e.g., 100 lU / mL IL-2, 290 lU / mL IL-4, 25 lU / mL TGFp, and 10 pg / mL anti- IFNy antibody). In other specific embodiments, OTX medium contains OPTMIZER™ medium or OPTMIZER™ Pro medium, 1.8 % immune cell serum replacement (“ICSR”), and OTX cytokines (e.g., 100 lU / mL IL-2, 290 lU / mL IL-4, 25 lU / mL TGFp, and 10 pg / mL anti-IFNy antibody).

[0067] “OTX cells,” “OUTLAST cells,” “OTX T cells,” “OUTLAST T cells,” “OTX CAR-T cells,” or “OUTLAST CAR-T cells” refer to the cells, T cells, or CAR-T cells cultured and expanded with the OTX medium (i.e., the expansion medium containing the OTX cytokines) for a sufficient time to exhibit an OUTLAST phenotype. An “OUTLAST phenotype” or an “OTX phenotype” as used herein refers to any phenotypic characteristics of a T cell or a T cell population cultured in the OTX medium (OTULAST medium, expansion medium provided herein) for a sufficient duration of time that distinguish them from control T cells or a control T cell population not cultured in the OTX medium for a sufficient duration. The OUTLAST phenotype or the OTX phenotype includes, and can be assessed by the presence of, any of secretion of IL-9; expression of a surface marker CCR4, CCR7, CD62L, or CD45RA; increased viability; increased persistence; downregulation of exhaustion; increased killing activity; increased tumor control; increased mitogenesis; an early memory T cell phenotype; prolonged survival; less differentiation into an effector phenotype; less T cell exhaustion; increased proliferation or activation in the presence or absence of TGFp or in the absence of IL-2 in medium; decreased levels of phosphorylated SMAD3; decreased potentiation of the TGFP signaling pathway in response to stimuli; and an increased ratio of CD4+T cells over CD8+T cells.

[0068] “OTX conditioning” or “OUTLAST conditioning” as used herein refers to culturing, expanding, and otherwise producing T cells or engineered T cells (e.g., CAR-T cells, gene edited T cells) using the OTX medium as provided in the present disclosure.

[0069] The basal growth medium of the expansion medium can be any medium that supports growth of T cells. The basal growth medium can comprise glucose, salts, amino acids, and vitamins. For example, the basal growth medium can contain a Roswell Park Memorial Institute (RPMI) 1640 medium, X-VIVO™-15 medium, CTS™ OPTMIZER™ Serum Free Medium, CTS™ OPTMIZER™ Pro Serum Free Medium, and / or IMMUNOCULT™-XF T Cell Expansion Medium. In some embodiments, the basal growth medium comprises the RPMI 1640 medium with ATCC modification (Gibco A1049101), the components of which are set forth in Table 1.

[0070] The serum or serum replacement of the expansion medium can be any serum or serum replacement that supports growth and expansion of T cells. “Serum replacement” as used herein refers to a composition used as substitute for a serum to support cell growth, differentiating, and / or expansion in cell culture. A serum replacement can comprise amino acids, vitamins, transferrin, insulin, collagen precursors, albumin, and / or lipid. For example, the serum can be fetal bovine serum (FBS). The serum replacement can be human AB serum, CTS™ Immune Cell Serum Replacement, and / or PHYSIOLOGIX™ Xeno-Free Serum Replacement. The expansion medium can contain 5-15% FBS (e.g., about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% FBS) or 1.5-5% serum replacement (e.g., about 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, or 5.0% serum replacement).

[0071] II. Method for Culturing T cells

[0072] Provided herein is a method of culturing and expanding a cell population, e.g., a T cell population. The method includes contacting the T cell population with expansion medium containing a basal growth medium, a serum or serum replacement, and one or more cytokines and / or cytokine inhibitors, such that the T cell population is expanded. The compositions of the expansion medium are described in the present disclosure.

[0073] The T cell population can comprise one or more primary T cells, such as one or more human primary T cells. The T cell population can consist essentially of one or more CD4+and / or CD8+T cells. The T cell population can include TILs or consist essentially of TILs.

[0074] The T cell population can be cultured in a conventional platform (e.g., flask, bioreactor). Alternatively, the T cell population can be cultured in a system (e.g., bioreactor) comprising a gas permeable membrane culture surface, e.g., a G-REX® gas permeable membrane platform (e.g., flask, bioreactor). The T cell population can be cultured in any desired scale, e.g., in a small scale (e.g., pilot scale) or a large scale (e.g., for clinical use). A cell culture platform, flask, or bioreactor based on a gas permeable membrane technology, such as a G-REX® gas permeable membrane platform, flask, or bioreactor can provide unlimited oxygen and nutrients to the cell culture. Conventional bioreactor platform technologies developed for large scale mammalian cell expansion are typically constrained by the mechanics of delivering oxygen to an expanding cell population. These systems often utilize complex mechanisms to enhance oxygen delivery, such as stirring, rocking, or perfusion, which adds to expense and increases their overall risk of failure. On the other hand, a gas permeable membrane-based platform, flask, or bioreactor such as a G-Rex bioreactor provides a more physiologic environment and avoids the risk and cost associated with more complex systems, providing a more robust, interacting cell population. By removing the need to actively deliver oxygen, gas permeable membrane-based platforms, flasks, and bioreactors can hold larger medium volumes (more nutrients) which allow the cells to reach a maximum density without complexity or need for medium exchange. This platform approach is scaled to meet the needs of research through commercial production with a direct, linear correlation between small and large devices. The T cell population can be cultured in the expansion medium for up to about 14 days (e.g., 1 day, 2 days 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, or longer than 14 days). The expansion medium that are in contact with the T cell population can be replaced with fresh expansion medium as needed, such as every 2-3 days.

[0075] A T cell population (e.g., unmodified T cell population, gene edited CAR-T cell population) may obtain an OUTLAST phenotype (e.g., secretion of IL-9; expression of a surface marker CCR4, CCR7, CD62L, or CD45RA; increased viability; increased persistence; increased downregulation of exhaustion; increased killing activity; increased tumor control; increased mitogenesis; an early memory T cell phenotype; prolonged survival; less differentiation into an effector phenotype; less T cell exhaustion; increased proliferation or activation in the presence or absence of TGFp or in the absence of IL-2 in medium; decreased levels of phosphorylated SMAD3; decreased potentiation of the TGFP signaling pathway in response to stimuli; an increased ratio of CD4+ T cells over CD8+ T cells) after contacting (e.g., culturing) them with the expansion medium for at least 3 days. Further, when a T cell population is initially cultured in a conventional medium (i.e., containing IL-2 and no other cytokines or cytokine inhibitors), subsequent contacting (e.g., culturing) with the OTX medium for at least 7 days may allow for the T cell population to obtain the OUTLAST phenotype. Thus, the method provided herein can include culturing the T cell population in the expansion medium for at least 3 days, optionally followed by culturing the T cell population in a control medium, or continuing to culture the T cell population in the expansion medium. The method provided herein can include culturing the T cell population in the expansion medium for at least 7 days optionally after culturing the T cell population in a control medium. The control medium can be a conventional medium or any non-OTX medium suitable for culturing T cells.

[0076] The method provided herein can further comprise, after culturing the T cell population in the expansion medium (e.g., for up to about 14 days), freezing the T cell population and / or using the T cell population (e.g., administering the T cell population to a subject).

[0077] The method provided herein can further comprise one or more of the following steps, prior to contacting the T cell population with the expansion medium: thawing a plurality of cells comprising the T cell population from a frozen stock, selecting from the plurality of cells one or more CD4+and / or CD8+T cells as the T cell population, contacting the T cell population with an activating reagent such that the T cell population is activated, introducing a polynucleotide encoding a chimeric antigen receptor (CAR) and / or a T cell receptor (TCR) into the T cell population such that one or more cells of the T cell population expresses the CAR or the TCR, and contacting the T cell population with one or more gene editing reagents such that a mutation is introduced at a target site in a genome of one or more cells of the T cell population. Said step of selecting one or more CD4+and / or CD8+T cells can be performed within one day after the step of thawing a plurality of cells; said step of contacting the T cell population with an activating reagent is performed within one day after the step of selecting one or more CD4+and / or CD8+T cells. Said step of introducing a polynucleotide encoding a CAR and / or a TCR into the T cell population can be performed about 1-2 days after the step of contacting the T cell population with an activating reagent. Said step of contacting the T cell population with one or more gene editing reagents can be performed about 3 days after the step of contacting the T cell population with an activating reagent and / or about 1-2 days after the step of introducing a polynucleotide encoding a CAR and / or a TCR into the T cell population. Said step of contacting the T cell population with expansion medium can be performed within one day after the step of introducing a polynucleotide encoding a CAR and / or a TCR into the T cell population or the step of contacting the T cell population with one or more gene editing reagents.

[0078] FIG. 1 depicts an embodiment of a method for culturing a T cell population provided herein. Process 100 begins with step 104 to select CD4+and / or CD8+cells step. Step 102 of thawing cryopreserved cells can be performed before step 104, for example immediately or within one day before step 104. The cryopreserved cells can be primary cells, such as leukocytes that were obtained from a subject (e.g., a cancer patient) e.g., by apheresis. Following step 104, process 100 proceeds to step 106 to activate T cells, e.g., immediately or within a day. In step 106, T cells can be activated e.g., by contacting the T cell population with an activating reagent. Following step 106, process 100 proceeds to step 108 to introduce a polynucleotide encoding a CAR and / or a TCR into the T cell population for expression, e.g., in about 1-2 days. CAR-T cells and / or engineered T cells can be thereby generated. Following step 108, process 100 can optionally proceed to step 110 to contact the T cell population with one or more gene editing reagents, e.g., in about 1-2 days after step 108 (introducing a polynucleotide encoding a CAR and / or a TCR into the T cell population) or about 3 days after step 106 (activating T cells). Step 112 to culture the T cell population in the expansion medium to allow expansion of the T cell population can follow step 108 (introducing a polynucleotide encoding a CAR and / or a TCR into the T cell population) or step 110 (gene editing), e.g., immediately or within one day. Step 112 can last up to 14 days with replacement of the expansion medium with fresh expansion medium every 1-3 days as needed to achieve an optimal growth and / or expansion of the T cell population. Following step 112, process 100 proceeds to step 114 to harvest, formulate, cry opreserve, or use the expanded T cell population, including administering the cells to a subject.

[0079] The expansion medium provided herein can also be used for any steps described above. For example, the expansion medium can be used for activating the T cell population, transducing the T cell population, introducing one or more gene editing reagents into the T cell population (or CAR-T cells), freezing the T cell population, administering the T cell population to a subject, and / or otherwise using the cells, in addition to culturing the T cell population for expansion. In specific embodiments, at any time during culturing the T cell population according to the method provided herein, including prior to freezing, resting or using the cells therapeutically or otherwise (such as administering the T cells to a subject, for example by infusing the T cells to the subject), the expanding cells are not contacted with a medium containing IL-2 as the only cytokine.

[0080] In some embodiments, the expanded T cell population according to the methods provided herein secretes IL-9. IL-9 secreting T cells can be referred to as “T9” cells.

[0081] The T cell population cultured according to the methods provided herein can have distinct characteristics (i.e., OUTLAST phenotype) as compared to a control T cell population, e.g., cultured in a control medium not comprising the expansion medium. For example, the cultured T cell population can secrete IL-9; express surface marker CCR4, CCR7, CD62L, and / or CD45RA; have increased viability, persistence, downregulation of exhaustion, killing activity, and / or tumor control in vivo and / or in vitro., have increased mitogenesis in response to a target antigen; have an early memory T cell phenotype or prolonged survival; have less differentiation into the effector phenotype (i.e., have a younger phenotype) or less T cell exhaustion; have enhanced proliferation or activation regardless of suppression by TGFP; have decreased potentiation of the TGFp signaling pathway in response to stimuli; have decreased levels of phosphorylated SMAD3; do not require IL-2 for proliferation (have increased proliferation or activation in the absence of IL-2 in medium); and / or have an increased ratio of CD4+T cells over CD8+T cells, relative to a control T cell population. Further, expression of one or more genes associated with an early memory T cell phenotype or prolonged survival can be increased, and / or expression of one of more genes associated with a T cell effector phenotype, T cell exhaustion, and / or suppression of T cell responses to tumors can be decreased in the cultured T cell population relative to a control T cell population cultured in a control medium not comprising the expansion medium. In some embodiments, the one or more genes associated with an early memory T cell phenotype are one or more of RUNX2, CCR7, CTSL, MYB, and SELL, and / or the one or more genes associated with a T cell effector phenotype and / or a T cell exhaustion phenotype is one or more of LAG3, LAIR1, FASLG, IFNG, KLRG1, TBX21, SLAMF7, GZMA, and GZMB. Expression of one or more of RUNX2, CCR7, CTSL, MYB, SELL, IL-9, IKZF2, CCR4, BATF3, SMAD2, , JUN, IRF4, USP18, MX1, IFNGR2, CD4, and TCF7 can be increased, and / or expression of one or more of LAG3, LAIR1, FASLG, IFNG, KLRG1, TBX21, SLAMF7, GZMA, GZMB, PRF1, PTGER2, , AD0RA2A, BID, TGFBR1, ID2, and CD8a can be decreased in the cultured T cell population relative to a control T cell population cultured in a control medium not comprising the expansion medium.

[0082] In some embodiments, expression of PTGER2 is decreased in the cultured T cell population relative to a control T cell population cultured in a control medium not comprising the expansion medium. PTGER2 encodes EP2, a receptor for prostaglandin E2 (PGE2). Without wishing to be bound by theory, PGE2, a known negative regulator of immune response in the tumor microenvironment can be present at high concentrations in tumor tissue from patients and leads to impaired IL-2 sensing in human CD8+TILs via the PGE2 receptors EP2 and EP4. PGE2 inhibits IL-2 sensing in TILs by downregulating the fL-2Ryc chain, resulting in defective assembly of IL- 2RP-IL2RYC membrane dimers. This results in impaired IL-2-mT0R adaptation and PGCla transcriptional repression, causing oxidative stress and ferroptotic cell death in tumor-reactive TILs. Inhibition of PGE2 signaling to EP2 and EP4 during TIL expansion for adoptive cell therapy can result in increased IL-2 sensing, leading to enhanced proliferation of tumor reactive TILs and enhanced tumor control once the cells were transferred in vivo. Morotti et al. Nature 24 April 2024. Culturing and / or expanding T cells (e.g., TILs) according to the methods provided herein (e.g., culturing / expanding in OTX medium) can downregulate PTGER2 in the T cells (e.g., TILs), and thus can enhance IL-2 sensing and amplify the IL-2 response in the T cells (e.g., TILs), thereby promoting the expansion of effector T cells with enhanced therapeutic potential, e.g., enhanced tumor reactivity and tumor control.

[0083] In some embodiments, the T cell population cultured according to the methods provided herein has increased viability in culture and / or upon freezing and thawing, relative to a control T cell population. A “control T cell population” can be a T cell population cultured in a control medium that is not the expansion medium, or cultured according to a control method that is not the method provided herein. One having skills in the art can select an appropriate control. For example, the expanded T cell population according to the methods provided herein can have viability in culture and / or upon freezing and thawing that is increased by about 1-100%, 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%, 20-200%, 30-200%, 40-200%, 50- 200%, 60-200%, 70-200%, or more than 200% (e.g., by about 1-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100%, 100-150%, 150-200%, or more than 200%), e.g., increased by about 1%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, or at least about 1%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, or 200% relative to a control T cell population. In specific embodiments, in the cultured T cell population, the viability is statistically significantly increased by up to about 15% relative to a control T cell population. Cell viability can be measured by any standard methods, including a VICELL™ cell viability analyzer, an MTT assay, a dye exclusion assay (e.g., using trypan blue), or a live / dead assay (e.g., using calcein-AM, propidium iodide, and / or a Hoechst dye).

[0084] In some embodiments, the T cell population cultured according to the methods provided herein has increased persistence, downregulation of exhaustion, killing activity upon exposure to target cells, tumor control, and / or mitogenesis in response to a target antigen relative to a control T cell population. “Persistence” as used herein refers to how long a T cell (such as a CAR-T cell, engineered T cell) lasts, maintaining its functionality (such as binding to and / or killing of a target cell) upon exposure to target cells or infusion into a subject. “Exhaustion” as used herein refers to a T cell’s loss of function, such as its ability to kill target cells. For example, the expanded T cell population according to the methods provided herein can have persistence, downregulation of exhaustion, killing activity, tumor control, and / or mitogenesis that is increased by about 1-100%, 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%, 20-90%, 30- 90%, 40-90%, 50-90%, 60-90%, 70-90%, 100-1000%, 200-1000%, 300-1000%, 400-1000%, 500- 1000%, 600-1000%, 700-1000%, 800-1000%, 200-900%, 300-900%, 400-900%, 500-900%, 600- 900%, 700-900%, or more than 1000% (e.g., by about 1-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100%, 100-200%, 200-300%, 300-400%, 400-500%, 500- 600%, 600-700%, 700-800%, 800-900%, 900-1000%, or more than 1000%), e.g., by about 1%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more, or at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or at least about 1%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more, or at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000% relative to a control T cell population. In specific embodiments, in the cultured T cell population, the persistence is statistically significantly increased by up to about 100%, the exhaustion is statistically significantly downregulated by up to about 80%, the volume of a tumor contacted by the cultured T cell population is statistically significantly decreased, or eradicated (to 0%), and / or the mitogenesis is statistically significantly increased by up to about 50% in response to a target antigen relative to a control T cell population. Persistence of T cells can be measured by any standard methods, including an assay to quantitate live T cells in tumor or an in vivo T cell persistence test. Killing activity of T cells can be measured by any standard methods, including cytotoxicity assay (e.g., ELOSPOT assay), or flow cytometry to measure intracellular cytokines or cytotoxic granules, or the cell surface CAR / TCR expression. Exhaustion of T cells can be measured by any standard methods, including an assay to measure expression of exhaustion markers (e.g., PD-1, LAG3, KLRG1) and / or activation markers (e.g., CD25) upon antigen stimulation or tumor challenge. Tumor control can be measured by any standard methods, including measuring tumor volume. Mitogenesis can be measured by any standard methods, including a MITOTRACKER™ total mitochondrial dye assay. Example assays for measuring persistence, downregulation of exhaustion, killing activity, tumor control, and / or mitogenesis are described in the Examples provided herein.

[0085] In some embodiments, the T cell population cultured according to the methods provided herein has an increased early memory T cell phenotype or prolonged survival. For example, the expanded T cell population according to the methods provided herein can have an early memory T cell phenotype or survival that is increased by about 1-100%, 10-100%, 20-100%, 30-100%, 40- 100%, 50-100%, 60-100%, 70-100%, 80-100%, 20-90%, 30-90%, 40-90%, 50-90%, 60-90%, 70- 90%, 100-1000%, 200-1000%, 300-1000%, 400-1000%, 500-1000%, 600-1000%, 700-1000%, 800-1000%, 200-900%, 300-900%, 400-900%, 500-900%, 600-900%, 700-900%, or more than 1000% (e.g., by about 1-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80- 90%, 90-100%, 100-200%, 200-300%, 300-400%, 400-500%, 500-600%, 600-700%, 700-800%, 800-900%, 900-1000%, or more than 1000%), e.g., by about 1%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more, or at least 1%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more, or at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000% relative to a control T cell population. The T cell phenotype can be measured by any standard methods, including detecting cellular phenotypic markers and sorting cells by flow cytometry. An early memory T cell phenotype or prolonged survival can also be assessed by measuring gene expression associated with these phenotypes, including RUNX2, CCR7, CTSL, MYB, SELL, IL-9, IKZF2, CCR4, BATF3, SMAD2, , JUN, IRF4, USP18, MX1, IFNGR2, CD4, and TCF7, by standard methods in the art (e.g., quantitative PCR).

[0086] In some embodiments, the T cell population cultured according to the methods provided herein has less differentiation into the effector phenotype (i.e., has a younger phenotype, such as naive and central memory phenotype) or less T cell exhaustion. For example, the expanded T cell population according to the methods provided herein can have a percentage of effector memory or effector cells that is reduced by about 1-20%, 5-20%, 10-20%, 15-20%, 1-10%, 5-10% (e.g., by about 1-5%, 5-10%, 10-15%, 15-20%), e.g., by about 1%, 5%, 10%, 15%, or 20%, or at least 1%, 5%, 10%, 15%, 20% relative to a control T cell population. The expanded T cell population according to the methods provided herein can have T cell exhaustion that is reduced by about 1- 100%, 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%, 90- 100%, 1-50%, 10-50%, 20-50%, 30-50%, 40-50% (e.g., by about 1-10%, 10-20%, 20-30%, 30- 40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100%), e.g., by about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control T cell population. In specific embodiments, in the cultured T cell population, the differentiation into an effector phenotype is statistically significantly decreased relative to a control T cell population down to 0%. T cell phenotype can be measured by any standard methods, including detecting cellular phenotypic markers and sorting cells by flow cytometry. An effector T cell phenotype or T cell exhaustion can also be assessed by measuring gene expression associated with these phenotypes, including LAG3, LAIR1, FASLG, IFNG, KLRG1, TBX21, SLAMF7, GZMA, GZMB, PRF1, PTGER2, , AD0RA2A, BID, TGFBR1, ID2, and CD8a, by standard methods in the art (e.g., quantitative PCR).

[0087] In some embodiments, the T cell population cultured according to the methods provided herein has enhanced proliferation or activation regardless of suppression by TGFp or absence of IL- 2 in the medium, or has decreased potentiation of the TGFP signaling pathway in response to stimuli. For example, the expanded T cell population according to the methods provided herein can have proliferation or activation in the presence of TGFp or in the absence of IL-2 that is increased by about 1-100%, 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 80- 100%, 20-90%, 30-90%, 40-90%, 50-90%, 60-90%, 70-90%, 100-1000%, 200-1000%, 300- 1000%, 400-1000%, 500-1000%, 600-1000%, 700-1000%, 800-1000%, 200-900%, 300-900%, 400-900%, 500-900%, 600-900%, 700-900%, or more than 1000% (e.g., by about 1-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100%, 100-200%, 200-300%, 300-400%, 400-500%, 500-600%, 600-700%, 700-800%, 800-900%, 900-1000%, or more than 1000%), e.g., by about 1%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more, or at least 1%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more, or at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000% relative to a control T cell population. The expanded T cell population according to the methods provided herein can have potentiation of the TGFp signaling pathway that is reduced by about 1-100%, 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%, 90-100%, 1-50%, 10-50%, 20-50%, 30-50%, 40-50% (e.g., by about 1-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100%), e.g., by about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control T cell population. In specific embodiments, in the cultured T cell population, the proliferation is statistically significantly increased by up to about 50% in the presence of TGFP, or by up to about 50% in the absence of IL-2, and / or the activation response is statistically significantly increased by up to 200% in the presence of TGFp or by up to 500% in the absence of TGFp, relative to a control T cell population. T cell proliferation can be measured by any standard methods including a an assay using a proliferation dye (e.g., CELL TRACE™ proliferation dye) and an assay to quantitate viable T cells. T cell activation can be measured by any standard methods, including measuring levels of activation markers (e.g., CD25, CD71) following an antigen challenge. The potentiation of the TGFP signaling pathway can be measured by any standard method, including quantitation of phosphorylated SMAD3 in cells by Western blotting. Example assays are described in the Examples provided herein. In some embodiments, the T cell population cultured according to the methods provided herein has decreased levels of phosphorylated SMAD3 relative to a control T cell population before or during activation, thereby blunting TGFP signaling. The T cell population cultured according to the methods provided herein can have phosphorylated SMAD3 levels that are reduced by about 1-100%, 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%, 90-100%, 1-50%, 10-50%, 20-50%, 30-50%, 40-50% (e.g., by about 1-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100%), e.g., by about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control T cell population.

[0088] In some embodiments, the T cell population cultured according to the methods provided herein has an increased ratio of CD4+T cells over CD8+T cells, relative to a control T cell population. Without wishing to be bound by theory, a higher ratio of CD4+T cells over CD8+T cells can be advantageous for certain T cell therapies. For example, the expanded T cell population according to the methods provided herein can have a CD4+ / CD8+ratio that is increased (as expressed by subtraction of a CD4+ / CD8+ratio in a control T cell population from that of the T cell population provided herein) by about 1-80%, 10-80%, 20-80%, 30-80%, 40-80%, 50-80%, 60- 80%, 70-80%, 1-90%, 10-90%, 20-90%, 30-90%, 40-90%, 50-90%, 60-90%, or 70-90% (e.g., by about 1-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90- 100%), e.g., by about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control T cell population. In specific embodiments, in the cultured T cell population, the ratio of CD4+T cells over CD8+T cells is statistically significantly increased by up to about 80%, relative to the control T cell population. The CD4+ / CD8+ratio in a T cell population can be measured by any standard methods, including cell labeling and flow cytometry.

[0089] In some embodiments, expression of one or more genes associated with an early memory T cell phenotype or prolonged survival is increased (e.g., these genes have positive Z scores), and / or expression of one of more genes associated with a T cell effector phenotype, T cell exhaustion, and / or suppression of T cell responses to tumors is decreased (e.g., these genes have negative Z scores) in the cultured T cell population relative to a control T cell population cultured in a control medium not comprising the expansion medium. The upregulation and downregulation of genes described herein in T cells cultured in the expansion medium (e.g., OTX medium) can occur in any T cells cultured in the expansion medium, regardless of the presence or absence of gene modification or antigen stimulation. For example, an increase in expression of one or more genes associated with an early memory T cell phenotype or prolonged survival and / or a decrease in expression of one of more genes associated with a T cell effector phenotype, T cell exhaustion, and / or suppression of T cell responses to tumors can be observed in a population of unmodified T cells as well as modified T cells (e.g., gene edited CAR-T cells), either in the presence or absence of repeated antigen challenge (chronic restimulation), so long as they are cultured in the expansion medium of the present disclosure for a sufficient time. Accordingly, culturing T cells in the expansion medium can confer the OUTLAST phenotype to the T cells regardless of the characteristics of the T cells, e.g., modified or unmodified, or stimulated or unstimulated. An “unmodified” cell or an “untransformed” cell as used herein refers to a cell in which the cell characteristics such as the gene or polypeptide expression profiles have not been modified, such as by introducing an exogenous polynucleotide of interest for expression of a polypeptide of interest (e.g., a CAR, a TCR), or introducing one or more gene editing reagents for modification of the genome. An “modified” cell, a “transformed” cell, an “engineered” cell, or an “edited” as used herein refers to a cell in which the cell characteristics such as gene or polypeptide expression profiles have been modified, such as by introducing an exogenous polynucleotide of interest for expression of a polypeptide of interest (e.g., a CAR, a TCR), or introducing one or more gene editing reagents for modification of the genome.

[0090] For example, one or more genes associated with an early memory T cell phenotype or prolonged survival, and genes such as RUNX2, CCR7, CTSL, MYB, SELL, IL-9, IKZF2, CCR4, BATF3, SMAD2, , JUN, IRF4, USP18, MX1, IFNGR2, CD4, and TCF7 can have positive Z scores (e.g., about 0.1 to about 1.7) or their expression can be increased by about 1-100%, 10-100%, 20- 100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%, 20-90%, 30-90%, 40-90%, 50-90%, 60-90%, 70-90%, 100-1000%, 200-1000%, 300-1000%, 400-1000%, 500-1000%, 600- 1000%, 700-1000%, 800-1000%, 200-900%, 300-900%, 400-900%, 500-900%, 600-900%, 700- 900%, or more than 1000% (e.g., by about 1-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100%, 100-200%, 200-300%, 300-400%, 400-500%, 500-600%, 600-700%, 700-800%, 800-900%, 900-1000%, or more than 1000%), e.g., by about 1%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more, or at least 1%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more, or at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000% in the expansion medium cultured T cells (unmodified, engineered, unstimulated, stimulated) relative to a control T cell population. Additionally or alternatively, one or more genes associated with suppression of T cell responses to tumors (e.g., PTGER2), a T cell effector phenotype and / or a T cell exhaustion phenotype, and genes such as LAG3, LAIR1, FASLG, IFNG, KLRG1, TBX21, SLAMF7, GZMA, GZMB, PRF1, PTGER2, , ADORA2A, BID, TGFBR1, ID2, and CD8a can have negative Z scores (e.g., about -0.1 to about -1.2) or can be decreased by about 1-100%, 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%, 90-100%, 1-50%, 10-50%, 20-50%, 30-50%, 40-50% (e.g., by about 1-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80- 90%, or 90-100%), e.g., by about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% in the expansion medium cultured T cell population (unmodified, engineered, unstimulated, stimulated) relative to a control T cell population.

[0091] 1. Expansion of T Cells

[0092] The methods provided herein can comprise culturing the T cell population (e.g., including CAR-T cells, TILs, mutated T cells, engineered T cells) such that the T cell population can be expanded, and / or the T cell population is cultured in the expansion medium for up to about 14 days.

[0093] In some embodiments, said T cell population (e.g., CAR-T cells, TILs, mutated T cells, engineered T cells) remain at an expansion stage for about 14 days, or up to about 14 days (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days). An “expansion stage” as used herein refers to a stage (or phase) of cells in which cells undergo cell divisions and increase in number. The T cells can be expanded for a certain period, e.g., about 3-14 days, 4-14 days, 5-14 days, 6-14 days, 7-14 days, 8-14 days, 9-14 days, 10-14 days, 11-14 days, 12-14 days, 13-14 days, or more, e.g., about 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, or more, without being rested, frozen, or used therapeutically or otherwise (e.g., administered to a subject). The T cells can continue expanding (or remain in the expansion stage) for a certain period in the expansion medium provided herein. For example, the T cells can remain in the expansion stage for a certain period without being contacted by a medium comprising IL-2 as the only cytokine.

[0094] To maintain expanding cells healthy, the culture medium generally needs to be replaced with a fresh medium more often than non-expanding cells, and the culture generally needs to be expanded to a larger culture surface, as further described herein. In some embodiments, the expansion medium is replaced with fresh expansion medium every 2-3 days. The amount of expansion medium and the frequency of medium change (replacement) depend on the scale of the culture (e.g., the number of the T cells), rate of proliferation, and / or culture condition (e.g., using a G-REX® gas permeable membrane bioreactor or a conventional bioreactor). One of skills in the art can determine the optimal amount of expansion medium and / or the frequency of medium change based on the cell health and / or desired outcome. For example, cells can be expanded on day 1 and periodically thereafter (such as every 2-3 days thereafter) until day 10, 12, or 14. For example, on day 1, T cells can be expanded, with the goal of keeping T cells at 1 to 2 million per ml of medium, or in some cases 0.5 million per ml. To avoid splitting T cells too quickly, if a culture is more than 2 million per ml, the medium volume can be doubled with fresh medium and cells, and the culture can be moved to a bigger vessel, or split between 2 wells. On days 3, 5, 6, 8, 10, 12, and / or 12, T cells can be similarly expanded. The phenotype of the T cells (e.g., expression of CD3, CD70, and / or CAR) can be monitored using flow cytometry. Most T cell cultures can expand for 10 to 14 days after being activated, then the need to expand cultures can progressively decline for several days till the cells stop proliferating. At this point T cells would need to either be restimulated or frozen (or cryopreserved).

[0095] To freeze or cryopreserve cells, cells can be counted and aliquoted appropriately, e.g., at 10 million cells per ml, e.g., in 1 ml or 0.5 ml aliquots, and frozen to -80°C in an appropriate medium (e.g., CS10 medium), and stored in liquid nitrogen (i.e., at -196°C). The cells can be frozen and cryopreserved when a desired cell number or concentration is achieved. The cells can be frozen and cryopreserved at any time point when or before they stop proliferating (or expanding), for example 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, or 14 days after activation of the cells and / or initiation of the cell culture.

[0096] 2. Thawing Cells

[0097] The cells for use in the methods provided herein (e.g., mixture of cells including the T cell population; T cells; CAR-T cells; engineered T cells) for use in the methods provided herein can be thawed from a frozen stock prior to being contacted with the activating reagent. In some embodiments, the methods provided herein comprise, thawing the cells comprising T cell population that have been frozen prior to contacting the T cell population with the activating reagent. The cells that have been frozen can be previously isolated leukocytes, e.g., primary human leukocytes, or previously isolated T cells, e.g., primary human T cells, that have been frozen in an appropriate medium (e.g., CS10 medium), and stored in liquid nitrogen (i.e., at -196°C), e.g., at 10 million cells per ml, e.g., in 1 ml or 0.5 ml aliquots. The cells can be those previously obtained (e.g., harvested by apheresis) from a subject, e.g., a cancer patient. To ensure highest viability of thawed cells, up to 2 vials can be thawed at a time. After thawing, the next step of the methods provided herein (e.g., culturing the thawed cells in the expansion medium, or selecting one or more CD4+and / or CD8+T cells) can be performed within one day. Selecting CD4+and / or CD8+T cells The methods provided herein can comprise selecting from cells (e.g., mixture of cells obtained from a subject, e.g., by apheresis, including the T cell population) one or more CD4+and / or CD8+T cells. Any selection method can be used. For example, CD4+and / or CD8+T cells can be selected by contacting the cells with magnetic beads coupled with an anti- CD4+antibody and an anti-CD8+antibody, and selecting CD4+and / or CD8+T cells that are bound to the magnetic beads. After the selection, the next step of the methods provided herein (e.g., activating the selected cells, or culturing the selected cells in the expansion medium) can be performed within one day. Activating T Cell Population

[0098] The methods provided herein can comprise contacting the T cell population with an activating regent to activate the T cell population. For T cell activation to be initiated, two signals are required: TCR recognition of MHC class II peptide and a simultaneous costimulatory signal delivered by the same APC. If both signals are received, the T cell goes into the Gi phase of the cell cycle and begins to produce IL-2. The skilled artisan recognizes that T cells utilize co-stimulatory signals that are antigen non-specific to become fully activated. In particular cases they are provided by the interaction between co-stimulatory molecules expressed on the membrane of APC and the T cell. Exemplary costimulatory molecules include one or more of the following in any combination: CD3; CD28; CTLA-4; ICOS; B7-H2; PD-1; B7-H3; PD-L2; B7-H4; PDCD6; BTLA; ; Integrin alpha 4 beta 1; CD96; LAG-3; CD160; CRTAM; TCL1A; DAP12; TIM-4; TSLP; EphB6; TSLP R; and HLA-DR. The effector domain is a signaling domain that transduces the event of receptor ligand binding to an intracellular signal that partially activates the T lymphocyte. Absent appropriate co-stimulatory signals, this event is insufficient for useful T cell activation and proliferation. A nonlimiting example of an effector domain of this disclosure is the effector domain of the T cell receptor zeta chain.

[0099] The T cells (that express CD3 and CD28) can be activated via co-stimulation of CD3 and CD28, for example by using CD3 / CD28 activation reagent, e.g., a CD3 / CD28 crosslinking antibody, MACS® GMP T Cell TRANSACT™ CD3 / CD28 activation reagent (Miltenyi Biotec) or IMMUNOCULT™ CD3 / CD28 activation reagent (Stemcell). Without wishing to be bound by theory, a CD3 / CD28 activation reagent can contain CD3 and CD28 agonists and crosslinking molecules (such as colloidal polymeric matrix) and, upon addition to the cells, can crosslinks CD3 and CD28 on the T cell surface to simulate the T cell. A CD3 / CD28 activation agent can be added at about 1-10% v / v, such as 1-2%, 2-3%, 3-4%, 4-5%, 5-6%, 6-7%, 7-8%, 8-9%, or 9-10% v / v to a T cell culture to activate the T cell population. For example, a CD3 / CD28 activation agent can be added at about 1.8% v / v to a-large scale T cell culture, or at about 5.4% v / v to a non-large scale T cell culture. Cells can be left for up to 3 days before needing to split, add new medium, and / or add IL-2.

[0100] According to the methods provided herein, 2-3 days after administration of the activation reagent to the T cell population, reduction in cell number, due to activation induced cell death (AICD) can be observed. Subsequently, 3-4 days after administration of the activation reagent to the T cell population, the cells start proliferating. This can be noticeable as the culture medium begin to get more exhausted and turn yellow.

[0101] The T cell population can be thawed, selected for CD4 and / or CD8 expression, and contacted with the activating reagent on the same day. The selection of the cells can be immediately prior, 1-5 minutes prior, 5-10 minutes prior, 10-30 minutes prior, 30-60 minutes prior, 1-2 hours prior, 2-3 hours prior, 3-6 hours prior, or 6-12 hours prior to contacting the thawed cells with the activating reagent. For example, T cell activation (by contacting the T cells with the activating reagents) can be performed immediately after (such as 1-30 minutes after) T cells are thawed, selected, and plated in an optimal density (e.g., 1 million per ml). In some embodiments, the concentration of the T cells are critical for achieving optimal activation results. In specific embodiments, cells to be activated are selected for CD4 and / or CD8 expression.

[0102] After the activation, the next step of the methods provided herein (e.g., introducing a CAR and / or TCR construct into the activated T cells for expression, or culturing the activated T cells) can be performed in about 1-2 days.

[0103] 3. Introducing CAR or TCR into T Cells for Expression

[0104] In some embodiments, a T cell population provided herein can be genetically engineered to produce T cell receptor (TCR) or a chimeric antigen receptor (CAR) that is exposed on the cell surface. For example, a polynucleotide encoding a CAR and / or a TCR into the T cell population such that one or more T cells of the population express the CAR and / or TCR. The CAR can be specific for cancer cells in the subject. CARs are proteins that avow the T cells to recognize a specific protein (antigen) on tumor cells (e.g., a solid tumor cell from a subject having cancer). For example, naive T cells can be transfected with and grown to express CARs such that T cells expressing CARs can target and kill tumors via tumor-associated antigens. The CAR of the disclosure may employ one, two, three, four, or more costimulatory molecules in any combination. The one or more costimulatory molecules in the chimeric receptor can come from the B7 / CD28 family, TNF superfamily (such as M83), or the signaling lymphocyte activation molecule (SLAM) family. Exemplary costimulatory molecules include one or more of the following in any combination: B7-1 / CD80; CD28; B7-2 / CD86; CTLA-4; B7-H1 / PD-L1; ICOS; B7-H2; PD-1; B7- H3; PD-L2; B7-H4; PDCD6; BTLA; 4-1BB / TNFRSF9 / CD137; CD40 Ligand / TNFSF5; 4-1BB Ligand / TNFSF9; GITR / TNFRSF18; BAFF / BLyS / TNFSF13B; GITR Ligand / TNFSF18; BAFF R / TNFRSF13C; HVEM / TNFRSF14; CD27 / TNFRSF7; LIGHT / TNFSF14; CD27 Ligand / TNFSF7; OX40 / TNFRSF4; CD30 / TNFRSF8; 0X40 Ligand / TNFSF4; CD30 Ligand / TNFSF8;

[0105] TAC / TNFRSF13B; CD40 / TNFRSF5; 2B4 / CD244 / SLAMF4; CD84 / SLAMF5; BLAME / SLAMF8; CD229 / SLAMF3; CD2 CRACC / SLAMF7; CD2F-10 / SLAMF9; NTB-A / SLAMF6;

[0106] CD48 / SLAMF2; SLAM / CD150; CD58 / LFA-3; CD2; Ikaros; CD53; Integrin alpha 4 / CD49d; CD82 / Kai-1; Integrin alpha 4 beta 1; CD90 / Thyl; Integrin alpha 4 beta 7 / LPAM-l; CD96; LAG-3; CD160; LMIR1 / CD300A; CRTAM; TCL1A; DAP12; TIM- 1 / KIM- 1 / HA VCR; Dectin- 1 / CLEC7 A; TIM-4; DPPIV / CD26; TSLP; EphB6; TSLP R; and HLA-DR.

[0107] In some embodiments, the CAR is specific for an antigen expressed on a cancer cell. The antigen according to the present disclosure can be any antigen expressed (e.g., overexpressed, specifically expressed) on cancer cells that is now known or later identified, including for examples, antigens listed in the following references: Novellino et al. 2005 Cancer Immunology, Immunotherapy 54(3): 187-207; Vigneron et al. 2013 Cancer Immunity 13:15; Finn. 2017 Cancer Immunol Res 5(5):347-354; and the database maintained at cancerresearch.org / scientists / meetings- and-resources / peptide-database, the entire contents of each of which are incorporated by reference herein.

[0108] In some embodiments of the present disclosure, the overexpression of a target cancer antigen by cancer cells allows these cells to be targeted in vitro and in vivo by CAR-expressing primary T cells, wherein the CAR is specific for the target cancer antigen. In some embodiments, incorporation of endodomains from both CD28 and 0X40 molecules mediates costimulation of the T lymphocytes, inducing T cell activation, proliferation, and cytotoxicity against target antigenpositive cancer and / or cancer initiating cells (CICs).

[0109] In particular embodiments of the disclosure, there are methods for killing cancer cells using genetically manipulated T-cells that express a chimeric antigen receptor (CAR) directed against a target cancer antigen. In some embodiments, engagement (antigen binding) of this CAR leads to activation of the linked T-cell receptor C chain and the costimulatory molecules CD28 and 0X40.

[0110] In particular embodiments of the disclosure, the CAR receptor comprises a single-chain variable fragment (scFv) that recognizes the target cancer antigen. The skilled artisan recognizes that scFv is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, connected with a short linker peptide of ten to about 25 amino acids. The linker may be rich in glycine for flexibility and / or it may have serine or threonine for solubility, in certain cases. The scFv may be generated by methods known in the art. For instance, a FMC63 scFv-based CAR followed by 4-1BB and CD3 intracellular domain, or a GPC3 scFv-based CAR followed by 4- IBB and CD3 intracellular domain can be constructed into a lentiviral vector for transducing the T cell population.

[0111] In certain aspects, one can use cytokine exodomains or other ligand / receptor molecules as exodomains to provide targeting to the tumor cells.

[0112] In some embodiments, the cancer cell is a cell of B cell lymphoma, T cell lymphoma, myeloma, leukemia, hematopoietic neoplasia, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, uterine cancer, cervical cancer, endometrial cancer, adenocarcinoma, breast cancer, pancreatic cancer, colon cancer, anal cancer, renal cancer, bladder cancer, prostate cancer, ovarian cancer, primary or metastatic melanoma, squamous cell carcinoma, basal cell carcinoma, brain cancer, angiosarcoma, hemangiosarcoma, head and neck carcinoma, thyroid carcinoma, soft tissue sarcoma, bone sarcoma, testicular cancer, gastrointestinal cancer, stomach cancer, glioblastoma, small cell lung cancer, non-small cell lung cancer, or any combination thereof, as well as any other cancer or malignant neoplasm now known or later identified (see, e.g., Rosenberg 1996 Ann. Rev. Med. 47:481-491, the entire contents of which are incorporated by reference herein).

[0113] Appropriate means for preparing a transduced population of lymphocytes expressing a selected CAR construct is well known to a skilled artisan, and includes a viral mediated CAR gene delivery system, such as retrovirus or lentivirus, and a transposon / transposase systems that include a non-viral mediated CAR gene delivery system, such as Sleeping Beauty (SB11), piggyBac, and Tol2. As one nonlimiting example, these cells can be transduced with a viral vector or transfected with a nonviral vector or nucleic acid construct that contains a nucleotide sequence encoding a CAR under conditions whereby the nucleotide sequence is expressed in the T cell and the CAR produced in the cell is transported to the cell surface. Transduced cytotoxic lymphocytes can be grown in T cell supportive polarizing conditions described herein that are suitable for a population of cells that will be introduced into a subject such as a human.

[0114] In specific embodiments, the methods comprise contacting the activated T cell population with a viral vector comprising a CAR or TCR construct to transduce the T cell population and produce engineered T cells. The viral vector can be a lentiviral vector. To achieve efficient transduction with a viral vector (such as a lentiviral vector), T cells can be activated in advance of the transduction. Conducting viral (such as lentiviral) transduction 1 to 2 days post T cell activation can generally achieve efficient transduction. Clinical-grade or GMP-compliant viral particles containing a CAR or a TCR can be prepared for example by using commercially available services. T cells can be passively transduced with virus (such as lentivirus) by first removing a volume of medium and then adding the equal volume of virus on cells. Cells can also be transduced by using g-force from spinning to put the viral particles in closer contact with the nucleus. After viral (e.g., lentiviral) transduction, medium can be completely changed out by spinning down cells, aspirating supernatant, and resuspending T cells in fresh complete medium (e.g., expansion medium) at an optimal concentration. Cells can start proliferating almost immediately thereafter and can be needed to be expanded every day or two for the next several days. For example, if 1 million cells are in 1 ml medium in a 48 well plate, by the next day they generally need to be transferred to a 24 well plate and diluted with an extra ml of medium or split into 2 wells of a 48 well plate. The cells can remain at the expansion stage for up to about 14 days (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days).

[0115] After introducing CAR or TCR into the T cell population, the next step of the methods provided herein (e.g., contacting the T cells with gene editing reagents) can be performed in about 1-2 days. Additionally or alternatively, after introducing CAR or TCR into the T cell population, culturing of the T cells with the expansion medium can be started immediately or within a day.

[0116] 4. Gene Editing

[0117] The methods provided herein can comprise contacting the T cell population (e.g., including CAR-T cells or engineered T cells) with one or more gene editing reagents to introduce a mutation in a target site in a genome of the T cells. A “gene editing reagent” as used herein refers to a set of one or more molecules or a construct comprising or encoding the one or more molecules for introducing one or more mutations in the genome. An exemplary gene editing system or editing reagents comprise a nuclease and / or a guide RNA. Also provided herein is a construct (e.g., a DNA construct, a recombinant DNA construct) for introducing one or more mutations in T cells. A construct can comprise an editing system or polynucleotides encoding editing reagents (e.g., nuclease, guide RNA, base editor) each operably linked to a promoter.

[0118] As used herein, the terms “nuclease” and “endonuclease” are used interchangeably to refer to naturally-occurring or engineered enzymes, which cleave a phosphodiester bond within a polynucleotide chain. The cleavage could be a single strand cleavage or a double strand cleavage. In certain embodiments, the nuclease lacks cleavage activity and is referred to as nuclease dead. Nucleases that can be used in precise genome-editing technologies to modulate the expression of the native sequence include, but are not limited to, meganucleases designed against the plant genomic sequence of interest (D’Halluin et al 2013 Plant Biotechnol J 11 : 933-941); Cas9 endonuclease; Cast 2a (Cpfl) endonuclease; ortholog of Cas 12a endonuclease; Cmsl endonuclease; transcription activator-like effector nucleases (TALENs); zinc finger nucleases (ZFNs); and a deactivated CRISPR nuclease (e.g., a deactivated Cas9, Casl2a, or Cmsl endonuclease) fused to a transcriptional regulatory element (Piatek el al. 2015 Plant Biotechnol J 13:578-589). In some embodiments, the editing system or the editing reagents comprise a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), and / or a clustered regularly interspaced short palindromic repeats (CRISPR) nuclease. In some embodiments, the editing reagents comprise a CRISPR nuclease. In some embodiments, the CRISPR nuclease is a Casl2a nuclease, herein used interchangeably with a Cpfl nuclease, e.g., a McCpfl nuclease. In some embodiments, the CRISPR nuclease is a Casl2a nuclease ortholog, e.g., Lb5Casl2a, CMaCasl2a, BsCasl2a, BoCasl2a, MlCasl2a, Mb2Casl2a, TsCasl2a, and MAD7 endonucleases.

[0119] A nuclease system can introduce insertion, substitution, or deletion of genetic elements at a predefined genomic locus by causing a double-strand break at said predefined genomic locus and, optionally, providing an appropriate DNA template for insertion.

[0120] The editing system, editing reagents, or construct described herein can comprise one or more guide RNAs (gRNAs), or gRNA cassette, to drive mutations at the target locus of the T cell genome. “Guide RNA” as used herein refers to a RNA molecule that function as guides for RNA- or DNA-targeting enzymes, e.g., nucleases. In some instances, a gRNA can comprise a targeting region (i.e., spacer) that is complementary to a targeted sequence as well as another region that allows the gRNA to form a complex with a nuclease (e.g., a CRISPR nuclease) of interest. For example, a CRISPR system comprises a CRISPR nuclease (e.g., CRISPR-associated (Cas) endonuclease or variant or ortholog thereof, such as Cas 12a or Cas 12a ortholog) and a guide RNA. A CRISPR nuclease associates with a guide RNA that directs nucleic acid cleavage by the associated endonuclease by hybridizing to a recognition site in a polynucleotide. The guide RNA directs the nuclease to the target site and the endonuclease cleaves DNA at the target site. The guide RNA comprises a direct repeat and a guide sequence, which is complementary to the target recognition site. In certain embodiments, the CRISPR system further comprises a tracrRNA (transactivating CRISPR RNA) that is complementary (fully or partially) to the direct repeat sequence present on the guide RNA. The CRISPR-Casl2a system may comprise at least one guide RNA (gRNA) operatively arranged with the ortholog endonuclease for genomic editing of a target DNA binding the gRNA. The system may comprise a CRISPR-Casl2a expression system encoding the Cas 12a ortholog nucleases and crRNAs (CRISPR RNAs) for forming gRNAs that are coactive with the Casl2a nucleases. A “TALEN” nuclease is an endonuclease comprising a DNA-binding domain comprising a plurality of TAL domain repeats fused to a nuclease domain or an active portion thereof from an endonuclease or exonuclease, including but not limited to a restriction endonuclease, homing endonuclease, and yeast HO endonuclease. A “zinc finger nuclease” or “ZFN” refers to a chimeric protein comprising a zinc finger DNA-binding domain fused to a nuclease domain from an endonuclease or exonuclease, including but not limited to a restriction endonuclease, homing endonuclease, and yeast HO endonuclease.

[0121] In some embodiments, the one or more gene editing reagents comprise a CRISPR / Cas endonuclease (e.g., a Cas9, Casl2a, or Cmsl endonuclease, or an ortholog thereof). T cells can be contacted with one or more gene editing reagents after (such as 1-3 days after) being activated or after (such as 1-2 days after) a CAR and / or TCR construct is introduced into the cells (e.g., by a viral vector, e.g., a lentiviral vector). Gene editing reagents can be introduced into the T cells in any means known in the art, including electroporation and transfection. In specific embodiments, gene editing reagents are introduced into the T cells by electroporation.

[0122] In specific embodiments, the one or more gene editing reagents introduce a loss-of-function mutation into a CD70 gene in the genome of the CAR-T cells. CD70 is the membrane-bound ligand of the CD27 receptor, and belongs to the tumor necrosis factor receptor superfamily. CD70 is expressed by diffuse large B-cell and follicular lymphoma and also by the malignant cells of Hodgkin lymphoma, Waldenstrom macroglobulinemia, and multiple myeloma, and by human T- lymphotropic virus type 1- and EBV-associated malignancies. In addition, CD70 is expressed by nonhematologic malignancies such as renal cell carcinoma and glioblastoma. Physiologically, CD70 expression is transient and restricted to a subset of highly activated T, B, and dendritic cells. Without wishing to be bound by theory, CAR-T cells having knockout of the CD70 show improved properties including potency and persistence over CAR T cells where the CD70 gene remains intact. CAR-T cells with CD70 KO showed resistance to exhaustion upon repeated exhaustion in culture, as well as a reduction in apoptosis, increased proliferation, and improved target cell lysis upon sequential rechallenges. In comparing CTX130, an investigational allogeneic CAR-T therapy in patients with CD70-expressing tumors, e.g., clear cell renal cell carcinoma and B and T cell malignancies, with and without CD70 KOs, CAR-T cells with CD70 KO showed increased potency (e.g., cytotoxic activity, proliferation), enhanced ability to withstand multiple tumor challenges in vivo, and increased resistance to overexpression of PD-L1 on target cells. KO of CD70 conferred benefit to CAR-T cells that far exceeded KO of other checkpoint related genes (e.g., PD1, TIM3, LAG3, TIGIT), and this benefit was present regardless of the antigen being targeted (Dequeant et al. 2021 Cancer Res 81 (13_Supplement): 1537).

[0123] In specific embodiments, 6.5 pg of CRISPR Cas9 ribonucleoproteins per million cells are electroporated into the T cells to generate a mutation that cause loss of function of CD70. Five (5) million electroporated cells can be moved to one well of a 6 well Grex plate (G-Rex® gas permeable membrane 6 Well Plate - P / N 80240M) containing 40 ml culture medium.

[0124] After contacting the T cell population with one or more gene editing reagents, the next step of the methods provided herein (e.g., culturing of the T cells with the expansion medium) can be started immediately or within a day.

[0125] III. T Cells of the Present Disclosure

[0126] In some embodiments, the T cell population comprise primary human T cells. The T cell population can be selected from cells (e.g., white blood cells) obtained, e.g., by apheresis, from a subject (e.g., a human subject). Selection of T cells can be based on CD4 and / or CD8 positivity. In some embodiments, the T cell population is CD3 positive. CD3 is a multimeric protein complex, known as the T3 complex, and is composed of four distinct polypeptide chains; epsilon (a), gamma (y), delta (5) and zeta (Q, that assemble and function as three pairs of dimers (ay, aS, ( ). The CD3 complex serves as a T cell co-receptor that associates noncovalently with the T cell receptor (TCR) (Smith-Garvin et al. 2009 Ann Rev Immunol 27, 591-619). The CD3 protein complex is a defining feature of the T cell lineage, and is involved in activating both the cytotoxic T cell (CD8+naive T cells) and T helper cells ( CD4+naive T cells). In some embodiments, the T cell population is CD28 positive. CD28 is the receptor for CD80 (B7.1) and CD86 (B7.2) proteins. CD28 provides costimulatory signals required for T cell activation and survival.

[0127] In some embodiments, the culture and expansion methods provided herein produce IL-9- secreting (T9) T cells (e.g., CAR-T cells, engineered T cells). Without wishing to be bound by theory, antigen-specific IL-9-secreting T cells, i.e., CD4+Th9 or CD8+Tc9 cells, are distinct subsets with stronger antitumor efficacy in murine tumor models compared to Thl, Thl7, or Tcl / CTLs. IL-9-secreting T cells (T9 cells) express different cytokine profiles and low cytolytic proteins and exhaustion markers compared to other subsets of T cells, and remain as hyperproliferative T cells and exhibit a greater antitumor activity than other subset of T cells against hematologic and solid tumors in vivo. The CD4+Th9 or CD8+Tc9 cells of the present disclosure can be produced from tumor-infiltrating and / or tumor-draining lymph node T cells.

[0128] In some embodiments, the T cell population provided herein comprise a plurality of CD4+Th9 cells (e.g., a population of CD4+Th9 cells). A CD4+Th9 cell provided herein may have specificity for a cancer cell and / or may be primed (e.g., with a cancer antigen-loaded APC) to have specificity for a cancer cell (e.g., a cancer cell in a subject for which the primed CD4+Th9 cell is to be administered). The CD4+Th9 cell may be programmed as and / or exhibit the phenotype for a mature effector T cell. In some embodiments, the CD4+Th9 cell may exhibit and / or maintain a mature effector cell signature with cytolytic activity as strong as Thl cells and / or that may persist as long as Th 17 cells in vivo. In some embodiments, the CD4+Th9 cell may exhibit cytotoxicity that is at least about 60% or more (e.g., 60%-140% or more) of the cytotoxicity of Thl cells and / or that is greater than about 100% of the cytotoxicity Thl7 cells. Cytolytic activity may be measured using an in vitro and / or in vivo cytolytic assay known to those of skill in the art. In some embodiments, the CD4+Th9 cell may exhibit an expression level of Id2, Eomes, Id3, 112, and / or a granzyme (e.g., Gzma, Gzmb, Gzmd, Gzme, Gzmk, Gzmg, and / or Gzmn) that is similar (e.g., within 10%) and / or increased compared to the expression level of the same gene in a Thl and / or Thl7 cell. The CD4+Th9 cell may be put under conditions to express Id2, Eomes, Id3, 112, and / or a granzyme (e.g., Gzma, Gzmb, Gzmd, Gzme, Gzmk, Gzmg, and / or Gzmn), optionally at an increased level compared to the expression level of the same gene in a Thl and / or Thl7 cell, and / or may be put under conditions to overexpress Id2, Eomes, Id3, 112, and / or a granzyme (e.g., Gzma, Gzmb, Gzmd, Gzme, Gzmk, Gzmg, and / or Gzmn). In some embodiments, the CD4+Th9 cell may exhibit an expression level of Id2, Eomes, Id3, 112, and / or a granzyme (e.g., Gzma, Gzmb, Gzmd, Gzme, Gzmk, Gzmg, and / or Gzmn) that is increased by about 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to the expression level of the same gene in a Thl and / or Thl7 cell. In some embodiments, the CD4+Th9 cell may exhibit an expression level of Id2, Eomes, Id3, 112, and / or a granzyme (e.g., Gzma, Gzmb, Gzmd, Gzme, Gzmk, Gzmg, and / or Gzmn) that is increased by at least 30% or more compared to the expression level of the same gene in a Thl and / or Thl7 cell. In some embodiments, the CD4+Th9 cell may exhibit an increased expression level (e.g., by at least 30%) of Eomes compared to the expression level of Eomes in a Thl and / or Thl7 cell. The CD4+Th9 cell may not carry and / or may not exhibit the molecular signature of a T cell exhaustion phenotype (such as in a Thl cell).

[0129] The CD4+Th9 cell may express and / or be capable of expressing a hyperproliferative phenotype. “Hyperproliferative” and grammatical variations thereof as used herein in reference to a T cell (e.g., a Th9, Thl, or Thl7 cell) refers to the cell expressing Ki67 and a plurality of the T cells (e.g., a Th9 cell population) in which greater than 50% of the cells in the plurality are Ki67+(with Ki67+meaning that the cell(s) expresses Ki67). In some embodiments, the CD4+Th9 cell or a plurality of the CD4+Th9 cells is hyperproliferative. In some embodiments, the percentage of Ki67+cells in the plurality of CD4+Th9 cells is increased compared to the percentage of Ki67+cells in a plurality of Thl and / or Thl7 cells (e.g., a population of similar or comparable size).

[0130] The CD4+Th9 cell, upon administration to a subject, may exhibit and / or exert an antitumor response in the subject and the antitumor response may be complete. In some embodiments, the CD4+Th9 cell, upon administration to a subject, reduces or completely eliminates a tumor such as a large established tumor, and there is no tumor relapse for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or more. In some embodiments, the CD4+Th9 cell administered to the subject is and / or upon administration becomes a Ki67+cell. In some embodiments, a plurality of the CD4+Th9 cells comprises at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more CD4+Th9 cells that are Ki67+cells and / or upon administration become Ki67+cells.

[0131] T cells and / or CAR-T cells produced (e.g., cultured, expanded) according to the present disclosure can be used to treat tumor, such as solid tumor. In specific embodiments, an anti-CD70 CAR is introduced and / or CD70 is knocked out (e.g., by gene editing) in the T cells of the present disclosure, and the T cells are expanded using the OTX medium (OUTAST medium). Such engineered T cells (e.g., engineered CAR-T cells) can be used to treat tumor, e.g., renal cell carcinoma. Without wishing to be bound by theory, OTX conditioning can reprogram T cells during ex vivo expansion for superior activity against solid tumors as compared to conventional culturing process. For example, OTX-conditioned T cells can show: upregulation of genes associated with an early memory T cell phenotype and downregulation of genes associated with effector phenotype or T cell exhaustion; improved metabolic fitness during prolonged stimulation; natural resistance to TGFp mediated suppression; enhanced activation responses to low antigen densities; and increased persistence in vivo that allows for tumor control in concert with reduced exhaustion. OTX conditioning is easy to implement into existing processes (e.g., cell based therapies) and is applicable to a wide range of cell therapy (e.g., T cell-based cell therapy) modalities.

[0132] As one nonlimiting example, effector T cells or memory T cells are prepared as follows: Naive T cells, tumor-infiltrating T cells, and / or T cells isolated from peripheral blood mononuclear cells (PBMCs) are cultured with interleukin-2 (IL-2), interleukin-7 (IL-7), and / or interleukin- 15 (IL- 15), in any combination.

[0133] In some embodiments, the T cell population provided herein comprise a plurality of CD8+Tc9 cells (e.g., a population of CD8+Tc9 cells). Differentiation of CD8+T cells under T helper 9- polarizing conditions can induce the development of an IL-9 producing CD8+T (Tc9) cell subset which elicits a greater antitumor response against large established tumors than classic type-1 CD8+cytotoxic T cells that are presently used in clinical protocols. Methods of making IL-9 secreting CD8+Tc9 cells are known in the art, including for example, as described in U.S. Pat. No. 9,694,033, the entire contents of which are incorporated by reference herein. In an exemplary embodiment of producing a population of CD8+Tc9 cells, Tc9 cells are produced by priming naive CD8+CD62L+T cells by contacting the naive cells with irradiated peptide-loaded splenic APCs in the presence of Tc9-polarized medium including IL-4 (10 ng / ml; R&D Systems), TGFP (1 ng / ml; R&D Systems), anti-IFN-y monoclonal antibodies (mAbs; 20 pg / ml; eBioscience) and anti-IL-12 mAbs (10 pg / ml; eBioscience). Beginning 2 days after priming, cell culture are expanded in fresh Tc9-polarized medium supplemented with 50 ng / ml IL-2 (50 ng / ml; R&D Systems) for additional 3 days before further use such as administration to a subject in need thereof for the treatment of cancer.

[0134] In some embodiments, the T cells provided herein can be primed with cancer antigen- loaded APCs to have specificity for cancer cells in the subject. Priming, or stimulation, of the T cells is performed in the presence of a T9 supportive environment, which results in the production a population of CD8+Tc9 or CD4+Th9 lymphocytes which secrete IL-9. For example, T cells can be primed as follows: peripheral blood mononuclear cells (PBMCs), naive T cells, unselected T cells and / or tumor-infiltrating T cells are contacted with an immunogenic peptide and / or loaded APCs, coated or soluble anti-CD3 / anti-CD28 mAbs, or anti-CD3 / anti-CD28 conjugated beads to prime the T cells. T cells are also primed in the presence of any Th9 polarization conditions. Examples of polarization conditions include one or more of the following agents in any combination: IL-2, IL-4, TGFP family cytokines, IL-ip, GITRL, OX40L, anti-GITR agonist mAbs, anti-OX40 agonist mAbs, TNF-a, IL-6, IL-7, IL-15 and / or anti-IFN-y monoclonal antibodies.

[0135] In one embodiment, a T9 supportive environment can include a standard T cell culture growth medium such as RPML1640 or AIM-V with the addition of an effective amount of T9 polarizing cytokines and agents. CD8+cytotoxic (Tc) cells or CD4+helper (Th) cells can differentiate into multiple effector subsets (e.g., Tel, Tc2, Thl, Th2) capable of secreting different cytokine patterns. In accordance with the present disclosure, T9 polarizing cytokines and agents are those cytokines and agents capable of differentiating or “polarizing” T cells into a specific Tc or Th effector subset with the specific phenotype of IL-9 secretion. c9 polarizing cytokines for use in the present disclosure include but are not limited to IL-4 and TGFp. T9 polarizing agents can include neutralizing agents such as INF-y and IL- 12 neutralizing agents.

[0136] T9 supportive environment can include about 1 to about 100 lU / ml of IL-4. In a certain embodiment the T9 supportive environment can include about 10 lU / ml of IL-4. A T9 supportive environment can include about 0.1 to about 10 lU / ml of TGFp. In a certain embodiment the T9 supportive environment can include about 1 lU / ml of TGFp.

[0137] T9 supportive environment can include a neutralizing amount of an INF-y neutralizing agent and / or a neutralizing amount of an IL- 12 neutralizing agent. A neutralizing amount is an amount of an agent sufficient to decrease the activity or amount of a substance to a level that is undetectable using standard methods. For example, a neutralizing amount of an IL- 12 or INF-y neutralizing agent is the amount of agent which decreases the biological activity of IL-12 or INF-y, for example to an IL- 12 or INF-y activity level below that which can be detected using a standard immunoassay. Such agents can thus be used to inhibit IL-12 or INF-y activity. Examples of such agents, include, but are not limited to anti-IL-12 or anti-INF-y antibodies and soluble IL- 12 or INF- y receptors.

[0138] In some embodiments a T9 supportive environment can include about 1 pg / ml to about 100 pg / ml of anti-IL-12 monoclonal antibodies. In certain embodiments, the IL- 12 neutralizing agent includes about 10 pg / ml of anti-IL-12 monoclonal antibodies. In some embodiments a T9 supportive environment can include about 1 pg / ml to about 100 pg / ml of anti-INF-y monoclonal antibodies. In one embodiment, the INF-y neutralizing agent includes about 20 pg / ml of anti-INF-y monoclonal antibodies.

[0139] In some embodiments, T cell populations described herein are incubated in the T9 supportive environment with the appropriate immunogenic peptide-loaded APCs for a time period sufficient to prime the naive cells. Preferably, however, the responder (T9) to stimulator (APC) ratio is in the range of about 10: 1 to 100:1. The T9 / APC culture may be maintained for as long a time as is necessary to stimulate a therapeutically useable or effective number of T9 cells.

[0140] In some embodiments, primed T9 cells may be effectively separated from the APC using one of a variety of known methods. For example, monoclonal antibodies specific for the APCs, for the peptides loaded onto the stimulator cells, or for the T9 (or a segment thereof) may be utilized to bind their appropriate complementary ligand. Antibody-tagged cells may then be extracted from the admixture via appropriate means, e.g., via well-known immunoprecipitation or immunoassay methods.

[0141] In some embodiments, the primed T9 cells are further allowed to proliferate in a second fresh T9 supportive environment further including IL-2. For example, the expansion step can include adding IL-2 to the T9 supportive culture medium (e.g., wherein the concentration of IL-2 is about 1 lU / ml to about 100 lU / ml or preferably about 50 lU / ml). In an exemplary embodiment, the primed population of T9 cells are allowed to proliferate in a second T9 supportive environment for about 3 days prior to therapeutic use.

[0142] Cells to be employed in the methods and compositions of this disclosure can be obtained from a subject (e.g., a human subject). In some embodiments, the cells can be from the same subject to whom the treatmen t(s) will be administered (i.e., the cells are autologous cells). Accordingly, in some embodiments, the T9 cells administered to the subject are derived from autologous naive CD8+or CD4+T cells obtained from the subject. For example, a blood sample can be obtained from a subject and naive CD8+or CD4+T cells isolated from the sample. The naive CD8+T cells can be contacting the population of naive CD8+T cells with an immunogenic Tumor derived peptide, in the presence of a T9 supportive environment to produce T9 IL-9 secreting cells and then administered to the same subject. In other embodiments, the cells can be from a subject that is not the same subject to whom the treatment s) will be administered (e.g., allogeneic cells). T9 cells administered to the subject can be derived from naive CD8+or CD4+T cells obtained from a donor (e.g., either a matched sibling donor or an HLA-mismatched donor that is identified either through a registry or from a non-matched family donor, such as an haplo-identical donor — usually a parent or child of the subject)

[0143] In some embodiments naive T cells or unselected T cells can be isolated from a blood sample and / or spleen of a subject, such as a donor or recipient subject, using standard methods including, e.g., Ficoll density gradient centrifugation followed by negative selection to remove undesired cells. Methods of isolating naive T cells are known to those of skill in the art and include FACS sorting of cells. Naive T cells or unselected T cells can also be obtained from a subject using an apheresis procedure.

[0144] In some embodiments, a population of PBMC, naive T cells or unselected T cells is contacted with an immunogenic peptide, coated or soluble anti-CD3 / anti-CD28 monoclonal antibodies, or anti-CD3 / anti-CD28 conjugated beads in order to prime the T cells. An immunogenic peptide for use in the disclosure can be prepared synthetically, or by recombinant DNA technology or isolated from natural sources such as whole viruses or tumors. The T cells produced are typically specific for an antigen present on a tumor (e.g., a solid tumor). Therefore, in certain embodiments, the immunogenic peptide is isolated or derived from a tumor (e.g., a subject's cancerous solid tumor).

[0145] In some embodiments, the desired immunogenic peptide can be loaded into the binding pockets of MHC molecules on the surface of antigen presenting cells (APCs) using standard methods. In some embodiments, the APCs of this disclosure can be loaded with a total cell or membrane preparation from cancer cells instead of or in combination with a molecularly defined antigen preparation.

[0146] In an exemplary embodiment, the APCs are irradiated antigen presenting dendritic cells which become peptide-loaded antigen dendritic cells when loaded with a desired immunogenic peptide. Typically, the antigen presenting cells are irradiated so APCs will not proliferate in response to T cell produced cytokines or other cytokines added to the culture.

[0147] The cultures described herein can typically be incubated under conditions of temperature and the like that are suitable for the growth and differentiation of T lymphocytes. For the growth of human T lymphocytes, for example, the temperature will generally be at least about 25°C, and in some embodiments, at least about 30°, and in some embodiments, about 37°C. Purified T9 cells produced according to the methods provided herein are also provided. The term “purified” does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified cell population of T9 cells is one in which the percentage of T9 cells in a population of cells (e.g., in culture) is more pure than T9 cells in their natural environment, such as within a human subject. In particular examples, substantially purified populations of T9 cells refers to populations of T9 cells that are at least 50%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98% or 99% pure. In one embodiment, a substantially purified population of T9 cells is composed of at least about 70%, such as at least about 80%, such as at least about 90% T9 cells. That is, the population of T9 cells includes less than about 20%, such as at least about 10%, of other T lymphocytes such as Tel or Thl cells. The purity of a T9 population can be measured based on cell surface characteristics (e.g. as measured by fluorescence activated cell sorting) or by cytokine secretion profile (e.g. as measured by an ELISA assay), as compared to a control.

[0148] T9 cell populations produced as described herein can be further characterized by the cytokines secreted or expressed by a portion of the cells. In certain embodiments, Tc9 cell populations produced as described herein can secrete IL-4, IL-10 and IL-17. Tumor exposure can enhance secretion of IL-9 and / or TFN-y by Th9 or Tc9 cells provided herein. Further, Th9 CAR-T cells can secrete lower amounts of IL-2 and TNF-a than Thl CAR-T cells, and Tc9 CAR-T cells can secrete significantly higher levels of IL-2 and TNF-a than Tel CAR-T cells, especially after coculture with tumor cells.

[0149] T9 cell populations of the present disclosure can display less exhausted phenotype compared to a T1 cell population. For example, in some embodiments, greater than about 40% of cells in a CD8+Tc9 population can secrete IL-7Ra, less than about 10% can be PD-1+and / or KLRG1+, less than about 25% are LAG-3+, and less than 15% of the population can be 2B4+.

[0150] The secretion of cytokines can be measured using standard bioassays, such as an ELISA. For example, fluorescence activated cell sorting can be utilized. Alternatively the supernatant content is tested for secretion of cytokines. In one embodiment, an assay, such as a bioassay, and ELISA, or a radioimmunoassay, is performed to test the cytokine secretion profile of the cells.

[0151] The methods disclosed herein can further include cryo-preserving the generated T9 lymphocytes.

[0152] In some aspects, the present disclosure provides T cells produced by the methods provided herein. The cells provided herein can be used for the treatment of any type of cancer in a subject. As used herein, the term “cancer” includes any type of cancer. A “cancer” in a subject refers to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Often, cancer cells will be in the form of a tumor, but such cells may exist alone within a subject, or may be a non-tumorigenic cancer cell, such as a leukemia cell. In some circumstances, cancer cells will be in the form of a tumor; such cells may exist locally within an subject, or circulate in the blood stream as independent cells, for example, leukemic cells. In one embodiment, the cancer may be tumorigenic cancer, i.e. a cancer associated with a tumor, or a skin lesion such as in melanoma. In addition, populations of the cells produced by the methods provided herein can be cryopreserved and / or thawed prior to clinical or experimental use.

[0153] In certain embodiments, prior to treatment, the patients are selected for having a particular cancer, or for being at risk of a particular cancer. The presence of cancer can be determined by means well known to clinicians. Initial assessment of cancer is based on symptoms presented by the patient. In addition, there are follow-up diagnostic procedures, including, but not limited to PET scans, CAT scans, biopsies, and bio-marker assessments.

[0154] Examples of cancer include, but are not limited to, renal cell carcinoma (RCC), breast cancer, melanoma, adrenal gland cancer, biliary tract cancer, bladder cancer, brain or central nervous system cancer, bronchus cancer, blastoma, carcinoma, a chondrosarcoma, cancer of the oral cavity or pharynx, cervical cancer, colon cancer, colorectal cancer, esophageal cancer, gastrointestinal cancer, glioblastoma, hepatic carcinoma, hepatoma, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, non-small cell lung cancer, osteosarcoma, ovarian cancer, pancreas cancer, peripheral nervous system cancer, prostate cancer, sarcoma, salivary gland cancer, small bowel or appendix cancer, small-cell lung cancer, squamous cell cancer, stomach cancer, testis cancer, thyroid cancer, urinary bladder cancer, uterine or endometrial cancer, and vulval cancer.

[0155] In some embodiments the cancer treated by the T cells provided herein is a solid tumor. Examples of solid tumors, such as sarcomas and carcinomas, include, but are not limited to: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovioma, mesothelioma, Ewing’s tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms’ tumor, cervical cancer, testicular tumor, bladder carcinoma, and CNS tumors (such as a glioma, astrocytoma, medulloblastoma, craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma and retinoblastoma). In particular embodiments, the cancer is a carcinoma solid tumor. For example, the solid tumor can include a renal cell carcinoma, ovarian cancer, breast cancer, colon cancer (adenocarcinoma) or malignant melanoma.

[0156] In some embodiments, the cancer treated by the T cells provided herein is melanoma. The term “melanoma” as used herein includes all types of melanoma, including, for example, melanoma skin cancer, ocular melanoma, and mucosal melanoma.

[0157] Pharmaceutical compositions comprising the cells of the present disclosure are also provided. The compositions of the present disclosure can include adjuvants, other medicinal agents, pharmaceutical agents, carriers, diluents, immunostimulatory cytokines, and pharmaceutically acceptable carriers. For example, the adjuvant can be in the composition of this disclosure or the adjuvant can be in a separate composition comprising the suitable adjuvant and a pharmaceutically acceptable carrier. Active agents may be provided in lyophilized form in a sterile aseptic container or may be provided in a pharmaceutical formulation in combination with a pharmaceutically acceptable carrier, such as sterile pyrogen-free water or sterile pyrogen-free physiological saline solution.

[0158] “Pharmaceutically acceptable” as used herein means that the compound or composition is suitable for administration to a subject to achieve the treatments described herein, without unduly deleterious side effects in light of the severity of the disease and necessity of the treatment. The pharmaceutically acceptable carrier can be selected on the basis of the chosen route of administration and standard pharmaceutical practice for adoptive transfer protocols. Suitable carriers and their formulation are described, for example, in the book Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 1985).

[0159] In some embodiments, the cells are formulated by first harvesting them from their culture medium, and then washing and concentrating the cells in a medium and container system suitable for administration (a “pharmaceutically acceptable” carrier) in a treatment-effective amount. Suitable infusion medium can be any isotonic medium formulation, typically normal saline, Normosol R (Abbott) or Plasma-Lyte A (Baxter), but also 5% dextrose in water or Ringer's lactate can be utilized. The infusion medium can be supplemented with human serum albumen.

[0160] The active agents described above (e.g., a T9 cell) may be formulated for administration in a pharmaceutical carrier in accordance with known techniques. See, e.g., Remington, The Science And Practice of Pharmacy (latest edition). In the manufacture of a pharmaceutical formulation according to the disclosure, the active compound (including the physiologically acceptable salts thereof) is typically admixed with, inter alia, an acceptable carrier. The carrier must, of course, be acceptable in the sense of being compatible with any other ingredients in the formulation and must not be deleterious to the subject. The carrier may be a liquid and is preferably formulated with the compound as a unit-dose formulation which may contain from 0.01 or 0.5% to 95% or 99% by weight of the active compound. The carrier may be sterile or otherwise free from contaminants that would be undesirable to administer or deliver to a subject.

[0161] Formulations of the present disclosure suitable for parenteral administration comprise sterile aqueous and non-aqueous injection solutions of the active compound, which preparations are preferably isotonic with the blood of the intended subject. These preparations may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended subject.

[0162] The T9 cell compositions described herein and pharmaceutical compositions including the cell populations can be administered to a subject to treat cancer. As less-exhausted younger cells, the sufficient lineage plasticity of T9 cells allows these cells subsequently to differentiate into long- lasting fFN-y-producing Tl-like effector cells upon administration to a subject. Without being bound by theory, it is believed that IL-9 secreted by the T9 cells promotes the migration of T9 cells into tumor tissues enabling the cells to exert long-lasting antitumor therapeutic effect. Therefore, another aspect of the disclosure relates to a method of treating a cancer in a subject. The method includes administering to a subject a therapeutically effective amount of a population of T9 cells produced as disclosed herein, wherein the population of T9 cells secretes IL-9.

[0163] In certain embodiments, a subject in need thereof is administered tumor specific IL-9 producing T9 cells; e.g. cancer specific IL-9 producing cells that are primed using a desire immunogenic tumor derived peptide in the presence of a T9 supportive environment and expanded in vitro as described herein. The expanded T cells (e.g., T9 cells) can be administered to patient.

[0164] Pharmaceutical compositions of the application can be administered by any means that achieve their intended purpose by any medically appropriate procedure, including but not limited to, intravenous, intratumor, intraperitoneal and / or intra-arterial administration. In certain embodiments, administration is intravenous or intratumoral. Cells or active agents may be administered systemically, or locally at the site of the cancer.

[0165] Dosage of the agents and compositions of this disclosure for the methods of use described herein will depend, among other things, on the condition of the subject, the particular disorder being treated, the route of administration, the nature of the therapeutic agent employed, and the sensitivity of the subject to the particular agent(s).

[0166] When administered to a human subject, the cells are generally in a volume of a liter or less, can be 500 ml or less, 250 ml or less, or 100 ml or less. The density of the cells is typically about 1 * 106to about 1 * 1012cells / ml, e.g., about 1 x 106to about 1 x 107cells / ml, about 1 x 106to about I x lO8cells / ml, about I x lO6to about I x lO9cells / ml, about I x lO6to about I x lO10cells / ml, about 1 * 106to about 1 * 1011cells / ml, about 1 x 107to about 1 x 1012cells / ml, about 1 x 107to about 1 x 1011cells / ml, about I x lO7to about I x lO10cells / ml, about I x lO7to about I x lO9cells / ml, or about I x lO7to about I x lO8cells / ml. In specific embodiments, the density of the cells is about 1 x 106to about 2x l06cells / ml. About 1 x io8to about 1 x io11T9 cells, e.g., about I x lO8to about I x lO11cells, about 1 x 108to about I x lO10cells, about 1 x 108to about I x lO9cells, about 1 x 109to about I x lO11cells, about 1 x 109to about I x lO11cells, or about I x lO10to about 1 x 1011cells can be utilized for the treatment of cancer in adult humans. In addition, populations of T9 lymphocytes can be cryopreserved and thawed prior to administration to a recipient as provided herein.

[0167] Tumor cell resistance to chemotherapy and radiotherapy agents represents a major problem in clinical oncology. One goal of current cancer research is to find ways to improve the efficacy of chemo- and radiotherapy by combining it with gene therapy. For example, the herpes simplex- thymidine kinase (HS-tK) gene, when delivered to brain tumors by a retroviral vector system, successfully induced susceptibility to the antiviral agent ganciclovir. In the context of the present disclosure, it is contemplated that cell therapy could be used similarly in conjunction with chemotherapeutic, radiotherapeutic, or immunotherapeutic intervention, in addition to other pro- apoptotic or cell cycle regulating agents.

[0168] In some embodiments, administration of the T cells provided herein to a subject precede and / or follow the other agent treatment(s) by intervals ranging from minutes to weeks. In embodiments where the other agent and the therapy of the present disclosure are applied separately to the subject, one would generally ensure that a significant period of time did not expire between each delivery, such that the agent and inventive therapy would still be able to exert an advantageously combined effect on the cell. In such instances, it is contemplated that one may contact the cell with the multiple modalities within about 12-24 hours of each other and, more preferably, within about 6-12 hours of each other. In some situations, it may be desirable to extend the time period for treatment significantly, however, where several days (2, 3, 4, 5, 6 or 7) to several week(s) (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations.

[0169] It is expected that the treatment cycles would be repeated as necessary. It also is contemplated that various standard therapies, as well as surgical intervention, may be applied in combination with the inventive cell therapy.

[0170] In some embodiments, prior to administration of the cells of this disclosure to a subject, the subject's immune system, such as T cells, can be non- selectively or selectively depleted, or ablated, by any method known in the art, for example, selective depletion or ablation of T cells or a specific subset of T cells. Exemplary treatments to induce lymphopenia in a subject prior to cell administration can include but are not limited to the administration of chemotherapeutics and / or total body irradiation.

[0171] In one embodiment, the subject’s immune system is depleted or ablated by the administration of an induction chemotherapy regimen comprising a therapeutically effective amount of etoposide, doxorubicin, vincristine, cyclophosphamide, and prednisone (EPOCH). In another embodiment, fludarabine can also be administered to improve the depletion of T cells.

[0172] In the treatment of cancers or tumors, the agents and compositions of the present disclosure may optionally be administered in conjunction with other, different, cytotoxic agents such as chemotherapeutic or antineoplastic compounds or radiation therapy useful in the treatment of the disorders or conditions described herein (e.g., chemotherapeutics or antineoplastic compounds). The other compounds may be administered prior to, concurrently and / or after administration of the antibodies or antigen binding fragments thereof of this disclosure. As used herein, the word “concurrently” means sufficiently close in time to produce a combined effect (that is, concurrently may be simultaneously, or it may be two or more administrations occurring before or after each other).

[0173] IV. Cell Culture Composition Containing Expansion Medium and a Population of Cells

[0174] A cell culture composition comprising the cell culture medium provided herein and a cell population cultured therein is provided.

[0175] The cell population of the cell culture composition can comprise a T cell population. The T cell population can comprise any T cells, including one or more primary human T cells, one or more tumor infiltrating lymphocytes (TILs), and / or one or more CD4+and / or CD8+T cells.

[0176] Additionally or alternatively, the cell population can include hematopoietic stem cells, which can differentiate into blood or immune cells including T cells.

[0177] The cell population may have been modified, such as by introducing an exogenous polynucleotide of interest or one or more gene editing reagents. A polynucleotide encoding a chimeric antigen receptor (CAR) and / or a T cell receptor (TCR) may have been introduced into the cell population such that one or more cells of the cell population express the CAR and / or the TCR. Alternatively, the cell population may not been modified e.g., with an exogenous polynucleotide of interest or one or more gene editing reagents. A “modified,” “transformed,” “engineered,” or “edited” cell or cell population as used herein refers to a cell or cell population in which the cell / cell population characteristics such as gene or polypeptide expression profiles have been modified, such as by introducing an exogenous polynucleotide of interest for expression of a polypeptide of interest (e.g., a CAR, a TCR), or introducing one or more gene editing reagents for modification of the genome. In contrast, an “unmodified” or “untransformed” cell or cell population as used herein refers to a cell or cell population in which the cell / cell population characteristics such as the gene or polypeptide expression profiles have not been modified, such as by introducing an exogenous polynucleotide of interest for expression of a polypeptide of interest (e.g., a CAR, a TCR), or introducing one or more gene editing reagents for modification of the genome.

[0178] The T cell population may not have been stimulated by an antigen. Alternatively, the T cell population may have been stimulated by an antigen.

[0179] The T cell population of the cell culture composition can have unique functionality, including any characteristics provided elsewhere in the present disclosure. For example, the T cell population of the cell culture can secrete IL-9; express CCR4, CCR7, CD62L, and / or CD45RA; comprise increased viability, increased persistence, increased downregulation of exhaustion, increased killing activity, increased tumor control, and / or increased mitogenesis; comprise an early memory T cell phenotype or prolonged survival; comprise less differentiation into an effector phenotype or less T cell exhaustion; comprise increased proliferation or activation in the presence or absence of TGFp or in the absence of IL-2 in medium; comprise decreased levels of phosphorylated SMAD3; comprise decreased potentiation of the TGFP signaling pathway in response to stimuli; and / or comprise an increased ratio of CD4+T cells over CD8+T cells, relative to a control T cell population cultured in a control medium not comprising the cell culture medium.

[0180] In specific embodiments, in the T cell population of the cell culture composition, the viability is statistically significantly increased by up to about 15%; the persistence is statistically significantly increased by up to about 100%; the exhaustion is statistically significantly downregulated by up to about 80%; a volume of a tumor contacted by the cultured T cell population is statistically significantly decreased, or eradicated; the mitogenesis is statistically significantly increased by up to about 50% in response to a target antigen; the differentiation into an effector phenotype is statistically significantly decreased down to 0%; the proliferation is statistically significantly increased by up to about 50% in the presence of TGFP; the proliferation is statistically significantly increased by up to about 50% in the absence of IL-2; the activation response is statistically significantly increased by up to 200% in the presence of TGFP; the activation response is statistically significantly increased by up to 500% in the absence of TGFP; and / or the ratio of CD4+T cells over CD8+T cells is statistically significantly increased by up to about 80%, as compared to the control T cell population.

[0181] Expression of one or more genes associated with an early memory T cell phenotype or prolonged survival can be increased, and / or expression of one of more genes associated with a T cell effector phenotype, T cell exhaustion, or suppression of T cell responses to tumors can be decreased in the T cell population of the cell culture composition relative to a control T cell population cultured in a control medium not comprising the cell culture medium. For example, expression of PTGER2 can be decreased in the T cell population of the cell culture composition relative to a control T cell population cultured in a control medium not comprising the cell culture medium. Expression of one or more of RUNX2, CCR7, CTSL, MYB, SELL, IL-9, IKZF2, CCR4, BATF3, SMAD2, , JUN, IRF4, USP18, MX1, IFNGR2, CD4, and TCF7 can be increased, and / or expression of one or more of LAG3, LAIR1, FASLG, IFNG, KLRG1, TBX21, SLAMF7, GZMA, GZMB, PRF1, PTGER2, , AD0RA2A, BID, TGFBR1, ID2, and CD8a can be decreased in the T cell population of the cell culture composition relative to a control T cell population cultured in a control medium not comprising the cell culture medium.

[0182] V. General Considerations

[0183] Methods involving conventional molecular biology techniques are described herein. Such techniques are generally known in the art and are described in detail in methodology treatises, such as Current Protocols in Molecular Biology, ed. Ausubel et al., Greene Publishing and Wiley- Interscience, New York, 1992 (with periodic updates). Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the application pertains. Commonly understood definitions of molecular biology terms can be found in, for example, Rieger et al., Glossary of Genetics: Classical and Molecular, 5th Ed., Springer-Verlag: New York, 1991, and Lewin, Genes V, Oxford University Press: New York, 1994. The definitions provided herein are to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the application.

[0184] As used herein and in the claims, the singular forms include the plural reference and vice versa unless the context clearly indicates otherwise. Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.”

[0185] As used herein, “a,” “an” and “the” can mean one or more than one, depending on the context in which it is used. For example, “a” cell can mean one cell or multiple cells.

[0186] As used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0187] The term “about,” as used herein when referring to a measurable value such as an amount of a compound or agent of this disclosure, dose, time, temperature, and the like, is meant to encompass variations of ±20%, 10%, ±5%, ±1%, 0.5%, or even ±0.1% of the specified amount. As used herein, “one or more” means one, two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.

[0188] The terms “increase,” “increases,” “increased,” “increasing,” “improve,” “enhance,” and similar terms indicate an elevation in the specified parameter of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 300%, 400%, 500% or more.

[0189] The terms “reduce,” “reduces,” “reduced,” “reduction,” “inhibit,” and similar terms refer to a decrease in the specified parameter of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 100%.

[0190] A “statistically significant” increase or decrease in a parameter refers to an increase or decrease in the parameter that is significant as statistically analyzed. Any known statistical methods can be used for statistical analysis and determination of the presence of a statistically significant increase or decrease, including analysis of variance (ANOVA) test (e.g., paired 2 way ANOVA tests with a Sidak Post-hoc multiple comparison test), Student’ s t-test, multivariate analysis or variance (MANOVA), and analysis of covariance (ANCOVA).

[0191] The term “therapeutically effective amount” or “effective amount” can refer to that amount of a pharmaceutical composition that results in amelioration of symptoms (e.g., reduction in size or elimination of a tumor) and / or a prolongation of survival in a subject. A therapeutically relevant effect relieves to some extent one or more symptoms of a disease or condition or returns to normal either partially or completely one or more physiological or biochemical parameters associated with or causative of the disease or condition.

[0192] As used herein, the terms “treating” or “treatment” of a condition or disease can include: (1) inhibiting the disease or condition, i.e., arresting, delaying or reducing the development of the disease or condition and its symptoms; or (2) relieving the disease or condition, i.e., causing regression of the disease or condition and its clinical symptoms. The term “treatment” or “treating,” as used herein, does not encompass 100% cure of cancer. However, in one embodiment, the therapeutic methods described herein can result in 100% reversal of detectable disease.

[0193] As used herein, the terms “prophylactic” or “preventative” treatment can include preventing at least one symptom of the disorder, disease or condition, i.e., causing a clinical symptom to not significantly develop in a subject that may develop or be predisposed to the disease but does not yet experience or display symptoms of the disease or condition.

[0194] As used herein, the term “subject” can refer to any animal, including, but not limited to, humans and non-human animals (e.g., rodents, arthropods, insects, fish (e.g., zebrafish)), nonhuman primates, ovines, bovines, ruminants, lagomorphs, porcines, caprines, equines, canines, felines, ayes, etc.), which is to be the recipient of a particular treatment. Typically, the terms “patient” and “subject” are used interchangeably herein in reference to a human subject.

[0195] As used herein, “IL-9” refers to a 4-helix bundle cytokine that is produced by T-cells, typically by CD4+helper cells (e.g., activated Th2 cells, or Th9 cells) but as described herein, also in cytotoxic CD8+Tc9 cells. Alternative names for IL-9 include, but are not limited to, P40, HP40, T-cell growth factor p40, interleukin-9, or P40 cytokine.

[0196] As used herein, “adoptive cell transfer” is the process of passively transferring cells, particularly immune-derived cells, into a host with the goal of transferring the immunologic functionality and characteristics into the host. In some embodiments, IL-9 producing cells are used in adoptive cell transfer according to the methods described herein. In some embodiments, T9 cells are used in adoptive cell transfer according to the methods described herein.

[0197] As used herein, the term “peptide” is used to designate a series of residues, typically L- amino acids, connected one to the other typically by peptide bonds between the alpha-amino and carbonyl groups of adjacent amino acids.

[0198] An “immunogenic peptide” is a peptide which comprises an allele-specific motif such that the peptide will bind the major histocompatibility complex (MHC) allele and be capable of inducing a cytotoxic T lymphocyte (CTL) response. Thus, immunogenic peptides are capable of binding to an appropriate MHC molecule and inducing a cytotoxic T response against the antigen from which the immunogenic peptide is derived.

[0199] As used herein, the term “costimulatory molecule” refers to a molecular component that promotes activation, proliferation and effector function of a T cell after engagement of an antigen specific receptor.

[0200] As used herein, the term “cytoplasmic signaling domain” refers to the 5 component of a costimulatory molecule or cytokine receptor that exists inside the cell and is responsible for transducing the external signal received to the internal metabolic processes of the cell, thereby altering its phenotype and function.

[0201] The terms “administered,” “contacted,” “provided to” and “exposed,” when applied to a cell, are used herein to describe the process by which a therapeutic agent is delivered to a target cell and / or is placed in direct juxtaposition with the target cell, e.g., under conditions that facilitate binding of a CAR to a target cancer antigen in and / or on a target cancer cell. In some embodiments, chemotherapy and / or radiation therapy can also be included before, after and / or during the administering, contacting, exposing and / or providing to step to achieve cell killing or stasis. In some embodiments, multiple agents can be delivered to a cell in a combined amount effective to kill the cell or prevent it from dividing. All peptide sequences mentioned herein are written according to the usual convention whereby the N-terminal amino acid is on the left and the C-terminal amino acid is on the right. A short line (or no line) between two amino acid residues indicates a peptide bond.

[0202] “Antibody” or “antibodies” as used herein refers to all types of immunoglobulins, including IgG, IgM, IgA, IgD, and IgE. The term “immunoglobulin” includes the subtypes of these immunoglobulins, such as IgGl, IgG2, IgG3, IgG4, etc. The antibodies may be of any species of origin, including (for example) mouse, rat, rabbit, horse, or human, or may be chimeric or humanized antibodies. The term “antibody” as used herein includes antibody fragments which retain the capability of binding to a target antigen, for example, Fab, F(ab’)2, and Fv fragments, and the corresponding fragments obtained from antibodies other than IgG. Such fragments are also produced by known techniques. In some embodiments antibodies may be coupled to or conjugated to a detectable group or therapeutic group in accordance with known techniques.

[0203] Furthermore, the term “antibody” as used herein, is intended to refer to immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CHI, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain (CL1). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementary determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). In various embodiments of the antibody or antigen binding fragment thereof of the disclosure, the FRs may be identical to the human germline sequences, or may be naturally or artificially modified. Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyterminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0204] In general, the antibodies and antigen binding fragments thereof of the present disclosure possess very high affinities, typically possessing KD values of from about IO-8through about IO-12M or higher, for example, at least IO-8M, at least IO-9M, at least IO-10M, at least 10-11M, or at least 10'12M, when measured by binding to antigen presented on cell surface.

[0205] The antibodies and antigen binding fragments thereof of the present disclosure possess very high affinities, typically possessing ECso values of from about 10'8through about IO-12M or higher, for example, at least IO-8M, at least IO-9M, at least IO-10M, at least 10-11M, or at least IO-12M, when measured by binding to antigen presented on cell surface. The term “antigen-binding portion” or “antigen-binding fragment” of an antibody (or simply “antibody portion” or “antibody fragment”), as used herein, refers to one or more fragments, portions or domains of an antibody that retain the ability to specifically bind to an antigen. It has been shown that fragments of a full-length antibody can perform the antigen-binding function of an antibody. Examples of binding fragments encompassed within the term “antigen-binding portion” of an antibody include (i) an Fab fragment, a monovalent fragment consisting of the VL, VH, CL1 and CHI domains; (ii) an F(ab’)2 fragment, a bivalent fragment comprising two F(ab)’ fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CHI domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody;

[0206] (v) a dAb fragment (Ward et al. (1989) Nature 241 :544-546), which consists of a VH domain; and

[0207] (vi) an isolated complementary determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single contiguous chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. 1988 Science 242:423-426; and Huston et al. 1988 Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term “antigen-binding portion” of an antibody. Other forms of single chain antibodies, such as diabodies, are also encompassed (see e.g., Holliger et al. 1993 Proc. Natl. Acad. Sci. USA 90:6444- 6448).

[0208] The term “epitope” refers to an antigenic determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule known as a paratope. A single antigen may have more than one epitope. Epitopes may be either conformational or linear. A conformational epitope is produced by spatially juxtaposed amino acids from different segments of one (or more) linear polypeptide chain(s). A linear epitope is an epitope produced by adjacent amino acid residues in a polypeptide chain. In certain embodiments, an epitope may include other moieties, such as saccharides, phosphoryl groups, or sulfonyl groups on the antigen.

[0209] The following example is for the purpose of illustration only and is not intended to limit the scope of the claims, which are appended hereto. EXPERIMENTAL

[0210] Example 1: Culturing and Expansion of Primary Human T Cells

[0211] This example describes an example timeline and protocols for culturing and expanding CAR-T cells in small scale using cell culture medium and methods provided in the present disclosure.

[0212] T cells were isolated from Leukopaks purchased from Stem Cell Tech using an EasySep Human T cell Isolation Kit negative selection. Previously isolated primary T cells have been stored in CS10 (also referred to as cryostore) Medium in liquid nitrogen, at 10 million per ml, in 1ml or 0.5ml aliquots.

[0213] On day 0, T cells were thawed and activated with IMMUNOCULT™ CD3 / CD28 activation reagent. In general, only up to 2 vials were thawed at a time to ensure highest viability of thawed cells. Thawing was performed according to the following protocol:

[0214] • Add 4 ml of sterile FBS to a 50 ml conical in a sterilized tissue culture hood, and let come to room temperature.

[0215] • Get 1 ml aliquots of T cells from the liquid nitrogen tank and transfer to ice or dry ice.

[0216] • Gently thaw frozen cells in H2O bath till a pea sized chunk of ice remains.

[0217] • Spray the cryovial with 70% ethanol, then sterilely gently transfer the thawed cells to the 4 ml FBS.

[0218] • Add 35-45 ml lx PBS to the 50ml conical, slowly and dropwise. The cells are fragile and need to release the DMSO slowly and adjust to the new salt concentrations slowly or else they are shocked and will burst. Take time here as this can generate variability between donors or samples.

[0219] • Spin cells at 1350RPM for 5 minutes to pellet. Do not invert tubes or vortex.

[0220] • Aspirate off supernatant and resuspend pellet in 1ml of IxPBS using a P1000 pipet. Add 9ml lx PBS and take lOul for cell counting. Spin remainder of cells at 1350RPM for 5 minutes.

[0221] • Count cells. Then aspirate off supernatant and resuspend pellet in Complete Medium at 1 million cells per 0.975ml or at desired concentration.

[0222] Immediately after T cells were thawed and at 1 million per ml, cells to be activated were separated, and 25 pl of IMMUNOCULT™ CD3 / CD28 activation reagent (Stemcell) was added for every ml of T cells to be activated and pipetted to mix. Cells were then plated at 1 ml per well in a 48 well culture plate, or 2ml per well in a 24 well plate. Cells were able to be left for up to 3 days before needing to split, add new medium, or IL-2. IMMUNOCULT™ CD3 / CD28 activation reagent (Stemcell) is a complexed antibody solution which, upon addition to the cells, cross-links key activation receptors to simulate the T cells being presented antigen. For making CAR-T cells, 25ul of this reagent is added to every 1ml of cells, Two (2)-3 Days after administering the IMMUNOCULT™ CD3 / CD28 activation reagent, there were some loss in cell number from activation induced cell death (AICD), but after 3-4 days cells, the cells started expanding at a high rate.

[0223] On day 1, T cells were transduced with lentivirus containing CAR-T construct (e.g., anti CD70 CAR-T). T cells have to be activated to be efficiently transduced with lentivirus. Conducting lentiviral transduction 1 to 2 days post T cell activation can generally achieve efficient transduction.

[0224] Twenty-four (24) hours after thaw / activation, T cells were passively transduced with lentivirus by first removing a volume of medium and then adding the equal volume of virus on cells. After lentiviral transduction, medium was completely changed out by spinning down cells, aspirating supernatant, and resuspending T cells in fresh complete medium at 1 million cells per ml. Cells started to proliferate almost immediately thereafter and needed to be expanded every day or two for the next several days. If cells are in 1 ml medium in a 48 well plate, by the next day they generally needed to be transferred to a 24 well plate and diluted with an extra ml of medium or split into 2 wells of a 48 well plate.

[0225] On day 3, 6.5 pg of CRISPR Cas9 ribonucleoproteins per million cells were electroporated into the T cells to generate a mutation that cause loss of function of CD70. Five (5) million electroporated cells were moved to one well of a 6 well Grex plate (G-Rex® gas permeable membrane 6 Well Plate - P / N 80240M) containing 40 ml culture medium.

[0226] Three (3) to 4 days after adding the activation reagent to T cells, the cells started proliferating. This was noticeable as their medium will begin to get more exhausted and turn yellow. On Day 4, T cells were expanded, with the goal of keeping T cells at 1 to 2 million per ml of medium, or in some cases 0.5 million per ml. To avoid splitting T cells too quickly, if a culture is more than 2 million per ml, the medium volume was doubled with fresh medium and cells, and the culture was moved to a bigger vessel, or split between 2 wells.

[0227] On days 6, 8, 10, and 12, T cells were similarly expanded and the phenotype was monitored using flow cytometry. For example, on day 4, 6, 8, 10, and / or 12, 30 ml medium was removed from top of each well of cells, and replaced with 30 ml fresh medium, and / or the culture was moved to a bigger vessel as needed. Most T cell cultures expanded for 10 to 14 days after adding the activation reagent, then the need to expand cultures progressively declined for several days till the cells stop proliferating. At this point T cells would need to either be restimulated or frozen. On Day 14, as the T cells stopped proliferating, final flow cytometry reading was conducted to confirm the phenotype, and the T cells were frozen (i.e., cryopreserved). To freeze or cryopreserve cells, cells were counted and aliquoted appropriately, e.g., at 10 million cells per ml, e.g., in 1 ml or 0.5 ml aliquots, and frozen to -80°C in a CS10 medium (“cryostore” medium), and stored in liquid nitrogen (i.e., at - 196°C).

[0228] Throughout the course, T cells were cultured using the RPMI 1640 medium with ATCC modification (Gibco A1049101) + 10% FBS, further containing 100 lU / ml IL-2 (Stemcell), 290 lU / ml IL-4 (Stemcell), 25 lU / ml TGFP (Stemcell), andlO ug / ml anti-IFNy (B27 Clone, Bio X Cell). Unlike previous culturing examples, the medium composition was not switched with one containing IL-2 as the only cytokine two (2) days prior to freezing or cell infusion. Concentrations of all cytokines are optimized. Use of anti-IFNy antibody is unlikely necessary.

[0229] Table 1 sets forth the components of the RPMI 1640 medium with ATCC modification (A1049101).

[0230] Table 2 sets forth general culturing volumes used, while less volume of medium than indicated can be used to allow better gas exchange. The culturing volumes can be optimized for the specific culturing condition. Additionally or alternatively, T cells can be cultured on a gas permeable membrane culture surface, e.g., in a G-rex® gas permeable membrane platform (e.g., flask or bioreactor) and / or in a large-scale culture platform. A gas permeable membrane culturing platform (e.g., G-rex® gas permeable membrane platform) and / or large scale culture can be useful for preparing T cells for clinical use.

[0231] Table 1. Components of the RPMI 1640 medium with ATCC modification (A1049101)

[0232] Table 2. Culturing volumes

[0233] Example 2: Characteristics in vitro of T Cells Expanded with Expansion Medium of Present Disclosure (“OTX Medium”)

[0234] OTX cells are not reliant on IL-2 for proliferation

[0235] CAR-T cells from the same donors were expanded over 14 days in RPMI with 10% FBS and 25 lU / mL TGFP, 100 lU / mL IL-2, 290 ZU / mL IL-4, and 10 pg / mL anti-IFNy (an example “OTX medium”) or RPMI with 10% FBS and IL-2 as the only cytokine (“conventional T medium”). After expansion over 14 days in the OTX or conventional T medium, CAR-T cells were labeled with CELL TRACE™ proliferation dye and cultured further for 3 days in RPMI with 10% FBS with or without 100 lU / ml IL-2. As shown in FIG. 2A (further culture with lOOIU / ml IL-2) and FIG. 2B (further culture with or without lOOIU / ml IL-2), OTX CAR-T cells (CAR-T cells expanded in the OTX medium) did not require IL-2 for further proliferation (increased proliferation indicated by decreased CELLTRACE™ proliferation dye mean fluorescence intensity (MFI)), but Tconv cells (CAR-T cells expanded in the conventional T medium) could not proliferate without IL-2.

[0236] OTX cells resist TGFf suppression

[0237] Equal numbers of anti-CD70 OTX CAR-T cells (CAR-T cells expanded in the OTX medium described above in this Example) or conventional CAR-T cells (CAR-T cells expanded in the conventional T medium described above in this Example) from the same donors (4 independent primary T cell donors) were challenged with varying target to effector (T:E) doses of CD70+Raji cells, with or without TGFP supplemented medium (25 ng / ml). CAR-T cells were counted using flow cytometry counting beads after 4 days of coculture. As shown in FIGs. 3 A and 3B, proliferation of Tconv cells (CAR-T cells expanded in the conventional T medium; FIG. 3 A) was suppressed by TGFP at lower doses of antigen, but proliferation of OTX CAR-T cells (CAR-T cells expanded in the OTX medium; FIG. 3B) was not.

[0238] OTX cells show enhanced activation responses to low antigen levels, regardless of suppression by TGFf

[0239] Equal numbers of anti-CD70 OTX or conventional CAR-T cells (expanded in the OTX medium or conventional T medium, respectively) from the same donors (4 independent primary T cell donors) were challenged with varying target to effector (E:T) doses of CD70+Raji cells, with or without TGFP supplemented medium (25 ng / ml). After 4 days cells were analyzed for the activation markers CD25 (FIGs. 4A and 4B) and CD71 (FIGs. 5 A and 5B) using flow cytometry.

[0240] As shown in FIGs. 4A, 4B, 5A, and 5B, OTX CAR-T cells show enhanced activation responses to low antigen levels as assessed by the CD25 or CD71 levels, regardless of TGFP suppression.

[0241] OTX cells display increased mitogenesis in response to tarset antisen

[0242] Equal numbers of anti-CD70 OTX or conventional CAR-T cells (expanded in the OTX medium or conventional T medium, respectively) from the same donors (2 independent primary T cell donors) were stained with MITOTRACKER™ Green total mitochondrial dye, and analyzed by flow cytometry after expansion (Day 1). The OTX or conventional CAR-T cells were also challenged with 786-0 cells, at the ratio of one 786-0 cell to every three CAR-T cells every 4 or 3 days, then stained with MITOTRACKER™ Green total mitochondrial dye on the indicated day post coculture (Day 5 or Day 8). As shown in FIGs. 6A and 6B, OTX CAR-T cells showed enhanced mitogenesis relative to conventionally expanded CAR-T cells post antigen stimulation.

[0243] OTX conditionins senerates early memory T cell products that resists exhaustion

[0244] In unmodified T cells

[0245] Six (6) biological replicates of healthy human unmodified T cells from 5 healthy donors were expanded in presence the OTX medium or conventional medium for 10-14 days, and harvest cell products were subjected to transcriptome analysis using Nanostring. The OTX medium used in this study did not contain any anti-IFNy antibody. FIG. 7 shows a heatmap of genes differentially expressed in OTX-conditioned unmodified T cells. Genes that had at least two-fold higher expression of transcripts in cells expanded in the OTX medium (“OTX”) as compared to conventionally expanded T cells (“Tconv”) included CTSL, IL-9, RUNX2, MYB, IKZF2, CCR4, CCR7, BATF3, SMAD2, SELL (encoding CD62L). Genes that had at least two-fold lower expression of transcripts in OTX cells as compared to Tconv cells included PRF1, GZMB, PTGER2, KLRG1, TBX21 (encoding Tbet), SLAMF7, IFNG, LAIR1, AD0RA2A, and LAG3. Table 3 shows Z scores of the relevant genes. Of note, PTGER2, a checkpoint gene (encoding PGE2 receptor) that can suppress T cell responses to tumors e.g., by impairing IL-2 sensing in tumor microenvironment, was strongly downregulated in OTX-conditioned cells relative to conventional medium-conditioned cells, indicating an enhanced IL-2 sensing and IL-2 response ability of the OTX-conditioned T cells. Further, OTX-conditioned T cells overexpressed transcripts associated with an early memory T cell phenotype and / or prolonged survival (e.g., RUNX2, CCR7, CTSL, MYB, SELL), while conventional T cells overexpressed transcripts associated with an effector-like phenotype as well as T cell exhaustion (e.g., LAG3, LAIR1, FASLG, IFNG, KLRG1, TBX21, SLAMF7, GZMA, GZMB). In sum, OTX conditioning generates a unique transcriptional profile ideal for long-term T cell responses.

[0246] In gene edited CAR-T cells

[0247] Healthy donor T cells from 3 donors were gene edited to knock out CD70, transduced with a lentiviral vector to deliver an anti-CD70 CAR, and expanded in the OTX or conventional medium. The OTX medium used in this study did not contain any anti-IFNy antibody. Gene edited CAR-T cells were harvested after 10 days of culture and Nanostring transcriptome analysis was performed. FIG. 8 shows a heatmap of genes differentially expressed in OTX-conditioned gene edited CAR-T cells. Genes that had at least two-fold higher expression of transcripts in cells expanded in the OTX medium (“OTX”) as compared to conventionally expanded T cells (“Tconv”) included IL-9, CTSL, MYB, RUNX2, CCR4, IKZF2, BATF3, JUN, and IRF4. Genes that had at least two-fold lower expression of transcripts in OTX cells as compared to Tconv cells included PRF1, PTGER2, TBX21, KLRG1, LAIR1, GZMB, IFNG, SLAMF7, BID, LAG3, TGFBR1, and ID2. Table 4 shows Z scores of the relevant genes. In sum, similarly to the unmodified T cells, OTX-conditioned gene edited CAR-T cells overexpressed transcripts associated with an early memory T cell phenotype and / or prolonged survival, while conventionally expanded gene edited CAR-T cells overexpressed transcripts associated with an effector-like phenotype as well as T cell exhaustion. This suggests that the effects achieved by OTX conditioning occur independently of any gene modifications (as well as lack of gene modifications) employed to engineer the T cells.

[0248] In separate experiments, at the end of cell expansion, the same gene edited CAR-T cells were exposed to CD70-positive target cells. After three rounds of antigen challenge in conventional complete medium, the resulting CAR-T cells were harvested and Nanostring transcriptome analysis was performed. FIG. 9 shows a heatmap of genes differentially expressed in OTX-conditioned gene edited CAR-T cells after antigen challenge. Genes that had at least two-fold higher expression of transcripts in cells expanded in the OTX medium (“OTX”) as compared to conventionally expanded T cells (“Tconv”) included USP18, MX1, IFNGR2, CCR4, CD4, RUNX2, TCF7, and CCR7. Genes that had at least two-fold lower expression of transcripts in OTX cells as compared to Tconv cells included GZMB, PTGER2, IFNG, CD8a, TBX21, PRF1, and SLAMF7. Table 5 shows Z scores of the relevant genes. In sum, gene edited CAR-T cells that had been initially expanded under OTX conditions and were repeatedly stimulated overexpressed transcripts associated with earlier memory phenotype (e.g., TCF7, CCR7, RUNX2), while gene edited CAR-T cells that had been produced in conventional medium overexpressed transcripts associated with a effector-like cell phenotype (TBX21, IFNG, GZMB, PRF1, SLAMF7), showing similar trends observed in the unmodified T cells and gene edited CAR-T cells without chronic restimulation.

[0249] Table 3. Z scores of relevant genes in unmodified T cells conditioned with OTX medium and conventional medium OTX medium conditioned Conventional medium conditioned OTX medium conditioned Conventional medium conditioned

[0250] Table 4. Z scores of relevant genes in gene edited CAR-T cells conditioned with OTX medium and conventional medium

[0251] Table 5. Z scores of relevant genes in gene edited CAR-T cells conditioned with OTX medium or conventional medium and stimulated by three rounds of antigen challenge

[0252] The OUTLAST phenotype can emerge after 3 days in OTX medium

[0253] Unmodified T cells from 2 healthy donors were expanded in OTX medium ("OTX”) and / or conventional medium (“conv”) over 10 days. The OTX medium used in this study did not contain any anti-IFNy antibody. Cells remained in the same type of medium for the full 10 days (10 D Conv, 10 D OTX) or were started in one type of medium for the indicated number of days before being switched over into the opposite type of medium for the remainder of the expansion. To assess whether the OUTLAST phenotype (OTX phenotype) was achieved, cells were analyzed for expression of surface markers associated with the OUTLAST phenotype (CCR4, CCR7, CD62L) after 10 days in culture. Further, on day 10, cells were restimulated in unsupplemented medium and IL-9 secretion into the culture medium, an OUTLAST phenotype, was assessed 24 hours after restimulation.

[0254] FIGs. 10A-10D depict the IL-9 secretion, CCR4 expression, CCR7 expression, and CD62L expression, respectively, in T cells cultured in the OTX medium and / or conventional medium for the following durations:

[0255] • 3 days in conventional medium followed by 7 days in OTX medium (“3 D Conv + 7 D OTX”)

[0256] • 6 days in conventional medium followed by 4 days in OTX medium (“6 D Conv + 4 D OTX”)

[0257] • 8 days in conventional medium followed by 2 days in OTX medium (“8 D Conv + 2 D OTX”)

[0258] • 10 days in conventional medium (“10D Conv”: negative control)

[0259] • 3 days in OTX medium followed by 7 days in conventional medium (“3 D OTX + 7 D Conv”)

[0260] • 6 days in OTX medium followed by 4 days in conventional medium (“6 D OTX + 2 D Conv”)

[0261] • 8 days in OTX medium followed by 2 days in conventional medium (“8D OTX + 2 D Conv”)

[0262] • 10 days in OTX medium (“10 D OTX”: positive control)

[0263] Cells were deemed to exhibit an OUTLAST phenotype if a meaningful increase in the above readouts over the conventional T cell control (10 D Conv) was observed. The data show that the OUTLAST phenotype can be achieved in cells that started expansion in OUTLAST medium for 3 days and were finished expansion in conventional medium (3 D OTX + 7 D Conv), as well as by cells that were in OUTLAST conditions for more than 3 days at the beginning of expansion (6 D OTX + 4 D Conv, 8 D OTX + 2 D Conv, 10 D OTX). Further, the OUTLAST phenotype was observed in cells that were started in conventional medium and that were finished in OUTLAST medium for 7 days or longer (3 D Conv + 7 D OTX). Example 3: Characteristics in vivo of T Cells Expanded with Expansion Medium of Present

[0264] Disclosure (“OTX Medium”)

[0265] OTX CD70 CAR-T Cells Exhibit Superior Tumor Control and Superior Persistence

[0266] OTX CD70 CAR-T cells (“OTX”; expanded in the OTX medium described in Example 2), conventional CD70 CAR-T cells (“convT”; expanded in the conventional T medium described in Example 2), or control T cells (“Control”) each at a dose of 7.5 x 105cells, or a HBSS vehicle was administered to a subject on day 0. The subject was challenged with the tumor cells 786-0 at a dose of 1 x 106cells on day 0 and 35, and the tumor volume was monitored over time. At the end of the study on day 53, the percentage of CAR-T cells in the tumor was assessed as an indicator of T cell persistence.

[0267] As shown in FIG. 11, OTX CAR-T cells effectively controlled tumor volume in vivo compared to control groups, outperformed conventional CAR-T cells in tumor rechallenge, and exhibited superior durability as compared to conventional CAR-T cells. Further, as shown in FIGs. 12A and 12B, excised / homogenized secondary tumor samples showed significantly increased T cell (human CD3+cells) expansion and persistence within the tumor of mice that had received OTX CAR-T cells relative to mice that had received conventional CAR-T cells indicating superior expansion and durability.

[0268] OTX CAR-T Cells Exhibit Superior Downregulation of Exhaustion Markers

[0269] OTX CAR-T cells (“OTX”; expanded in the OTX medium described in Example 2), conventional CAR-T cells (“convT”; expanded in the conventional T medium described in Example 2), or control T cells (“Control”) each at a dose of 3 x 106cells were administered to a subject on day 0. The subject was challenged with the tumor cells 786-0 at a dose of 1 x 107cells on day 0 and 35.

[0270] On day 34, prior to tumor rechallenge, OTX T cells, conventional T cells, and control T cells were obtained from the blood of the subject and CD25 MFI was measured as an indicator of activation. As shown in FIG. 13, OTX CAR-T cells exhibited superior activation as compared to conventional CAR-T cells or control T cells.

[0271] On day 51, at the end of the study, OTX T cells, conventional T cells, and control T cells were obtained from the spleen of the subject and the presence of three independent exhaustion markers PD-1, LAG3, and KLRG1, as well as the activation marker CD25 were assessed. As shown in FIG. 14 A, OTX CAR-T cells exhibited a superior exhaustion marker profile indicating less exhaustion (superior downregulation of exhaustion) as compared to conventional CAR-T cells and control T cells, while the percentage of CD25 positive T cells was not significantly different across the group as shown in FIG. 14B.

[0272] In sum, the in vitro and in vivo studies provided herein demonstrate the following features of OTX-conditioned T cells relative to T cells not conditioned (cultured, produced) using the OTX medium: upregulation of genes associated with an early memory T cell phenotype and downregulation of genes associated with effector phenotype or T cell exhaustion; improved metabolic fitness during prolonged stimulation; natural resistance to TGFP mediated suppression; enhanced activation responses to low antigen densities; and increased persistence in vivo that allows for tumor control in a rechallenge xenograft model in concert with reduced exhaustion. The characteristics of OTX-conditioned T cells including the foregoing are collectively referred to as the “OTX phenotype” or “OUTLAST phenotype.”

[0273] Example 4: Development of Expansion Medium of Present Disclosure (“OTX Medium”)

[0274] Impact of IL-4 and TGFP cytokine concentrations on surface expression levels of CD45RA and CCR7

[0275] Unmodified T cells from 2 healthy donors were cultured in media containing 250 lU / mL of IL-2, and varying levels of IL-4 and TGFP at even intervals between the minimum and maximum levels provided, in addition to OTX reference conditions (containing IL-4 400 lU / ml and TGFP 25 lU / ml). Cells were activated on day 0, and harvested on day 9 of processing.

[0276] To assess whether the OUTLAST phenotype was achieved, CD4+and CD8+cells were analyzed for expression of surface markers associated with OUTLAST phenotype (CCR7, CD45RA). Table 6 shows mean fluorescence intensity (MFI) across both donors and technical replicates. Cells were considered of achieving OUTLAST phenotype if a meaningful increase from the control (“T Conv Reference” containing no IL-4 or TGFP) was measured. As shown in Table 6, TGFP concentrations of 4.5 lU / ml and above achieved the OUTLAST phenotype, and increasing concentrations of TGFP were associated increased expression of CCR7 and CD45RA. No clear impact of IL-4 concentration on the level of OUTLAST surface marker expression was detected.

[0277] Table 6. Effect of IL-4 and TGF cytokine concentrations on surface expression levels of

[0278] CD45RA and CCR7

[0279]

[0280] The combined cell growth, recovery, and final product attributes demonstrate that OPTMIZER1'1and OPTMIZER™ Pro medium yield high-quality OTX cells with comparable results

[0281] Initial medium screening studies utilized 3 donors and 5 different basal medium types. Cells were enriched using CD4 and CD8 microbeads using the CLINIMACS® Plus cell separation instrument and activated using TRANSACT™ CD3 / CD28 activation reagent. All OTX medium contained 1.8 % immune cell serum replacement (ICSR), 25 lU / mL TGFP, 100 lU / mL IL-2, 290 lU / mL IL-4, and 10 pg / mL anti-IFNy. As shown in FIG. 15 A, process growth and recovery of total viable cells, as measured by the VICELL™ cell viability analyzer, was lowest in the STEMXVIVO™ and EXCELLERATE™ basal medium conditions. Remaining medium types (IMMUNOCULT™, OPTMIZER™, and OPTMIZER™ Pro basal medium) exhibited variability donor-to-donor but had comparable growth.

[0282] Transduction and transfection efficiency were both assessed from the thawed drug product. The STEMXVIVO™ vasal medium conditions did not have sufficient cells to determine the transduction or transfection efficiency. As shown in FIG. 15B, EXCELLERATE™ basal medium negatively impacted the transfection efficiency.

[0283] Cell phenotyping was tested from the thawed drug product. The STEMXVIVO™ basal medium conditions did not have sufficient cells at harvest to test. In the EXCELLERATE™ basal medium conditions, the phenotype was not tested because of the poor transfection efficiency. As shown in FIGs. 15C and 15D, the remaining basal medium all showed comparable distribution of naive, central memory, effector memory, and effector cells in the CD4+and CD8+T Cells. Based on the combined data, it was concluded that OPTMIZER™ and OPTMIZER™ Pro medium consistently produced high-quality OTX cells, warranting their inclusion in further screening studies. Medium formulations targeting the OTX phenotype appear to perform comparably or superior to conventional T cell medium resardless of cytokine concentrations

[0284] Activation recovery, transduction recovery, transfection recovery, and population doubling label of OTX T cells (expanded under the OTX conditions) and conventional T cells (expanded in the conventional T medium) were studied. The data set included data from 9 donors within 4 studies. OTX conditions used both OPTMIZER™ and OPTMIZER™ Pro basal medium and a range of cytokine concentrations tested are as follows: 0-400 lU / mL TGFP, 0-1000 XU / mL IL-2, 0- 3000 lU / mL IL-4, and 0-10 pg / mL anti-IENy. Conventional T medium contained 100 lU / mL IL-2 as the only cytokine and used OPTMIZER™ Pro basal medium. With the exception of one study, these studies were designed using a Definitive Screening Design. T cells were isolated CD4+and CD8+cells that were activated with TRANSACT™ CD3 / CD28 activation reagent. Activation and transduction recovery were both calculated from the seeded total viable cells to the post-incubation total viable cell count. Transfection recovery was calculated from the transfection seed total viable cells to the first expansion feed cell count. The population doubling level was calculated from the transfection seed total viable cells to the harvest total viable cell count. The OTX medium performed comparably or superior to conventional T cell medium as measured by viable cell recovery and population doubling level from the VICELL™ cell viability analyzer regardless of cytokine concentrations.

[0285] Medium formulations targeting the OTX phenotype did not have significantly different transduction or transfection efficiency compared to the conventional T cell medium

[0286] Transduction and transfection efficiencies of OTX T cells (expanded under the OTX conditions) and conventional T cells (expanded in the conventional T medium) were studied. The data set included data from 9 donors within 4 studies and contains 5 conventional T cell medium, 57 OTX transduction, and 39 OTX transfection data points. Transduction and transfection efficiency are both tested from the thawed drug product. The OTX conditions used both the OPTMIZER™ and OPTMIZER™ Pro basal medium. The choice of OPTMIZER™ or OPTMIZER™ Pro basal medium did not have a significant impact on transduction efficiency in this dataset. However, within this dataset, the OTX conditions using OPTMIZER™ basal medium demonstrated a higher mean transfection efficiency (N = 3), which aligns with the findings observed in previous studies. Medium formulations targeting the OTX phenotype preferentially support the retention of naive and central memory CD4+and CD8+T cells and overall expansion of CD4+cells compared to conventional T cell medium

[0287] CD4:CD8 ratios and cell phenotypes of OTX T cells (expanded under the OTX conditions) and conventional T cells (expanded in the conventional T medium) were studied. The data were collected from the thawed drug product of 4 conventional T cell medium conditions and 63 OTX medium conditions. As shown in FIG. 16 A, the conventional T cell medium had a mean CD4:CD8 ratio of approximately 1 : 1 compared to 1.6: 1 in the OTX medium. As shown in FIG. 16B, of the expanded cells, the OTX medium retained a “younger” phenotype compared to the conventional T cell medium in both the CD4 and CD8 cells with less differentiation into the effector phenotype.

Claims

THAT WHICH IS CLAIMED:

1. A method of culturing a T cell population, the method comprising contacting the T cell population with an expansion medium, the expansion medium comprising a basal growth medium, a serum or serum replacement, and one or more cytokines and / or cytokine inhibitors, such that the T cell population is expanded.

2. The method of claim 1, wherein the one or more cytokines and / or cytokine inhibitors comprise IL-2, IL-4, TGFP, and / or an anti-IFNy antibody.

3. The method of claim 2, wherein said expansion medium comprise IL-2 at a concentration of 10 lU / ml or more; IL-4 at a concentration of 50 lU / ml or more; TGFP at a concentration of 3 lU / ml or more; and / or the anti-IFNy antibody at a concentration of 0-20 pg / ml.

4. The method of claim 2 or 3, wherein said expansion medium comprises TGFP at a concentration of 4.5 lU / ml or more.

5. The method of any one of claims 1-4, wherein said expansion medium does not comprise the anti-IFNy antibody or IL-4.

6. The method of any one of claims 1-5, wherein the T cell population is not contacted with a medium containing IL-2 and no other cytokines or cytokine inhibitors.

7. The method of any one of claims 1-6, wherein the basal growth medium comprises a Roswell Park Memorial Institute (RPMI) 1640 medium, X-VIVO™-15 medium, CTS™ OPTMIZER™ Serum Free Medium, CTS™ OPTMIZER™ Pro Serum Free Medium, and / or IMMUNOCULT™-XF T Cell Expansion Medium.

8. The method of any one of claims 1-7, wherein the serum or serum replacement comprises fetal bovine serum (FBS), human AB serum, CTS™ Immune Cell Serum Replacement, and / or PHYSIOLOGIX™ Xeno-Free Serum Replacement.

9. The method of any one of claims 1-8, wherein the T cell population comprises one or more primary human T cells.

10. The method of any one of claims 1-9, wherein the T cell population consists essentially of one or more CD4+and / or CD8+T cells.

11. The method of any one of claims 1-10, wherein the T cell population comprises one or more tumor infiltrating lymphocytes (TILs).

12. The method of any one of claims 1-11, wherein the expansion medium is replaced with fresh expansion medium every 2-3 days and / or the T cell population is cultured in the expansion medium for up to about 14 days.

13. The method of any one of claims 1-12, comprising culturing the T cell population in the expansion medium for at least 3 days optionally followed by culturing the T cell population in a control medium, or comprising culturing the T cell population in the expansion medium for at least 7 days optionally after culturing the T cell population in a control medium.

14. The method of any one of claims 1-13, the method further comprising, after culturing the T cell population in the expansion medium, freezing the T cell population and / or administering the T cell population to a subject.

15. The method of any one of claims 1-14, wherein the T cell population is cultured in a system comprising a gas permeable membrane culture surface.

16. The method of any one of claims 1-15, the method further comprising, prior to contacting the T cell population with the expansion medium:(a) thawing a plurality of cells comprising the T cell population from a frozen stock;(b) selecting from the plurality of cells one or more CD4+and / or CD8+T cells as the T cell population;(c) contacting the T cell population with an activating reagent such that the T cell population is activated;(d) introducing a polynucleotide encoding a chimeric antigen receptor (CAR) and / or a T cell receptor (TCR) into the T cell population such that one or more cells of the T cell population expresses the CAR or the TCR; and / or(e) contacting the T cell population with one or more gene editing reagents such that a mutation is introduced at a target site in a genome of one or more cells of the T cell population and level or activity of a gene of interest is altered in the one or more cells.

17. The method of claim 16, wherein: said step of selecting one or more CD4+and / or CD8+T cells is performed within one day after the step of thawing a plurality of cells; said step of contacting the T cell population with an activating reagent is performed within one day after the step of selecting one or more CD4+and / or CD8+T cells; said step of introducing a polynucleotide encoding a CAR and / or a TCR into the T cell population is performed about 1-2 days after the step of contacting the T cell population with an activating reagent; said step of contacting the T cell population with one or more gene editing reagents is performed about 3 days after the step of contacting the T cell population with an activating reagent and / or about 1-2 days after the step of introducing a polynucleotide encoding a CAR and / or a TCR into the T cell population; and / or said step of contacting the T cell population with expansion medium is performed within one day after the step of introducing a polynucleotide encoding a CAR and / or a TCR into the T cell population or the step of contacting the T cell population with one or more gene editing reagents.

18. The method of claim 16 or 17, wherein said step of selecting one or more CD4+and / or CD8+T cells comprises contacting the plurality of cells with magnetic beads coupled with an anti- CD4+antibody and an anti-CD8+antibody, and selecting CD4+and / or CD8+T cells that are bound to the magnetic beads.

19. The method of any one of claims 16-18, wherein one or more cells of the T cell population are CD3+and CD28+, and the activating reagent binds and crosslinks CD3 and CD28 on surface of the one or more cells of the T cell population.

20. The method of any one of claims 16-19, wherein introducing the polynucleotide encoding a CAR and / or a TCR into the T cell population comprises introducing a viral vector comprising the polynucleotide into the T cell population.

21. The method of claim 20, wherein the viral vector is a lentiviral vector, a retroviral vector, or an adenoviral vector.

22. The method of any one of claims 16-19, wherein the polynucleotide encoding a CAR and / or a TCR is introduced into the T cell population by transposition or mRNA transfection.

23. The method of any one of claims 16-22, wherein contacting the T cell population with the one or more gene editing reagents comprises electroporating the one or more gene editing reagents into one or more cells of the T cell population.

24. The method of any one of claims 1-23, wherein the cultured T cell population: secretes IL-9; expresses CCR4, CCR7, CD62L, and / or CD45RA; comprises increased viability, increased persistence, increased downregulation of exhaustion, increased killing activity, increased tumor control, and / or increased mitogenesis; comprises an early memory T cell phenotype or prolonged survival; comprises less differentiation into an effector phenotype or less T cell exhaustion; comprises increased proliferation or activation in the presence or absence of TGFP or in the absence of IL-2 in medium; comprises decreased levels of phosphorylated SMAD3; comprises decreased potentiation of the TGFP signaling pathway in response to stimuli; and / or comprises an increased ratio of CD4+T cells over CD8+T cells, relative to a control T cell population cultured in a control medium not comprising the expansion medium.

25. The method of claim 24, wherein in the cultured T cell population: the viability is statistically significantly increased by up to about 15%; the persistence is statistically significantly increased by up to about 100%; the exhaustion is statistically significantly downregulated by up to about 80%; a volume of a tumor contacted by the cultured T cell population is statistically significantly decreased, or eradicated; the mitogenesis is statistically significantly increased by up to about 50% in response to a target antigen; the differentiation into an effector phenotype is statistically significantly decreased down to 0%; the proliferation is statistically significantly increased by up to about 50% in the presence of TGFP; the proliferation is statistically significantly increased by up to about 50% in the absence ofIL-2;the activation response is statistically significantly increased by up to 200% in the presence of TGFP; the activation response is statistically significantly increased by up to 500% in the absence of TGFP; and / or the ratio of CD4+T cells over CD8+T cells is statistically significantly increased by up to about 80%, as compared to the control T cell population.

26. The method of any one of claims 1-25, wherein expression of one or more genes associated with an early memory T cell phenotype or prolonged survival is increased, and / or expression of one of more genes associated with a T cell effector phenotype, T cell exhaustion, or suppression of T cell responses to tumors is decreased in the cultured T cell population relative to a control T cell population cultured in a control medium not comprising the expansion medium.

27. The method of any one of claims 1-26, wherein expression of PTGER2 is decreased in the cultured T cell population relative to a control T cell population cultured in a control medium not comprising the expansion medium.

28. The method of any one of claims 1-27, wherein expression of one or more of RUNX2, CCR7, CTSL, MYB, SELL, IL-9, IKZF2, CCR4, BATF3, SMAD2, JUN, IRF4, USP18, MX1, IFNGR2, CD4, and TCF7 is increased, and / or expression of one or more of LAG3, LAIR1, FASLG, IFNG, KLRG1, TBX21, SLAMF7, GZMA, GZMB, PRF1, PTGER2, ADORA2A, BID, TGFBR1, ID2, and CD8a is decreased in the cultured T cell population relative to a control T cell population cultured in a control medium not comprising the expansion medium.

29. The method of any one of claims 26-28, wherein the cultured T cell population is a population of unmodified T cells or engineered CAR-T cells in the presence or absence of antigen stimulation.

30. The method of any one of claims 15-29, wherein the CAR is bispecific for two antigens.

31. The method of any one of claims 15-30, wherein the CAR is specific for an antigen on a cancer cell.

32. The method of claim 31, wherein the cancer cell is a cell of B cell lymphoma, T cell lymphoma, myeloma, leukemia, hematopoietic neoplasia, thymoma, lymphoma, sarcoma, lungcancer, liver cancer, non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, uterine cancer, cervical cancer, endometrial cancer, adenocarcinoma, breast cancer, pancreatic cancer, colon cancer, anal cancer, renal cancer, bladder cancer, prostate cancer, ovarian cancer, primary or metastatic melanoma, squamous cell carcinoma, basal cell carcinoma, brain cancer, angiosarcoma, hemangiosarcoma, head and neck carcinoma, thyroid carcinoma, soft tissue sarcoma, bone sarcoma, testicular cancer, gastrointestinal cancer, stomach cancer, glioblastoma, small cell lung cancer, non-small cell lung cancer, or any combination thereof.

33. Cell culture medium for expanding a cell population, the cell culture medium comprising a basal growth medium, a serum or serum replacement, and one or more cytokines and / or cytokine inhibitors.

34. The cell culture medium of claim 33, wherein the one or more cytokines and / or cytokine inhibitors comprise IL-2, IL-4, TGFP, and / or an anti-IFNy antibody.

35. The cell culture medium of claim 34, the cell culture medium comprising the IL-2 at a concentration of 10 lU / ml or more; the IL-4 at a concentration of 50 lU / ml or more; the TGFP at a concentration of 3 lU / ml or more; and / or the anti-IFNy antibody at a concentration of 0-20 pg / ml.

36. The cell culture medium of claim 34 or 35, wherein said expansion medium comprises TGFP at a concentration of 4.5 lU / ml or more.

37. The culture medium of any one of claims 33-36, wherein the culture medium does not comprise the anti-IFNy antibody or IL-4.

38. The cell culture medium of any one of claims 33-37, wherein the cell culture medium does not contain IL-2 as the only cytokine and / or cytokine inhibitors.

39. The cell culture medium of any one of claims 33-38, wherein the basal growth medium comprises a Roswell Park Memorial Institute (RPMI) 1640 medium, X-VIVO™-15 medium, CTS™ OPTMIZER™ Serum Free Medium, CTS™ OPTMIZER™ Pro Serum Free Medium, and / or IMMUNOCULTTM-XF T Cell Expansion Medium.

40. The cell culture medium of any one of claims 33-39, wherein the serum or serum replacement comprises fetal bovine serum (FBS), human AB serum, CTS™ Immune Cell Serum Replacement, and / or PHYSIOLOGIX™ Xeno-Free Serum Replacement.

41. A cell culture composition comprising the cell culture medium of any one of claim33-40 and a cell population cultured therein.

42. The cell culture composition of claim 41, wherein the cell population comprises a T cell population.

43. The cell culture composition of claim 42, wherein the T cell population comprises one or more primary human T cells, one or more tumor infiltrating lymphocytes (TILs), and / or one or more CD4+and / or CD8+T cells.

44. The cell culture composition of any one of claims 41-43, wherein the cell population comprises hematopoietic stem cells.

45. The cell culture composition of any one of claims 41-44, wherein the cell population has been modified by introducing an exogenous polynucleotide of interest or one or more gene editing reagents.

46. The cell culture composition of claim 45, wherein a polynucleotide encoding a chimeric antigen receptor (CAR) and / or a T cell receptor (TCR) has been introduced into the cell population such that one or more cells of the cell population express the CAR and / or the TCR.

47. The cell culture composition of any one of claims 41-44, wherein the cell population has not been modified with an exogenous polynucleotide of interest or one or more gene editing reagents.

48. The cell culture composition of any one of claims 42-47, wherein the T cell population has not been stimulated by an antigen.

49. The cell culture composition of any one of claims 42-48, wherein the T cell population has been stimulated by an antigen.

50. The cell culture composition of any one of claims 42-49, wherein the T cell population: secretes IL-9; expresses CCR4, CCR7, CD62L, and / or CD45RA; comprises increased viability, increased persistence, increased downregulation of exhaustion, increased killing activity, increased tumor control, and / or increased mitogenesis;comprises an early memory T cell phenotype or prolonged survival; comprises less differentiation into an effector phenotype or less T cell exhaustion; comprises increased proliferation or activation in the presence or absence of TGFP or in the absence of IL-2 in medium; comprises decreased levels of phosphorylated SMAD3; comprises decreased potentiation of the TGFP signaling pathway in response to stimuli; and / or comprises an increased ratio of CD4+T cells over CD8+T cells, relative to a control T cell population cultured in a control medium not comprising the cell culture medium.

51. The cell culture composition of claim 50, wherein in the T cell population: the viability is statistically significantly increased by up to about 15%; the persistence is statistically significantly increased by up to about 100%; the exhaustion is statistically significantly downregulated by up to about 80%; a volume of a tumor contacted by the cultured T cell population is statistically significantly decreased, or eradicated; the mitogenesis is statistically significantly increased by up to about 50% in response to a target antigen; the differentiation into an effector phenotype is statistically significantly decreased down to 0%; the proliferation is statistically significantly increased by up to about 50% in the presence of TGFP; the proliferation is statistically significantly increased by up to about 50% in the absence of IL-2; the activation response is statistically significantly increased by up to 200% in the presence of TGFP; the activation response is statistically significantly increased by up to 500% in the absence of TGFP; and / or the ratio of CD4+T cells over CD8+T cells is statistically significantly increased by up to about 80%, as compared to the control T cell population.

52. The cell culture composition of any one of claims 42-51, wherein expression of one or more genes associated with an early memory T cell phenotype or prolonged survival isincreased, and / or expression of one of more genes associated with a T cell effector phenotype, T cell exhaustion, or suppression of T cell responses to tumors is decreased in the T cell population relative to a control T cell population cultured in a control medium not comprising the cell culture medium.

53. The cell culture composition of any one of claims 42-52, wherein expression of PTGER2 is decreased in the T cell population relative to a control T cell population cultured in a control medium not comprising the cell culture medium.

54. The cell culture composition of any one of claims 42-53, wherein expression of one or more of RUNX2, CCR7, CTSL, MYB, SELL, IL-9, IKZF2, CCR4, BATF3, SMAD2, JUN, IRF4, USP18, MX1, IFNGR2, CD4, and TCF7 is increased, and / or expression of one or more of LAG3, LAIR1, FASLG, IFNG, KLRG1, TBX21, SLAMF7, GZMA, GZMB, PRF1, PTGER2, ADORA2A, BID, TGFBR1, ID2, and CD8a is decreased in the T cell population relative to a control T cell population cultured in a control medium not comprising the cell culture medium.

Images