Ketogenic diet and BHB enhance proliferation and Anti-tumor efficacy of adoptively transferred t cells

Abstract

In various aspects and embodiments, the disclosure provides methods for treating a patient with a CAR-T therapy or improving chimeric antigen receptor (CAR)-T therapy, the methods including administering to a subject in need thereof a therapeutically effective amount of a CAR-T therapy and a ketogenic composition comprising a ketogenic diet, beta-hydroxybutyrate (BHB), a BHB salt, a BHB precursor, a ketone diester, or a combination thereof.

Description

[0001] Attorney Docket No: 046483-7485W01(04066)

[0002] KETOGENTC DIET AND BHB ENHANCE PROLIFERATION AND ANTI-TUMOR EFFICACY OF ADOPTIVELY TRANSFERRED T CELLS

[0003] CROSS-REFERENCE TO RELATED APPLICATION

[0004] The present application is entitled to priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 715,446 filed on November 1, 2024, which is herein incorporated by reference in its entirety.

[0005] BACKGROUND

[0006] The development of the chimeric antigen receptor (CAR) and its successful clinical use to direct T cells against specific types of cancers has been an important advancement in cancer immunotherapy. Despite the efficacy of some of these approaches, most patient responses are transient. Chronic T cell exhaustion is a significant challenge in cancer immunotherapy, making it difficult for T cells to effectively clear tumors. Although monoclonal antibodies targeting immune checkpoints like PD-1 have shown promise, their benefits are often limited to a select group of patients with solid tumors, and they can come with substantial off-tumor side effects. Additionally, these therapies typically struggle to show effectiveness against hematological malignancies. While CAR-T cell therapy has provided hope, especially for patients with refractory B cell lymphoma, CAR-T cells have proven so far ineffective in solid tumor settings due to their complex tumor microenvironments.

[0007] Therefore, there is a pressing need to develop strategies to safely, broadly, and feasibly enhance CAR-T cell therapies for hematological and solid tumors.

[0008] SUMMARY OF THE INVENTION

[0009] In one aspect, methods for treating a patient with a CAR-T therapy or improving chimeric antigen receptor (CAR)-T therapy are provided, the methods, comprises administering to a subject in need thereof a therapeutically effective amount of a CAR-T therapy and a ketogenic composition comprising a ketogenic diet, beta-hydroxybutyrate (BHB), a BHB salt, a BHB precursor, a ketone diester, or a combination thereof. Attorney Docket No: 046483-7485W01(04066)

[0010] In some embodiments, the ketogenic composition comprises D-beta-hydroxybutyric (D- BHB) acid, L-beta-hydroxybutyric (L-BHB) acid, racemic beta-hydroxybutyric acid or a salt, monoester, polyester, or mixture thereof.

[0011] In some embodiments, the ketogenic composition comprises a BHB salt or BHB ester.

[0012] In some embodiments, the ketogenic composition comprises a BHB mineral salt, a BHB organic salt, or a combination thereof.

[0013] In some embodiments, the ketogenic composition comprises a BHB mineral salt. In certain embodiments, the BHB mineral salt is sodium BHB, potassium BHB, calcium BHB, magnesium BHB, lithium BHB, or a mixture thereof.

[0014] In some embodiments, the ketogenic composition comprises a BHB organic salt. In certain embodiments, the BHB organic salt is arginine BHB, lysine BHB, histidine betahydroxybutyrate, ornithine BHB, creatine BHB, agmatine BHB, citrulline BHB, or a mixture thereof.

[0015] In some embodiments, the ketogenic composition comprises a BHB sodium salt, a BHB potassium salt, a BHB calcium salt, a BHB magnesium salt, or a mixture thereof.

[0016] In some embodiments, the ketogenic composition comprises a BHB precursor selected from the group consisting of 1,3-butanediol (BDO), ethyl acetoacetate, ethyl betahydroxybutyrate, and mixture thereof.

[0017] In some embodiments, the ketogenic composition comprises a ketone diester. In certain embodiments, the ketone diester is R, S 1,3-butanediol di acetoacetate or R,S 1,3 butanediol acetoacetate diester.

[0018] In some embodiments, the ketogenic composition further comprises a medium chain fatty acid ester thereof, or derivative thereof. In certain embodiments, the medium chain fatty acid, ester thereof, or derivative thereof is selected from the group consisting of coconut oil, coconut milk powder, fractionated coconut oil, palm oil, palm kernel oil, caprylic acid, isolated hexanoic acid, isolated octanoic acid, isolated decanoic acid, ethoxylated triglyceride or derivative thereof, enone triglyceride or derivative thereof, aldehyde triglyceride or derivative thereof, monoglyceride or derivative thereof, diglyceride or derivative thereof, triglyceride or derivative thereof, medium chain triglyceride salts, salt derivatives thereof, and mixtures thereof. Attorney Docket No: 046483-7485W01(04066)

[0019] In some embodiments, the ketogenic composition is administered to the subject before CAR-T therapy. In some embodiments, the ketogenic composition is administered to the subject during the course of CAR-T therapy. In some embodiments, the ketogenic composition is administered to the subject before and during CAR-T therapy.

[0020] In some embodiments, the ketogenic composition is administered to the subject once per day, twice per day, or three times per day for a period of at least one week. In certain embodiments, the ketogenic composition is administered to the subject once per day, twice per day, or three times per day for a period of at least two weeks. In other embodiments, the ketogenic composition is administered to the subject once per day, twice per day, or three times per day for a period of at least one month.

[0021] In some embodiments, the beta-hydroxy butyrate (BHB), BHB salt, BHB precursor, or ketone diester is administered to the subject in an amount between about 0.01-50 g / kg / day. In some embodiments, the beta-hydroxybutyrate (BHB), BHB salt, BHB precursor, or ketone diester is administered to the subject in an amount between about 0.1-20 g / kg / day. In some embodiments, the beta-hydroxybutyrate (BHB), BHB salt, BHB precursor, or ketone diester is administered to the subject in an amount between about 1-10 g / kg / day. In some embodiments, the beta-hydroxybutyrate (BHB), BHB salt, BHB precursor, or ketone diester is administered to the subject in an amount between about 2-5 g / kg / day.

[0022] In another aspect, a method for improving CAR-T therapy comprises administering to a subject in need thereof a therapeutically effective amount of CAR-T therapy, wherein the subject is in a state of ketosis before and / or after administration of the therapeutically effective amount of CAR-T therapy, wherein the subject is in a state of ketosis when the subject’s blood ketone levels are greater than about 0.5 mmol / L. In some embodiments, the subject is in a state of ketosis when the subject’s blood ketone levels are greater than about 1.0 mmol / L, greater than about 1.5, greater than about 2.0 mmol / L, greater than about 2.5 mmol / L, greater than about 3 mmol / L, greater than about 5.0 mmol / L, greater than about 7.5 mmol / L, greater than about 10.0 mmol / L, greater than about 12.5, greater than about 15.0, or any range thereof. In some embodiments, the subject is in a state of ketosis when the subject’s blood ketone levels are between about 0.5 mmol / L and about 16 mmol / L. In some embodiments, the subject is in the Attorney Docket No: 046483-7485W01(04066) state of ketosis following administration of a ketogenic composition described herein. In some embodiments, the subject is in the state of ketosis after being on a ketogenic diet.

[0023] In some embodiments, the subject is in the state of ketosis before and after administration of the therapeutically effective amount of CAR-T therapy. In some embodiments, the subject is in a state of ketosis for at least about 1-2 weeks before administration of the CAR-T therapy. In some embodiments, the subject is in a state of ketosis for at least 1 week following administration of the CAR-T therapy. In some embodiments, the subject is in a state of ketosis for at least 2 weeks following administration of the CAR-T therapy. In some embodiments, the subject is in a state of ketosis for at least 3 weeks following administration of the CAR-T therapy. In some embodiments, the subject is in a state of ketosis for at least 4 weeks following administration of the CAR-T therapy. In some embodiments, the subject is in a state of ketosis for at least 6 weeks following administration of the CAR-T therapy. In some embodiments, the subject is in a state of ketosis for at least 8 weeks following administration of the CAR-T therapy. In some embodiments, the subject is in a state of ketosis for at least 10 weeks following administration of the CAR-T therapy. In some embodiments, the subject is in a state of ketosis for at least 12 weeks following administration of the CAR-T therapy.

[0024] In some embodiments, the subject is in a state of ketosis for at least 2 weeks following administration of a ketogenic composition described herein. In some embodiments, the subject is in a state of ketosis for at least 3 weeks following administration of a ketogenic composition described herein. In some embodiments, the subject is in a state of ketosis for at least 4 weeks following administration of a ketogenic composition described herein. In some embodiments, the subject is in a state of ketosis for at least 6 weeks following administration of a ketogenic composition described herein. In some embodiments, the subject is in a state of ketosis for at least 8 weeks following administration of a ketogenic composition described herein. In some embodiments, the subject is in a state of ketosis for at least 10 weeks following administration of a ketogenic composition described herein. In some embodiments, the subject is in a state of ketosis for at least 12 weeks following administration of a ketogenic composition described herein. Attorney Docket No: 046483-7485W01(04066)

[0025] In some embodiments, the subject in the state of ketosis has increased serum level(s) of BHB, acetoacetate, and / or acetone compared to a subject that is not in a state of ketosis. In some embodiments, the subject in the state of ketosis has increased serum level(s) of one or more metabolites compared to a subject that is not in a state of ketosis, wherein the one or more metabolites are selected from the group consisting of BHB, docosanoic acid, a-linolenic acid, linoleic acid, erucic acid, tetradecanedioic acid, 10-hydroxy decanoic acid, stearic acid, 1- norleucine, arachidic acid, 1-norleucine, nicotinamide 1 -oxide, d-saccharic acid, n-acetyl-d- alloisoleucine, uric acid, methylcysteine, oleamide, palmitoylcarnitine, dl-P-leucine, oleic acid, and combinations thereof.

[0026] In some embodiments, the increased serum level(s) are increased by at least 50% compared to a subject before being in a state of ketosis. In some embodiments, the increased serum level(s) are increased by at least 2-fold compared to a subject before being in a state of ketosis. In some embodiments, the increased serum level(s) are increased by at least about 5-fold to 20-fold compared to a subject before being in a state of ketosis. In some embodiments, the subject that is not in a state of ketosis is the subject being administered the CAR-T cells.

[0027] In some embodiments, the ketogenic composition is administered to the subject once per day, twice per day, or three times per day for a period of at least one week. In certain embodiments, the ketogenic composition is administered to the subject once per day, twice per day, or three times per day for a period of at least two weeks. In other embodiments, the ketogenic composition is administered to the subject once per day, twice per day, or three times per day for a period of at least one month.

[0028] In some embodiments, the beta-hydroxy butyrate (BHB), BHB salt, BHB precursor, or ketone diester is administered to the subject in an amount between about 0.01-50 g / kg / day. In some embodiments, the beta-hydroxybutyrate (BHB), BHB salt, BHB precursor, or ketone diester is administered to the subject in an amount between about 0.1-20 g / kg / day. In some embodiments, the beta-hydroxybutyrate (BHB), BHB salt, BHB precursor, or ketone diester is administered to the subject in an amount between about 1-10 g / kg / day. In some embodiments, the beta-hydroxybutyrate (BHB), BHB salt, BHB precursor, or ketone diester is administered to the subject in an amount between about 2-5 g / kg / day. Attorney Docket No: 046483-7485W01(04066)

[0029] In another aspect, a method of producing a cell population comprising T-cells comprises culturing T cells in cell culture media comprising beta-hydroxybutyrate (BHB), a BHB salt, a BHB precursor, a ketone diester, or a combination thereof.

[0030] In some embodiments, the cell culture media comprises BHB in an amount between about 0.5-10 mM. In some embodiments, the cell culture media comprises BHB in an amount between about 0.5-5 mM. In some embodiments, the cell culture media comprises BHB in an amount between about 2-10 mM. In some embodiments, the cell culture media comprises BHB in an amount between about 2-5 mM.

[0031] In some embodiments, the T-cells are cultured in media comprising D-beta- hydroxybutyric (D-BHB) acid, L-beta-hydroxybutyric (L-BHB) acid, racemic betahydroxybutyric acid or a salt, monoester, polyester, or mixture thereof.

[0032] In some embodiments, the T-cells are cultured in media comprising a BHB salt or BHB ester.

[0033] In some embodiments, the T-cells are cultured in media comprising a BHB mineral salt, a BHB organic salt, or a combination thereof.

[0034] In some embodiments, the T-cells are cultured in media comprising a BHB mineral salt. In certain embodiments, the BHB mineral salt is sodium BHB, potassium BHB, calcium BHB, magnesium BHB, lithium BHB, or a mixture thereof.

[0035] In some embodiments, the T-cells are cultured in media comprising a BHB organic salt. In certain embodiments, the BHB organic salt is arginine BHB, lysine BHB, histidine beta- hydroxybutyrate, ornithine BHB, creatine BHB, agmatine BHB, citrulline BHB, or a mixture thereof.

[0036] In some embodiments, the T-cells are cultured in media comprising a BHB sodium salt, a BHB potassium salt, a BHB calcium salt, a BHB magnesium salt, or a mixture thereof.

[0037] In some embodiments, the T-cells are cultured in media comprising a BHB precursor selected from the group consisting of 1,3 -butanediol (BDO), ethyl acetoacetate, ethyl beta- hydroxybutyrate, and mixture thereof. Attorney Docket No: 046483-7485W01(04066)

[0038] In some embodiments, the T-cells are cultured in media comprising a ketone diester. In certain embodiments, the ketogenic composition is administered to the subject the ketone diester is R, S 1,3 -butanediol diacetoacetate or R,S 1,3 butanediol acetoacetate diester.

[0039] In some embodiments, the T-cells are cultured with a composition further comprising a medium chain fatty acid ester thereof, or derivative thereof. In certain embodiments, the medium chain fatty acid, ester thereof, or derivative thereof is selected from the group consisting of coconut oil, coconut milk powder, fractionated coconut oil, palm oil, palm kernel oil, caprylic acid, isolated hexanoic acid, isolated octanoic acid, isolated decanoic acid, ethoxylated triglyceride or derivative thereof, enone triglyceride or derivative thereof, aldehyde triglyceride or derivative thereof, monoglyceride or derivative thereof, diglyceride or derivative thereof, triglyceride or derivative thereof, medium chain triglyceride salts, salt derivatives thereof, and mixtures thereof.

[0040] In some embodiments, the cell population comprises an oxygen consumption rate (OCR) between about 1-200 pmol / min. In some embodiments, the cell population comprises an extracellular acidification rate (ECAR) of about 1-150 pmol / min.

[0041] In a further aspect, a method of manufacturing CAR-T cells from peripheral blood mononuclear cells (PBMCs) from a blood sample obtained from a subject, comprises: (a) producing a T-cell enriched population of cells from a population of immune cells isolated from a subject; (b) transforming the T-cell enriched population of cells with a vector encoding a chimeric antigen receptor (CAR); and (c) culturing the T-cells obtained in step (b) in media comprising beta-hydroxybutyrate (BHB), a BHB salt, a BHB precursor, a ketone diester, or a combination thereof.

[0042] In some embodiments, the cell culture media comprises BHB in an amount between about 0.5-10 mM. In some embodiments, the cell culture media comprises BHB in an amount between about 0.5-5 mM. In some embodiments, the cell culture media comprises BHB in an amount between about 2-10 mM. In some embodiments, the cell culture media comprises BHB in an amount between about 2-5 mM.

[0043] In some embodiments, the T-cells are cultured in media comprising a BHB salt or BHB ester. Attorney Docket No: 046483-7485W01(04066)

[0044] In some embodiments, the T-cells are cultured in media comprising a BHB mineral salt, a BHB organic salt, or a combination thereof.

[0045] In some embodiments, the T-cells are cultured in media comprising a BHB mineral salt. In certain embodiments, the BHB mineral salt is sodium BHB, potassium BHB, calcium BHB, magnesium BHB, lithium BHB, or a mixture thereof.

[0046] In some embodiments, the T-cells are cultured in media comprising a BHB organic salt. In certain embodiments, the BHB organic salt is arginine BHB, lysine BHB, histidine betahydroxybutyrate, ornithine BHB, creatine BHB, agmatine BHB, citrulline BHB, or a mixture thereof.

[0047] In some embodiments, the T-cells are cultured in media comprising a BHB sodium salt, a BHB potassium salt, a BHB calcium salt, a BHB magnesium salt, or a mixture thereof.

[0048] In some embodiments, the T-cells are cultured in media comprising a BHB precursor selected from the group consisting of 1,3 -butanediol (BDO), ethyl acetoacetate, ethyl betahydroxybutyrate, and mixture thereof.

[0049] In some embodiments, the T-cells are cultured in media comprising a ketone diester. In certain embodiments, the ketogenic composition is administered to the subject the ketone diester is R, S 1,3 -butanediol diacetoacetate or R,S 1,3 butanediol acetoacetate diester.

[0050] In some embodiments, the media further comprises a medium chain fatty acid, ester thereof, or derivative thereof. In some embodiments, the medium chain fatty acid, ester thereof, or derivative thereof is selected from the group consisting of coconut oil, coconut milk powder, fractionated coconut oil, palm oil, palm kernel oil, caprylic acid, isolated hexanoic acid, isolated octanoic acid, isolated decanoic acid, ethoxylated triglyceride or derivative thereof, enone triglyceride or derivative thereof, aldehyde triglyceride or derivative thereof, monoglyceride or derivative thereof, diglyceride or derivative thereof, triglyceride or derivative thereof, medium chain triglyceride salts, salt derivatives thereof, and mixtures thereof.

[0051] In a further aspect, the present invention provides a population of T-cells, wherein the population of T-cells comprises an oxygen consumption rate (OCR) between about 1-200 pmol / min and / or an extracellular acidification rate (ECAR) of about 1-150 pmol / min. In certain embodiments, the population of T-cells comprises CAR-T cells. Attorney Docket No: 046483-7485W01(04066)

[0052] BRIEF DESCRIPTION OF THE DRAWINGS

[0053] The foregoing and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings.

[0054] FIGs. 1A-1K: Ketogenic diet potentiates CART 19 via P-hydroxybutyrate. (1A) Experimental schema to screen various diets in Balb / c mice implanted with A20 murine diffuse large B cell lymphoma tumors. Diets were first replaced in each mouse cage. After 1 week, 2 x 106A20 cells were inoculated subcutaneously into the right flank mice, and on day 14 postimplantation 0.35 x io5CAR19+ T cells were infused via tail vein. (IB) Composition of macro- and micro- nutrients in each of six screened diets. Of note, the Western diet contains 0.15% cholesterol. (1C) Average tumor volumes (± SEM) at an aligned time points following CART19 infusion across all three independent diet screening cohorts (day 16 for cohort 1, day 16 for cohort 2, day 18 for cohort 3), chosen to permit comparable tumor burden between cohorts. P value (Control vs. KD): 0.0155 (ID) Merged overall survival of three independent diet screening cohorts. (1E-1F) Global metabolomic profding of serum from individual mice of representative cohort at day 7 post infusion. (IE) 2D PCA plot depicting clustering of serum metabolites compositions according to diet. (IF) Volcano plot of differentially enriched serum metabolites between ketogenic and control diet. (1G) 72 hr proliferation of human CART19 seeded with irradiated OCI-Lyl8 cells (effector: target = 0.25, n=3 technical replicates). Here, fold change of T cells over vehicle control wells is represented. (1H) Correlation of fold changes from (IF) and (1G). (II) Experimental schema to compare effect of BHB and ketogenic diet to a standard control in vivo using the A20 model in Balb / c. (1 J) Average tumor volumes (± SEM). (IK) Analysis of CD45.1+ tumor infdtrating CAR T cells at day 7 post-infusion. Left: Percent IL-2+IFNy+. Right: Percent PD-1+LAG3+. Statistical significance was determined by unpaired t- test (1C), one-way ANOVA with post hoc Tukey tests (1G, 1J, IK), q-value, Benjamini- Hochberg FDR-adjust (IF), or log-rank Mantel-Cox (ID).

[0055] FIGs. 2A-2J: Ketogenic diet potentiates CART19 via P-hydroxybutyrate. (2A) Experimental schema to screen various diets in Balb / c mice implanted with A20 mouse Attorney Docket No: 046483-7485W01(04066) lymphoma tumors. First, diets were replaced in each mouse cage. After 7 days, 2 x 106 A20 cells were inoculated subcutaneously into the right flank of mice, and on day 18 post-implantation, 0.35 x io64-lBB^ CAR19+ T cells were infused via tail vein. (2B) Percent composition of macro and micro- nutrients in each of six screened diets. Here, “Other” refers to minerals, vitamins, and antioxidants. Of note, the Western diet contains 0.15% cholesterol. See Table 1 for full compositions of each diet. In these studies, three independent experiments of diet screenings were performed. On average, n=5-14 Balb / c mice (1-3 cages) were used per experimental arm, per screening cohort. See Source Data for further details. (2C) Average tumor volume at an aligned time points following CART19 infusion across all three independent diet screening cohorts (day 16 for cohort 1, day 16 for cohort 2, day 18 for cohort 3), chosen to permit comparable tumor burden between cohorts. P value (Control vs. KD): 0.0155. (2D) Median overall survival of mice in diet screening of three independent cohorts. (2E) A separate in vivo experiment, including untransduced T cell (UTD) controls infused into mice receiving KD, was performed with four experimental arms: UTD+Control, UTD+KD, CART19+Control, and CART19+KD. This experiment followed the same timeline and doses as described in panel A. Shown is the average tumor volume at day 14 post-UTD / CART19 infusion. (2F) Proliferation (96 h) of human CART19 seeded with irradiated OCI-Lyl8 cells (effector: target = 0.25, / ?=4 technical replicates) in the presence of 1 : 10 diluted serum from KD- or control diet-fed mice, collected 7 days post CART 19 infusion. See Methods for details on experimental preparation. (2G) 2D PCA plot depicting clustering of serum metabolite compositions according to diet. Here, serum was collected at day 7 post CART 19 infusion. (2H) Heatmap of global metabolomics used to drive clustering in FIG. 2G. Differentially detected metabolites in KD relative to all other diets are highlighted. (21) Volcano plot of differentially enriched serum metabolites between ketogenic and control diet. (2 J) Proliferation (72 h) of human 4- 1 BB^ CART19 seeded with irradiated OCI-Lyl8 cells (effector: target = 0.25, n=3 technical replicates) and individual KD-enriched metabolites, each at a final concentration of 1 mM. Here, fold change of T cells over respective vehicle control wells is shown. Statistical significance was determined by one-way ANOVA (2C, 2E, 2J), log-rank Mantel-Cox (2D), unpaired / -test (2F), or q-value, Benjamini -Hochberg FDR-adjusted (21). s.c., subcutaneous; i.p., intraperitoneal; i.v., Attorney Docket No: 046483-7485W01(04066) intravenous; CTX, cyclophosphamide; CT, control; KD, ketogenic diet; UTD, untransduced; TMNO, trimethylamine N-oxide; DHB, 2,5-dihydroxybenzaldehyde; ADG, 1,5-Anhydro-D- glucitol; Lino, linoleic acid; a-Lino, linolenic acid; Eru, erucic acid; Tetradec, tetradecanedioic acid; Doco, docosanoic acid. Plotted are means ± SD. ns.p > 0.05, *p < 0.05, **p < 0.01, ****p < 0.0001.

[0056] FIGs. 3A-3R: BHB enhances CAR-T cell antitumor function in human preclinical models. (3A) Schema to examine the effects of three different BHB treatment durations: (1) Early stop: from tumor-implantation to CAR T-cell infusion only, (2) Late start: after CAR T- cell infusion only, or (3) Whole treatment: throughout the whole course of treatment. (3B) Individual tumor curves. (3C) Overall survival of mice, n= 12 in Early stop group, w=13 mice in Late start group, =\ 3 mice in whole treatment group. (3D) Schema to evaluate in vivo effect of BHB on CART19 in immunodeficient mice (NOD SCID gamma, or NSG) bearing OCI-Lyl8 DLBCL tumors. Here, 5 * 106OCI-Lyl8 cells were subcutaneously implanted into the right flank of mice on day -11, and on day 0, a sub therapeutic dose of 3 x io64-lBB(^ CART 19 cells was infused. On day -3, daily oral gavage began of 280 pL of BHB (HVMN Ketone-IQ), or 80 mg per mouse. (3E) Individual tumor volumes following CART 19 infusion. (3F) Bioluminescent images of subcutaneous tumors on right flank in mice (left) and flux quantification (right) on day 17 post CART 19 infusion. (3H) Peripheral expansion of CAR T cells on days 7, 14, and 21 following infusion. (31) Serum concentration of IFNy on days 4 and 7 following infusion, measured using ELISA. (3J) Overall survival of mice. Here, w=4 mice in UTD groups, and z?=7 mice in CART19 groups. (3K) Schema using a B cell acute lymphoblastic leukemia (B-ALL) model in NSG mice. In this model, starting on day - 15, BHB was provided ad libitum in the drinking water for individual cages. Then, 1 x 106Nalm6 cells were infused on day -6 to establish B-ALL tumors, followed by infusion of 0.5 x 1064-lBB(^ CART19 cells on day 0. Mice were imaged using IVIS 1-2 times per week. (3L) Individual mouse BLI measurements. (3M) Representative BLI images (left) and flux quantification (right) on day 27 following infusion. (3N) Overall survival of mice. Here, H=5 mice per group for all groups. (30) Schema using a pancreatic adenocarcinoma (PDAC) model (AsPC-1) in NSG mice. Here 2x 106AsPC-1 cells were implanted subcutaneously on day -23, and BHB was provided ad libitum in Attorney Docket No: 046483-7485W01(04066) the drinking water for individual cages (200 mL BHB per 1 L) on day -14. On day 0, 0.5x106anti-mesothelin 4-lBB(^ CAR T cells (clone M5, CARTmeso) were infused into mice. (3P) Average tumor volumes ± SEM for each group. (3Q) Day 23 post infusion values of tumor size (left) and bioluminescence as measured by IVIS (right). (3R) Overall survival of mice. n=5 mice in UTD groups, and zz= 10 mice in CARTmeso groups. Statistical significance was determined by unpaired / -test (3F and 3M), one-way ANOVA with post hoc Tukey tests (3H, 31 and 3Q), or log-rank Mantel-Cox (3J, 3N and 3R). CR, complete response; BLI, bioluminescence intensity; Veh, vehicle. Plotted are means ± SEM or means ± SD. ns. p > 0.05, *p < 0.05, **p < 0.01, *** / ? < 0.001, **** / ? < 0.0001.

[0057] FIGs. 4A-4Q: BHB effectively fuels the TCA cycle in antigen-activated CAR T cells. (4A-4B) Carbon-tracing experiment using either uniform U-glucose or U-labelled BHB in antigen-activated 4-lBB(^ CAR T cells. (4A) Fraction of13C label in TCA metabolites antigen- activated human CART19 incorporated after 5 h culture with one of two mediums: (a) lOmM [U-13Ce]-glucose + 5mM BHB or (b) lOmM glucose + 5mM [U-13C4]-BHB. Metabolites were extracted and analyzed using the Agilent QTOF 6546 High-Resolution LC-MS. Here, CART19 were seeded with irradiated OCI-Lyl8 (effector: target = 0.25, n=3 technical replicates) for 72 h prior to a 5 h culture in labelled media. (4B) Oxygen consumption rate (OCR) plots (left) and quantification of OCR (right) of antigen-activated human CAR T cells treated with no BHB, 2 mM BHB or 5 mM BHB. (4C) OCR of murine CAR T cells cultured during 5 days of expansion in no BHB or 5mM BHB (Left). Right: data of each individual biological replicates are shown. (4D) Left: PCA of ATAC-seq results from murine CAR T treated with no or 5 mM BHB. Right: log2FC shows opening of regions like FOXO1, ZAP70, TCF7, and GZMB. (4E) Western blot demonstrating partial knockout ofBDHl. Quantification of knockout using ImageJ (right). (4F) Experimental design to generate BDH1 -deficient murine CART19 using CRISPR-Cas9. A20 tumors were established in Balb / c mice, and mice were placed on either standard water or water containing BHB (200 mL BHB / IL). Mock electroporated (Cas9 only) CAR T cells or BDH1 knockout CAR T cells were infused into A20-bearing mice. (4G) Average tumor volumes (± SEM) are shown. (4H) 24 hr fFNy production of human CART 19 seeded with irradiated OCL Lyl8 cells (effector: target = 0.25, n=5 technical replicates) in either hypoxic or normoxic Attorney Docket No: 046483-7485W01(04066) conditions. Statistical significance was determined by unpaired / -test (A), one-way ANOVA with post hoc Tukey test (4B, 4G, 4H), and paired / -test (4C). (41) Full isotopologue distributions of representative TCA intermediates reveal differential labeling patterns between glucose- and BHB-derived carbons. (4J-4N) Simultaneous tracer experiment with [l,6-13Ce]-glucose and [U-13C4]-BHB allowing parallel assessment of substrate utilization. Here, CD28^ CART19 were seeded with irradiated OCI-Lyl8 (effector: target = 0.25, / / =4 technical replicates) for 72 h prior to culture in labelled media containing lOmM [l,6-13Ce]-glucose and 5mM [U-13C4]-BHB. CART19 cells were collected prior to culture (-10 min) and at 0, 10, 70, 150, and 300 min (5 h) of culture for mass spectrometry analysis. (4J). Schematic of experiment. (4K). Uptake of glucose (m+2) and BHB (m+4) into antigen-activated CAR T cells over 5 hours. (4L). Heatmap summarizing pool contributions from glucose and BHB across key metabolites at isotopic steady state (5h). (4M-4N). Kinetic labeling of citrate (4M) and glutamate (4N) from either glucose (m+1) or BHB (m+2), demonstrating dynamic incorporation of tracer-derived carbons into these TCA intermediates. (40) Agilent Seahorse XF analysis of mouse CAR T cells expanded for 5 days in vehicle or 5 mM BHB. From left to right: oxygen consumption rate (OCR), spare respiratory capacity (SRC), maximal O2 consumption (VO2 Max). (4P) ATP production, shown as total output and partitioned into glycoATP, mitoATP, spare glycoATP, and spare mitoATP. (4Q). OCR of antigen-stimulated human 4-lBB(^ CART19 cells. Here, human CART19 cells were co-cultured with irradiated OCI-Lyl8 cells (effector: target ratio = 0.25) with media containing vehicle, 2 mM BHB, or 5 mM BHB. At 120 h, CART19 cells were washed (l x PBS), and assessed using Agilent Seahorse XF (n=5 technical replicates). Statistical significance was determined by unpaired / -test (4A, 41, 40, 4P). two-way ANOVA (4K-4N), or one-way ANOVA with Tukey ’s post hoc test (4Q). Glc, glucose; Pyr, pyruvate; AcAc-CoA, acetoacetyl- CoA; R+A, rotenone and antimycin A. Plotted are means ± SD. ns. p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0058] FIGs. 5A-5K: BHB induces transcriptional and epigenomic reprogramming in CAR T cells. (5A-5H) Single cell RNA sequencing of circulating CAR T cells. (5A) In vivo design for single cell RNA-sequencing (scRNA-seq) on circulating CART19 cells. First, NSG mice were inoculated subcutaneously with 5 x 106CD19+OCI-Lyl8 cells and gavaged daily with oral BHB Attorney Docket No: 046483-7485W01(04066)

[0059] (80mg per mouse) or vehicle (water). On day 0 post-implantation, mice received a curative dose of 5 x 106human CART19 cells. Peripheral blood T cells were purified on day 17 following infusion, (w = 7 mice per arm), pooled amongst groups, and prepared for 3' scRNA-seq (10X Genomics). (5B) Expression of canonical T cell subset markers after UMAP data clustering.

[0060] (5C) UMAP projection split into six major circulating T cell subsets: activated CD4+(red), cytotoxic CD8 (blue), MKI67hlCD4 / CD8+(purple), CD8+TEM (grey), CD4 / 8+TCM (green), and gamma delta (y5, dark green) T cells. Here, a total of n = 2414 cells for Vehicle treatment and ii = 2267 cells for BHB treatment are analyzed after downstream filtering and quality control using Seurat. (5D) Proportional distribution of T cell clusters between vehicle and BHB groups. (5E) UMAP highlighting the proportion of GZMB+CD8+T cells (blue) in CART 19 amongst with vehicle (left) or BHB (right) treatment. (5F) Heatmap showing expression of canonical mitochondrial respiration genes across Activated CD4+, GZMB+CD8+and MKI67111CD4 / 8+clusters. (5G) Volcano plots of differentially expressed genes (BHB vs Vehicle, adjusted p < 0.05, |log2FC| > 0.25) across all clusters. (5H) GSEA across all clusters for HALLMARK oxidative phosphorylation (left) and REACTOME respiratory electron transport (right). (5I-5K) ATAC-seq of human CART19 cells stimulated on CD19-coated wells for 48 h in the presence of vehicle or 5 mM BHB (n=5 technical replicates) (51) PCA of ATAC-seq profiles. (5J) Genomic annotation of ATAC-seq peaks. (5K) Volcano plot of differential chromatin accessibility (p < 0.05, |log2FC| > 0.3), highlighting gained (orange) and lost (purple) regions in BHB-treated CART19 cells relative to vehicle treated CART19 cells. Statistical significance was determined by Seurat’s FindMarkers function (a two-sided, Wilcoxon rank-sum test with Benjamini- Hochberg correction, in (5G), or permutation-derived NES with FDR correction (5H). ATAC- seq, Assay for Transposase-Accessible Chromatin using sequencing; GSEA, gene-set enrichment analysis; NES, normalized enrichment score. UMAP, Uniform Manifold and Approximation;

[0061] PCA, principal component analysis; TEM, T effector memory; TC , T central memory.

[0062] FIGs. 6A-6J: BHB augments mitochondrial metabolism in peripheral T cells of healthy volunteers. (6A) Schema of healthy human volunteer trial. Following overnight fast, blood was drawn from ten healthy volunteers (time = Tl). Subsequently, each volunteer was administered 105 ml BHB (i.e., 30g, HVMN Ketone-IQ). Blood was drawn again at 90 mins (time = T2) Attorney Docket No: 046483-7485W01(04066) following BHB ingestion. (6B) Blood concentrations of glucose and BHB over time, measured using the KetoMojo PLUS meter. (6C) Volcano plot of untargeted serum metabolomics comparing T2 vs T1 across all ten donors (p < 0.05, |log2FC| > 0.3). (6D-6J) Multiple metabolic analyses of T cells isolated from healthy volunteer peripheral blood. (6D) Representative Seahorse plots of oxygen consumption rate (mean ± SEM) from two healthy volunteers. (6E-6F) Spare respiratory capacity (6E) and Max VO2 (6F) (n=10 volunteers, paired from T1 to T2) as assessed by the Agilent Seahorse XF. (6G) ATP production rate (mean ± SEM) (n=10 volunteers). (6H). Schema of NADH and proton gradient (AT) generation from ketolysis of BHB in the mitochondria. (61-6 J). Quantification of mitochondrial NADH levels (61) and membrane potential (A m, 6J) in peripheral blood T cells from four healthy volunteers at T1 and T2. For each volunteer and each time point, 15 randomly selected fields of view (FOVs) per dish were imaged at 37 °C using a Zeiss Observer 7 widefield fluorescence microscope with a 20x / 0.8 objective. Each dot in the violin plots represents one FOV. Statistical significance was determined by two-tailed unpaired Welch’s t-tests (6C), paired / -test (6E), or unpaired t-test (6F, 6G, 61 and 6J). a.u., arbitrary unit. NADH, Nicotinamide adenine dinucleotide; TMRE, tetramethylrhodamine, ethyl ester, ns. p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, **** / ? < 0.0001.

[0063] FIGs. 7A-7K: BHB improves patient CAR-T and healthy donor-derived T cell metabolism. BHB serum levels correlate with CART19 expansion. (7A) Serum from n=17 patients taken on day 7 following infusion with CTL019 (UPCC13413) was profiled for metabolic composition via mass spectrometry. (7B) A statistical correlation was found between BHB levels on day 7 and CAR T cell expansion (measured by copies per pg of DNA). (7C) CRS grading at day 7, split by BHB levels of 17=40 CART19 patients. (7D-7E) BHB enhances in vitro expansion of patient T cells. (7E) Fold change quantification of final cell count over initial cell count. (7F) Oxygen consumption rate of patient-expanded CART 19 under 0 and 2 mM BHB (left), and quantification of oxygen consumption rate (right). (7G) Healthy human donor trial. Three healthy donors were subsequently administered 100 ml BHB (Ketone-IQ). Blood was drawn before and after BHB ingestion. (7H) Circulating BHB and glucose levels were measured every 15 minutes until 90 minutes. (7I-7K) T cells were isolated and analyzed via Agilent Attorney Docket No: 046483-7485W01(04066)

[0064] Seahorse XF immediately. Oxygen consumption rate (71), ATP production rate (7J) and Max VO2 (7K). Statistical significance was determined by linear regression (7B), Fisher’s test (7C), paired / -test (7E), and unpaired / -test (7F, 71, 7J, 7K).

[0065] FIGs. 8A-8I: Influence of diet and diet-derived metabolites on CAR T antitumor function. (8A) Tumor growth curves in individual mice following CART 19 infusion. (8B) Average weight changes over time. (8C) BHB level measured 7 days post-infusion. Upper panel: peak area of BHB from metabolomics analysis. Lower panel: concentration of BHB. (8D) Global metabolomic changes in the serum collect on day 7 post infusion. (8E) Average weight changes in mice administrated control diet, ketogenic diet or BHB. (8F) Analysis of CD45.U spleen T cells at day 3 post-infusion. Left: Percent ZL-2+IFNy+. Right: percent PD-U LAG3+. (8G) Analysis of CD1 U cells at day 7 post-infusion. Left: Percent CD1 lb+. Middle: Percent MHCI+ CD1 lb+. Right: Percent MHClUCDl lb+. (8H) Individual tumor sizes of the cohort evaluating the effects of a ketogenic diet or BHB on tumor growth in A20 tumor-bearing mice. Diets were introduced in each mouse cage for 1 week prior to subcutaneous inoculation of 2 * 106A20 cells subcutaneously into the right flank of mice. (81) Overall survival analysis. n=8 mice in control group, n=10 mice in ketogenic diet group, and n=8 mice in BHB group. Statistical significance was determined by one-way ANOVA with post hoc Tukey tests (8C, 8F, 8G), two-way ANOVA with (8B and 8E), or log-rank Mantel-Cox (81).

[0066] FIGs. 9A-9J: Ketogenic diet improves CAR T-mediated tumor control and survival compared to other dietary interventions. (9 A) Average tumor volume at aligned time points across three independent diet-screening cohorts (day 16 for cohorts I and II, day 18 for cohort III), chosen to allow comparable tumor burden between cohorts. Only the Control vs. KD comparison reached statistical significance (p = 0.0155). (9B) Merged median survival of mice on various diets from three independent screening cohorts. (9C) Tumor growth curves from a representative cohort of A20-bearing Balb / c mice fed with control or KD from a representative cohort, following UTD or CART19 infusion. (9D) Merged overall survival of mice on ketogenic versus control diets from three independent screening cohorts. (9E) Comparison of CAR T cell infiltration between mice fed with control or KD from a representative cohort, following UTD or CART 19 infusion. (9F) Tumor growth curves of CART19-treated mice fed high-fiber, high-fat, Attorney Docket No: 046483-7485W01(04066)

[0067] Western, or high-protein diets (representative cohort). (9G) Merged overall survival of mice on each diet from three independent screening cohorts. (9H) Proportion of CAR+T cells amongst CD3+T cells in A20 tumors (detected using the anti-G4S stain on flow cytometry), resected from a subset of mice (w=4-5 mice per arm) 7 days post CART 19 infusion. (91) Tumor growth curves in mice fed control or KD following UTD / CART19 infusion. (9 J) Left: Peak area of BHB from bulk metabolomics of mice serum collected at 7 days post CART 19 infusion, derived using data from FIG. 2G; Right: BHB blood concentration from the same samples, now measured using the KetoMojo PLUS monitor. Statistical significance was determined by one-way ANOVA (9A and 9B), log-rank Mantel-Cox (9D and 9G), two-way ANOVA (9E), unpaired t-test (9H), or oneway ANOVA with post hoc Tukey tests (9J). ns. p > 0.05, **p < 0.01, ****p < 0.0001.

[0068] FIGs. 10A-10I: Effects of BHB on the phenotype and function of CART 19 cells. (10A) BHB enhances the cytotoxicity murine CART 19 cells in vitro. (10B) Pretreatment of tumor cells with 5 mM of BHB does not affect the anti-tumor efficacy of CART19 cells. (10C) BHB concentration in blood of mice treated with BHB for varying duration. (10D) Schema to study the effect of ex vivo BHB treatment on CAR T cell function. 0 or 5 mM of BHB was added to the cell culture media during CART 19 cell manufacturing. A total of 5 * 106OCI-Lyl8 cells were inoculated subcutaneously into the right flank of NSG mice on day -11, followed by infusion of 3 x 106 UTD, or CART19 cells pre-treated with or without BHB on day 0. (10E) Human CART19 cells were manufactured with 0 or 5 mM of BHB, with CCR7 and CD45RA markers were used to identify naive, central memory (TCM), effector memory (TEM) T cells and terminal effector memory T cells (TEMRA). (10F) Average tumor burden. (10G) Overall survival of mice. n=5 mice per group. (10H) Blood BHB levels of human DLBCL model. (101) Percent body weight changes of mice. Statistical significance was determined by unpaired t-test (A,B,H), one-way ANOVA with post hoc Tukey tests (10C), two-way ANOVA with (10J), or log-rank Mantel-Cox (10G).

[0069] FIGs. 11A-11G: BHB treatment induces global genetic and epigenetic changes in CART19 cells. (HA) PCA of RNA-seq results from antigen-activated human CART19 cells. (11B) Heatmap of significantly differentially expressed genes (p <= 0.05, |log2FoldChange| >= 0.1). (11C) Volcano plot of differentially expressed genes. (HD) PCA of ATAC-seq results Attorney Docket No: 046483-7485W01(04066) from antigen-activated human CART19 cells. (1 IE) Heatmap of differential accessibility at coding promoters (p <= 0.05, |log2FoldChange| >= 0.1). (11F) Volcano plot of differentially accessible gene promoters. (11G) Pathway analysis of gene promoters with reduced accessibility in the presence of BHB.

[0070] FIGs. 12A-12N: Comparison of ketogenic diet to BHB supplementation on CAR T antitumor function. (12A) In vitro mouse CART 19 cytotoxicity against A20 cells after 48 h of coculture and treatment with vehicle or 5 mM BHB (n=5 technical replicates). (12B) In vitro mouse CART19 proliferation after 72 h of coculture with irradiated A20 cells and treatment with vehicle or 5 mM BHB (n=5 technical replicates, effector: target = 0.25). (12C) A20 cell growth over 6 days, under treatment with vehicle, 2 mM, or 5 mM BHB in vitro. (12D-12F) In vivo experiment to assess the influence of BHB (via daily gavage, 80mg BHB per mouse) and KD ad libitum) in comparison to a standard control diet following CART 19 infusion using the A20 model in Balb / c mice. (12D) Experimental design. Mice were administered respective dietary interventions one week prior to subcutaneous inoculation of 2 x 106A20 cells into the right flank. Mice were infused with 0.35 x 106CAR19+ T cells 18 days following tumor implantation. (12E- 12F) Individual mouse tumor volumes following UTD (12E) or mouse CART19 (12F) infusion in mice. (12G) Overall survival of mice. (12H) Average percent body weight changes (± SEM) over time from the same cohort. (121) Tumor infdtrating CD45.1+T cells at day 7 post infusion. Left: Quantification of percent IL-2+IFNy of CD45.1+population following 5 hours of ex vivo stimulation with PMA / Ionomycin and Brefeldin A. Right: Quantification of percent PD-1+LAG3+of CD45.H population. (12J) Schema of low-dose CD45.1+CART19 (O.lx 106CAR19+T cells) infusion model to assess host T cell (CD45.2Q immune responses. (12K) Representative plots for PD-1 and LAG-3 double-positive fraction. (12L) Quantification of percent PD-1+LAG3+of CD45.2+population. (12M) Quantification of percent TNFa+IL-2+of CD45.2+population. (12N) Quantification of tumor CD1 lb+cells and characterization of their antigen presentation in A20 tumors at day 7 post CART19 infusion. Left: Fraction of CDl lb+cells amongst total tumor cells. Middle: Percent MHC1 of CD1 lb+population. Right: Percent MHCII of CD1 lb+population. Statistical significance was determined by unpaired / -test (12A and 12B), one-way ANOVA with post hoc Tukey tests (12C, 121, 12L, 12M, 12N), two-way Attorney Docket No: 046483-7485W01(04066)

[0071] ANOVA (12F), or log-rank Mantel-Cox (12G). Plotted are means ± SEM or means ± SD. ns. p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0072] FIGs. 13A-13O: BHB must be actively present in the circulation to confer a functional benefit to CAR T cells. (13A) Schema to examine effects of three different BHB treatment regimens, supplemented via drinking water (i.e., ad libitum), in a the A20 model of mouse lymphoma in Balb / c mice. Here, the three regimens are: (1) Early stop: from tumor-implantation to CAR T cell infusion only, (2) Late start: after CAR T cell infusion only, or (3) Continuous treatment: throughout the entire course of the experiment. (13B) BHB concentration in the blood of mice for each group one day before CAR T infusion. (13C) Individual mouse tumor burden following mouse CART19 infusion. (13D) Overall survival of mice. In this model, n=l 2 mice in Early stop group, w=12 mice in Late start group, w=13 mice in continuous treatment group. (13E- 13F) Effect of BHB treatment during human 4-lBB(^ CART19 manufacturing on CAR T cell phenotype and in vivo function. (13E) Schema for BHB treatment during human CART19 manufacturing and subsequent in vivo experiment in human lymphoma model (CD 19+ OCL Lyl8 DLBCL). Human CART 19 cells were manufactured from healthy donor T cells (as described in Methods) with vehicle or 5 mM BHB. (13F) CCR7 and CD45RA markers were used to identify naive, central memory (TCM), effector memory (TEM), and effector memoryexpressing CD45RA (TEMRA) T cells. Shown is a representative contour plot of post-expansion phenotype n=2 biological donors). (13G-13H) A total of 5 * 106OCI-Lyl8 cells were inoculated subcutaneously into the right flank of NSG mice on day -11, followed by infusion of 3 x 106UTD, or CART19 cells pre-treated with or without BHB during expansion on day 0. (13G) Average tumor volumes following CART 19 infusion (± SEM). Statistics are shown at day 16 post-infusion of CART19. (13H) Overall survival of mice. Here, n=5 mice per group. (131) Blood BHB levels of human DLBCL model from FIG. 3A at 7 days post CART19 infusion.

[0073] (13 J) Percent body weight changes of mice from FIG. 3 A. (13K-13L) In vitro effect of BHB (0- 100 mM) on growth rate of OCI-Lyl8 (DLBCL) (13K) and Nalm6 (B-ALL) (13L) cells after 72 hours of in vitro treatment. (13M) In vitro cytotoxicity of 4- 1 BBq-based (left) and CD28q- (right) CART 19 cells against OCI-Lyl8 cells following 72-hour treatment with vehicle, 2 mM, or 5 mM BHB. (13N) CD25 expression (activation) on CART19 cells after a 24 h stimulation on Attorney Docket No: 046483-7485W01(04066)

[0074] CD19-coated wells and 2 mM BHB treatment. Representative histograms (left) and CD25 MFI (x 104) of CD3+ cells (right). (130) AsPC-1 cell growth in vitro during treatment with 0 mM (vehicle), 2mM, or 5mM BHB. Statistics are shown at 60 h of culture. Statistical significance was determined by one-way ANOVA with post hoc Tukey tests (13B, 13G, 131, 13M, and 130), log-rank Mantel-Cox (13D and 13H), or unpaired / -test (13N). MFI, median fluorescent intensity. Plotted are means ± SEM or means ± SD. ns. p > 0.05, *p < 0.05, **p < 0.01, *** ? < 0 ooi, 0.0001.

[0075] FIG. 14: BHB carbons feed branch metabolites of the TCA. Fraction of13C label in TCA metabolites antigen-activated human CART19 incorporated after 5 h culture with one of two mediums: (a) lOmM13C-glucose (13C-glc) + 5mM BHB or (b) lOmM glucose + 5mM13C- BHB. Metabolites were extracted and analyzed using the Agilent QTOF 6546 High-Resolution LC-MS. Here, CART19 were seeded with irradiated OCI-Lyl8 (effector: target = 0.25, n=3 technical replicates) for 72 h prior to coculture with labelled media. The relative contributions of13C label from13C-glucose (13C-glu) or13-BHB to TCA cycle metabolite and related pathway pools are shown. Statistical significance was determined by unpaired / -test for each metabolite. Plotted are means ± SD. ns. p > 0.05, *p < 0.05, **p < 0.01.

[0076] FIGs. 15A-15K: BHB treatment induces transcriptional and epigenetic changes in CART19 to promote an effector, metabolically active state. (15A) Heatmap showing expression of canonical mitochondrial respiration genes in CD8+TEM, CD4 / 8+TCM, and 76 clusters. (15B) ATAC-seq results from mouse 4-lBB^ CART19 cells stimulated with anti-CD3 / CD28 beads and treated with vehicle or 5 mM BHB for 5 days ( =4 technical replicates per condition). Left: PCA of ATAC-seq analysis. Right: log2 fold change comparing chromatin accessibility of BHB- treated CAR T cells to vehicle-treated CAR T cells. (15C-15E) RNA sequencing of human 4- 1BB(^ CART 19 cells stimulated on CD19-coated wells and treated with vehicle or 5mM BHB for 48 h (n=4 technical replicates per condition). (15C) Left: PCA of RNA-seq analysis. Right: volcano plot of differentially expressed genes (p < 0.05, |log2FC| > 0.38). (15D) Human Metabolome Database (HMDB) enrichment analysis of genes upregulated in BHB-treated CART19 cells. (15E) Reactome pathway analysis of genes upregulated in BHB-treated CART19 cells. (15F-15H) CUT&RUN profiling of H3K27 acetylation signature (H3K27ac) in human 4- Attorney Docket No: 046483-7485W01(04066)

[0077] 1BB(^ CART19 cells stimulated on CD19-coated wells and treated with vehicle or 5mM BHB for 48 h («=3 technical replicates per condition). (15F) PCA of CUT&RUNseq. (15G) Genomic distribution of H3K27ac-marked regions. (15H) Scatter plot of differential H3K27ac signature showing enrichment (yellow) or depletion (purple) of H3K27ac regions in BHB-treated CART19 cells relative to vehicle (p < 0.05, |log2FC| > 0.3). (15I-15J) GREAT (Genomic Regions Enrichment of Annotations Tool) analysis of GO Biological Process terms for regions of increased chromatin accessibility identified by ATAC-seq (151) and upregulated H3K27ac regions identified by CUT&RUN-seq (15 J). (15K-15L) Overlap analyses of RNA-seq, ATAC- seq, and CUT&RUN-seq datasets (panels C-J). (15K) Overlap of upregulated genes identified by ATAC-seq (p < 0.05, |log2FC| > 0.1), H3K27ac CUT&RUN (p < 0.05, |log2FC| > 0.3), and RNA-seq (p < 0.05, |log2FC| > 0.38), showing convergence on HPGD. Statistical significance was determined by DESeq2 (15B, 15C and 15H).

[0078] FIGs. 16A-16J: BDH1 -deficient mouse CAR T cells are metabolically unresponsive to BHB supplementation. (16A) Schema of ketolysis, depicting the enzymatic conversion of BHB to acetoacetate by BDH1 in the mitochondria. Acetoacetate is subsequently converted to acetyl - CoA, which enters the TCA cycle. (16B) Western blot of or mouse CAR T cell protein lysates following CRISPR-Cas9 editing with & BdhI sgRNA (see Methods). Quantification of BDH1 knockout efficiency using ImageJ (right). (16C) Experimental design to generate BDH1- deficient mouse CART 19 using CRISPR-Cas9. A20 tumors were established in Balb / c mice (2 * 106A20 cells, subcutaneous injection), and mice were placed on either vehicle or BHB (200 mb BHB / 1 L) containing water. On day 18 post tumor implantation, 0.35 * 106mock electroporated (Cas9 only) CAR T cells or BDHl-deficient CAR T cells were infused into A20- bearing mice. In this model, n = 5 mice were used in each experimental arm. (16D) Mean (± SD) tumor volumes. (16E) Overall survival of mice. (16F) Left: Schema of generating Bdhl- deficient CART19 cells from a Bdhlfl / flmouse. Here, T cells were harvested from Bdhl0 11C57BL / 6 mouse spleen, activated using anti-CD3 / CD28 Dynabeads, and transduced with retroviruses bearing CAR19 and / or Cre-eGFP constructs. Right: transduction efficiency, showing both fraction of CAR+ cells (anti-G4S stain) and Cre+ cells (GFP). (16G) Oxygen consumption under vehicle or BHB supplementation for Cre+ and Cre- Bdhlfl / flCART19. Here, Attorney Docket No: 046483-7485W01(04066) after 5 days of manufacturing and expansion, mouse CART19 cells were supplemented with vehicle (PBS) or 5mM BHB for an additional 24 h prior to loading onto Agilent Seahorse XF plates. (16H) Spare respiratory capacity (161) Maximum O2 consumption. (16J) Luminescencebased cytotoxicity of mouse Cre+ or - Bdhlfl / ilCART19 cells, following 48 h of coculture with CD19+luciferase+ B16 cells (effector : target = 1 :3), in the presence or absence of 5mM BHB. Statistical significance was determined by two-way ANOVA (16D), log-rank Mantel-Cox (16E), or one-way ANOVA with post hoc Tukey tests (16H-16J). Plotted are means ± SEM or means ± SD. ns. p > 0.05, *p < 0.05, **p < 0.01.

[0079] FIGs. 17A-17G: BHB treatment enhances T cell metabolic fitness in patient-derived CART 19 and peripheral healthy volunteer T cells. (17A) OCR ± SD of T cells from ten volunteer, comparing pre- (time = Tl, 0 min) and post- (time = T2, 90 min) BHB ingestion via Agilent Seahorse XF. (17B) Representative live-cell fluorescence images of NADH and mitochondrial membrane potential (A'Fm, TMRE) in peripheral blood T cells at Tl and T2. Cells were imaged at 37 °C using a Zeiss Observer 7 widefield fluorescence microscope with a 20* / 0.8 objective. Brightfield, fluorescence, and pseudo-colored pixel intensity heatmap images are shown for each timepoint in a representative healthy volunteer. Scale bars: 25 pm. (17C) Intracellular cytokine staining of peripheral T cells following BHB ingestion in healthy volunteers. Here, peripheral blood mononuclear cells (PBMCs) were collected from healthy volunteers immediately before (Tl, 0 min) and 90 min after ingestion of 30g BHB (T2, 90 min). Cells were stimulated ex vivo with PMA / ionomycin (eBioscience, 500X) and Brefeldin A and subsequently stained for intracellular cytokines. Shown are the percentages of (left to right) CD8+IFNy, CD8+TNFa+, CD4+ZFNy+, and CD4+TNFa+T cells, at Tl and T2. (17D) Kinetics of ex vivo UTD and CART 19 cell expansion, under with or without 2mM BHB, from the leukapheresis products of three DLBCL patients who went on to receive CTL019 (NCT02030834). (17E) Fold change of cell counts amongst UTD and CART19 cells. (17F-17G) Expanded CART 19 cells of two patients from FIG. 17D were analyzed using the Agilent Seahorse XF assay. (17F-17G) Quantification of OCR (mean ± SD) and SRC (mean ± SD) of CART19 cells for Patient 1 (17F) and Patient 2 (17G) are shown. Statistical significance was Attorney Docket No: 046483-7485W01(04066) determined by paired t-test (17C and 17E), or paired t-test (17F and 17G). TMRE, tetramethylrhodamine, ethyl ester, ns. p > 0.05, *p < 0.05, ***p < 0.001.

[0080] FIGs. 18A-18F: Evaluation of systemic effects of BHB ingestion in patients and healthy volunteers. (18A) Serum from n=17 patients collected on day 7 following infusion with CTL019 (UPCC13413) was profded for metabolic composition via mass spectrometry. (18B) Statistical correlation between BHB levels and CAR T cell expansion (measured by copies per pg of DNA) on day 7. (18C) Cytokine release syndrome (CRS) grading at day 7, split by BHB levels of n=39 CART19 patients. Here, low is defined as below the median BHB peak area, and high is defined as above the median BHB peak area. (18D) Complete metabolic panel (CMP) of serum from healthy volunteers before (Tl) and 90 min after ingestion of 30g BHB (T2). Parameters include glucose, creatinine, estimated glomerular filtration rate (eGFR), blood urea nitrogen (BUN), total protein, albumin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and bilirubin. Here, the gray shaded areas indicate the clinical normal reference range for each parameter. (18E) CMP values from two healthy volunteers before (Day 0) and after two consecutive weeks (Day 14) of daily BHB ingestion (30g per day). (18F) Design schema for proof-of-concept trial to test the effect of BHB administration during commercial CART19 treatment in relap sed / refractory lymphoma (NCT0661034). The design of the trial includes administration of BHB over a period of 4-5 weeks, following lymphodepletion (LD). At each study visit (marked by “+” symbol), trial participants will have blood and stool samples collected for biobanking and future retrospective analyses of composition of peripheral immune cells and gut microbiota. Statistical significance was determined by linear regression (18B), Fisher’s test (18C), or paired / -test (18D). ns. p > 0.05, *p < 0.05.

[0081] FIGs. 19A-19D: Synergistic effects of BHB on solid tumor cell killing. In vitro killing assays of AsPC-1 (19A) and B16 (19B) cells following 60-hour treatment with vehicle, 2 mM, or 5 mM BHB. In vitro killing assays of SKOV3 (19C) and 4T1 (19D) cells following 60-hour treatment with vehicle, 1 mM, 2 mM, 5 mM or 10 mM BHB. Statistics were determined at 60 h by determined by one-way ANOVA with post hoc Tukey tests (19A-19D).

[0082] FIGs. 20A-20D: BDH1 knockdown abolishes the survival benefit of BHB. (20A) Manufacturing scheme for BDH1 knockdown human CART19 cells. Human T cells (CD4:CD8 Attorney Docket No: 046483-7485W01(04066) at 1 :1) were activated with anti-CD3 / CD28, transduced with anti-CD19 CAR and a BDH1 shRNA lentiviral vector, selected with puromycin (5 pg / mL), expanded, and cryopreserved. Statistical significance was determined by log-rank Mantel-Cox. (20B) In vivo study design. NSG mice bearing CD19+Nalm6 leukemia received BHB supplementation or vehicle beginning before tumor CART19 infusion. BDHl-shRNA CART19 cells were infused intravenously on day 0. Tumor burden was monitored by bioluminescence imaging (BLI) and survival was recorded. (20C) Tumor burden over time. Lines show group means with SEM. No significant difference between BDH1-KD CART19 with or without BHB at day 36 post infusion. (20D) Overall survival of the same cohorts. Statistical significance was determined by t-test (20C) and log-rank Mantel-Cox (20D).

[0083] FIGs. 21A-21B: Acetoacetate (AcAc) fails to rescue metabolic function in BDH1- deficient CAR T cells. SeahorseXF was performed on BDH1 -deficient CAR T cells after 24 h of culture with either vehicle, BHB, or AcAc. Despite AcAc being the product of BDH1 dehydrogenation of BHB, it fails to improve metabolic function, suggesting a role for NADH generation in the BDH1 -dependent BHB-mediated enhancement of oxygen consumption.

[0084] FIGs. 22A-22D: The role of Hcar2 and Hopx in BHB-regulated CAR T cell function. (22A) Human Protein Atlas expression of HCAR2 across cell types. (22B-22D) BHB enhances CAR T cell-mediated killing of tumor cells independent of Hcar2 or Hopx. B16-CD19 target cells were seeded onto xCELLigence E-plates and co-cultured with CD19-directed CAR T cells derived from (22B) wild-type, (22C) Hcar27", or (22D) Hopx / mice at an effector-to-target (E:T) ratio of 1:2 in the presence of 0, 2, or 5 mM BHB. Real-time changes in impedance (cell index) were recorded using the xCELLigence system. Data are presented as mean ± SEM of technical replicates.

[0085] FIGs. 23A-23B: BHB enhances proliferation without altering mTORCl signaling. (23A) Proliferation of CAR T cells cocultured for 96 h with irradiated OCI-Lyl8 cells (effectortarget = 0.25, n=3 technical replicates), under increasing concentrations of rapamycin, an mTORCl inhibitor, in the presence or absence of 2 mM BHB. Plotted are means + / - SEM. (23B) Immunoblot analysis of phosphorylated S6 kinase (p-S6K), total S6K, and P-actin in human Attorney Docket No: 046483-7485W01(04066)

[0086] CART19 cells stimulated on human CD19-coated wells with 5 mM BHB and / or 50 nM rapamycin for 1 h or 24 h.

[0087] DETAILED DESCRIPTION

[0088] Definitions

[0089] Unless otherwise defined, scientific and technical terms used herein have the meanings that are commonly understood by those of ordinary skill in the art. In the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The use of “or” means “and / or” unless stated otherwise. The use of the term “including,” as well as other forms, such as “includes” and “included,” is not limiting.

[0090] Generally, nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein is well-known and commonly used in the art. The methods and techniques provided herein are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. Enzymatic reactions and purification techniques are performed according to manufacturer’s specifications, as commonly accomplished in the art, or as described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well- known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0091] That the disclosure may be more readily understood, select terms are defined below.

[0092] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. Attorney Docket No: 046483-7485W01(04066)

[0093] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0094] “Activation,” as used herein, refers to the state of a T cell that has been sufficiently stimulated to induce detectable cellular proliferation. Activation can also be associated with induced cytokine production, and detectable effector functions. The term “activated T cells” refers to, among other things, T cells that are undergoing cell division.

[0095] As used herein, to “alleviate” a disease means reducing the severity of one or more symptoms of the disease.

[0096] The term “antigen” as used herein is defined as a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen.

[0097] Furthermore, antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA, which comprises a nucleotide sequence or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an “antigen” as that term is used herein. Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full-length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell, or a biological fluid.

[0098] As used herein, the term “autologous” is meant to refer to any material derived from the same individual to which it is later to be re-introduced into the individual.

[0099] As used herein “beta-hydroxybutyrate,” also known as 0HB or BHB, is a carboxylic acid having the general formula CH3CHOHCH2COOH which may be utilized by a patient's body as a Attorney Docket No: 046483-7485W01(04066) fuel source during instances of low glucose levels in the patient and is considered a ketone body. In the present disclosure, salt variants of beta-hydroxybutyrate are disclosed.

[0100] A “co-stimulatory molecule” refers to the cognate binding partner on a T cell that specifically binds with a co-stimulatory ligand, thereby mediating a co-stimulatory response by the T cell, such as, but not limited to, proliferation. Co-stimulatory molecules include, but are not limited to an MHC class I molecule, BTLA and a Toll ligand receptor.

[0101] A “co-stimulatory signal,” as used herein, refers to a signal, which in combination with a primary signal, such as TCR / CD3 ligation, leads to T cell proliferation and / or upregulation or downregulation of key molecules.

[0102] A “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal’s health continues to deteriorate. In contrast, a “disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal’s state of health.

[0103] The term “downregulation” as used herein refers to the decrease or elimination of gene expression of one or more genes.

[0104] “Effective amount” or “therapeutically effective amount” are used interchangeably herein, and refer to an amount of a compound, formulation, material, or composition, as described herein effective to achieve a particular biological result or provides a therapeutic or prophylactic benefit. Such results may include, but are not limited to an amount that when administered to a mammal, causes a detectable level of immune suppression or tolerance compared to the immune response detected in the absence of the composition of the invention. The immune response can be readily assessed by a plethora of art-recognized methods. The skilled artisan would understand that the amount of the composition administered herein varies and can be readily determined based on a number of factors such as the disease or condition being treated, the age and health and physical condition of the mammal being treated, the severity of the disease, the particular compound being administered, and the like. Attorney Docket No: 046483-7485W01(04066)

[0105] “Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0106] As used herein “endogenous” refers to any material from or produced inside an organism, cell, tissue, or system.

[0107] The term “epitope” as used herein is defined as a small chemical molecule on an antigen that can elicit an immune response, inducing B and / or T cell responses. An antigen can have one or more epitopes. Most antigens have many epitopes; i.e., they are multivalent. In general, an epitope is roughly about 10 amino acids and / or sugars in size. Preferably, the epitope is about 4- 18 amino acids, more preferably about 5-16 amino acids, and even more most preferably 6-14 amino acids, more preferably about 7-12, and most preferably about 8-10 amino acids. One skilled in the art understands that generally the overall three-dimensional structure, rather than the specific linear sequence of the molecule, is the main criterion of antigenic specificity and therefore distinguishes one epitope from another. Based on the present disclosure, a peptide used in the present invention can be an epitope.

[0108] As used herein, the term “exogenous” refers to any material introduced from or produced outside an organism, cell, tissue, or system.

[0109] The terms “expand” and “expanding” refer to increasing in number, as in an increase in the number of T cells. In one embodiment, the T cells that are expanded ex vivo increase in number relative to the number originally present in the culture. In another embodiment, the T cells that are expanded ex vivo increase in number relative to other cell types in the culture. The term "ex vivo " as used herein, refers to cells that have been removed from a living organism, Attorney Docket No: 046483-7485W01(04066)

[0110] (e.g., a human) and propagated outside the organism (e.g., in a culture dish, test tube, or bioreactor).

[0111] The term “expression” as used herein is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0112] “Expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., Sendai viruses, lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[0113] As used herein, “Fab” refers to a fragment of an antibody structure that binds to an antigen but is monovalent and does not have a Fc portion, for example, an antibody digested by the enzyme papain yields two Fab fragments and an Fc fragment (e.g., a heavy (H) chain constant region; Fc region that does not bind to an antigen).

[0114] As used herein, “F(ab')2” refers to an antibody fragment generated by pepsin digestion of whole IgG antibodies, wherein this fragment has two antigen binding (ab') (bivalent) regions, wherein each (ab') region comprises two separate amino acid chains, a part of a H chain and a light (L) chain linked by an S — S bond for binding an antigen and where the remaining H chain portions are linked together. A “F(ab')2” fragment can be split into two individual Fab' fragments.

[0115] The term “immune cell” refers to a cell which is capable of affecting or inducing an immune response upon recognition of an antigen. In some embodiments, the immune cell is a T- cell, a natural killer (NK) cell, a macrophage, a myeloid cell or a dendritic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. The cells may be autologous or allogeneic to the subject to which they are administered. In an embodiment, the present invention provides a population of CAR-expressing cells such as CAR- T -cells. Attorney Docket No: 046483-7485W01(04066)

[0116] The term “immune response” as used herein is defined as a cellular response to an antigen that occurs when lymphocytes identify antigenic molecules as foreign and induce the formation of antibodies and / or activate lymphocytes to remove the antigen.

[0117] The term “immunosuppressive” is used herein to refer to reducing overall immune response.

[0118] “Insertion / deletion,” commonly abbreviated “indel,” is a type of genetic polymorphism in which a specific nucleotide sequence is present (insertion) or absent (deletion) in a genome.

[0119] “Isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0120] “Ketosis” as used herein refers to a subject having blood ketone levels within the range of about 0.5 mmol / L and about 16 mmol / L. Ketone levels sustained above 0.5 mmol / L and ideally in the range of 1.0 to 3.0 mmol / L appear to offer the most therapeutic effects in humans. Ketosis may improve mitochondrial function, elevate Krebs cycle intermediates (e.g., succinate, fumarate), decrease ROS production, reduce inflammation, elevate adenosine, and increase the activity of neurotrophic factors associated

[0121] The term “knockout” as used herein refers to the ablation of gene expression of one or more genes from a cell.

[0122] A “lentivirus” as used herein refers to a genus of the Retroviridae family. Lentiviruses are unique among the retroviruses in being able to infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived from lentiviruses offer the means to achieve significant levels of gene transfer in vivo.

[0123] The term “medium chain triglycerides” (MCT) are molecules having a glycerol backbone attached to three medium chain fatty acids. Medium chain fatty acids range from 6 to 12 carbon atoms in length. Exemplary fatty acids are caprylic acid, also known as octanoic acid, Attorney Docket No: 046483-7485W01(04066) comprising 8 carbon molecules, and capric acid, also known as decanoic acid, comprising 10 carbon molecules.

[0124] By the term “modified” as used herein, is meant a changed state or structure of a molecule or cell of the invention. Molecules may be modified in many ways, including chemically, structurally, and functionally. Cells may be modified through the introduction of nucleic acids.

[0125] By the term “modulating,” as used herein, is meant mediating a detectable increase or decrease in the level of a response in a subject compared with the level of a response in the subject in the absence of a treatment or compound, and / or compared with the level of a response in an otherwise identical but untreated subject. The term encompasses perturbing and / or affecting a native signal or response thereby mediating a beneficial therapeutic response in a subject, preferably, a human.

[0126] In the context of the present invention, the following abbreviations for the commonly occurring nucleic acid bases are used. “A” refers to adenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refers to thymidine, and “U” refers to uridine.

[0127] The term “oligonucleotide” typically refers to short polynucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, C, G), this also includes an RNA sequence (i.e., A, U, C, G) in which “U” replaces “T.”

[0128] Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some versions contain an intron(s).

[0129] “Parenteral” administration of an immunogenic composition includes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrastemal injection, or infusion techniques.

[0130] The term “polynucleotide” as used herein is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric “nucleotides.” The Attorney Docket No: 046483-7485W01(04066) monomeric nucleotides can be hydrolyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR, and the like, and by synthetic means.

[0131] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides, and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

[0132] As used herein, the term “proliferation” refers to an increase in cell division, either symmetric or asymmetric division of cells. In particular aspects, “proliferation” refers to the symmetric or asymmetric division of T cells. “Increased proliferation” occurs when there is an increase in the number of cells in a treated sample compared to cells in a non-treated sample.

[0133] As used herein the term “state of ketosis” refers to a metabolic state in which the body, due to low carbohydrate availability, shifts from using glucose as its primary fuel source to burning fat for energy. In certain embodiments, a state of ketosis occurs when blood ketone levels exceed about 0.5 mrn / L. Exemplary ketones include ketone bodies produced in such a state, such as acetoacetate, beta-hydroxy butyrate, and acetone.

[0134] By the term “specifically binds,” as used herein with respect to an antibody, is meant an antibody which recognizes a specific antigen, but does not substantially recognize or bind other Attorney Docket No: 046483-7485W01(04066) molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. But such cross-species reactivity does not itself alter the classification of an antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antibody as specific. In some instances, the terms “specific binding” or “specifically binding,” can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope “A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled “A” and the antibody, will reduce the amount of labeled A bound to the antibody.

[0135] By the term “stimulation,” is meant a primary response induced by binding of a stimulatory molecule (e.g., a TCR / CD3 complex) with its cognate ligand thereby mediating a signal transduction event, such as, but not limited to, signal transduction via the TCR / CD3 complex. Stimulation can mediate altered expression of certain molecules, such as upregulation of interferon-gamma, and / or reorganization of cytoskeletal structures, and the like.

[0136] A “stimulatory molecule,” as the term is used herein, means a molecule on a T cell that specifically binds with a cognate stimulatory ligand present on an antigen presenting cell.

[0137] A “stimulatory ligand,” as used herein, means a ligand that when present on an antigen presenting cell (e.g., an aAPC, a dendritic cell, a B-cell, and the like) can specifically bind with a cognate binding partner (referred to herein as a “stimulatory molecule”) on a T cell, thereby mediating a primary response by the T cell, including, but not limited to, activation, initiation of an immune response, proliferation, and the like. Stimulatory ligands are well-known in the art and encompass, inter alia, an MHC Class I molecule loaded with a peptide, an anti-CD3 antibody, a superagonist anti-CD28 antibody, and a superagonist anti-CD2 antibody.

[0138] The term “subject” is intended to include living organisms in which an immune response can be elicited (e.g., mammals). A “subject” or “patient,” as used therein, may be a human or Attorney Docket No: 046483-7485W01(04066) non-human mammal. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline and murine mammals. Preferably, the subject is human.

[0139] The terms “T cell” or “T lymphocyte” are art-recognized and are intended to include thymocytes, naive T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. A T cell can be a T helper (Th) cell, for example a T helper 1 (Thl) or a T helper 2 (Th2) cell. The T cell can be a helper T cell (HTL; CD4+T cell) CD4+T cell, a cytotoxic T cell (CTL; CD8+T cell), a tumor infiltrating cytotoxic T cell (TIL; CD8+T cell), CD4+CD8+T cell, CD4 CD8' T cell, or any other subset of T cells. Other illustrative populations of T cells suitable for use in particular aspects include naive T cells and memory T cells.

[0140] As used herein, the term “T cell receptor” or “TCR” refers to a complex of membrane proteins that participate in the activation of T cells in response to the presentation of antigen. The TCR is responsible for recognizing antigens bound to major histocompatibility complex molecules. TCR is composed of a heterodimer of an alpha (a) and beta (0) chain, although in some cells the TCR consists of gamma and delta (y / 8) chains. TCRs may exist in alpha / beta and gamma / delta forms, which are structurally similar but have distinct anatomical locations and functions. Each chain is composed of two extracellular domains, a variable and constant domain. In some embodiments, the TCR may be modified on any cell comprising a TCR, including, for example, a helper T cell, a cytotoxic T cell, a memory T cell, regulatory T cell, natural killer T cell, and gamma delta T cell.

[0141] The term “therapeutic” as used herein means a treatment and / or prophylaxis. A therapeutic effect is obtained by suppression, remission, or eradication of a disease state.

[0142] “Transplant” refers to a biocompatible lattice or a donor tissue, organ, or cell, to be transplanted. An example of a transplant may include but is not limited to skin cells or tissue, bone marrow, and solid organs such as heart, pancreas, kidney, lung, and liver. A transplant can also refer to any material that is to be administered to a host. For example, a transplant can refer to a nucleic acid or a protein.

[0143] The term “transfected” or “transformed” or “transduced” as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A Attorney Docket No: 046483-7485W01(04066)

[0144] “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed, or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0145] To “treat” a disease as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.

[0146] A “vector” is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” includes an autonomously replicating plasmid or a virus. The term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, Sendai viral vectors, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.

[0147] Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0148] In one aspect, a method for improving chimeric antigen receptor (CAR)-T therapy is provided. The CAR-T therapy can be based on ex-vivo CAR-T therapy, where the CAR-T therapy is produced by ex-vivo generation of the CAR-T therapy or where the CAR-T therapy is produced by in vivo generation of the CAR-T therapy. Methods of producing CAR-T cells ex- vivo are well known in the art and any method can be used to generate the CAR-T cells ex-vivo. Attorney Docket No: 046483-7485W01(04066)

[0149] Exemplary non4imiting methods for producing ex-vivo CAR-T cells are also provided for herein.

[0150] Production of CAR-T cells in vivo are also well known and any method, whether utilizing a viral vector or a non-viral vector, can be used. Examples of viral vectors for generating CAR- T cells in vivo, can be, for example, a lentivirus. The lentivirus, for example, can be generated pseudotyped with a VSV-G protein to facilitate the targeting of the lentivirus with a target cell, such as a T-cell. The VSV-G protein can be mutated to reduce or abrogate its binding to its cognate receptor, the LDL receptor, and then the lentivirus can also comprise a targeting moiety that binds to the target cell, such as an immune cell. Examples of viral vectors and / or mutated VSV-G proteins can be found in PCT Publication No. WO 2022 / 183072, U.S. Patent Application Publication No. 2024 / 0218390, PCT Publication No. WO 2024 / 145593, PCT Publication No. WO 2024 / 145599, PCT Publication No. WO 2024 / 145605, PCT Publication No. WO 2024 / 145622, PCT Publication No. WO 2024 / 196734, PCT Publication No. WO 2024 / 196738, PCT Publication No. WO 2024 / 196754, PCT Publication No. WO2021 / 18376 and PCT Publication No WO 2023 / 114884, each of which is hereby incorporated by reference in its entirety.

[0151] Examples of non-viral vectors for producing CAR-T cells are also well known, such as using lipid nanoparticles (LNPs) to provide nucleic molecules encoding the CAR to the target cell. Non-limiting examples of such methods and compositions include those provided for in US20240299311, W02024040195A1, WO2024249954A1, WO2025096878A1 and WO2025179294A2, each of which is hereby incorporated by reference in its entirety.

[0152] Accordingly, in some embodiments, methods of treating a patient comprises administering to a subject in need thereof a therapeutically effective amount of CAR-T therapy and a ketogenic composition comprising beta-hydroxybutyrate (BHB), a BHB salt, a BHB precursor, a ketone diester, or a combination thereof. In some embodiments, method for improving chimeric antigen receptor (CAR)-T therapy are provided, the methods comprising administering to a subject in need thereof a CAR-T therapy and a ketogenic composition comprising beta-hydroxybutyrate (BHB), a BHB salt, a BHB precursor, a ketone diester, or a combination thereof. In some embodiments, the administering the CAR-T therapy to the patient Attorney Docket No: 046483-7485W01(04066) comprises administering a therapeutically effective amount of CAR-T cells. In some embodiments, administering the CAR-T therapy comprises administering a vector to the patient, wherein the vector comprises a nucleic acid molecule encoding a chimeric antigen receptor. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a lentivirus, such as a pseudotyped lentivirus, such as those provided for herein. In some embodiments, the vector is a lipid nanoparticle (LNP) comprising the nucleic acid molecule encoding the chimeric antigen receptor.

[0153] In another aspect, a method of producing a cell population comprising T-cells comprises culturing T cells in media comprising beta-hydroxybutyrate (BHB), a BHB salt, a BHB precursor, a ketone diester, or a combination thereof.

[0154] In a further aspect, a method of manufacturing CAR-T cells from peripheral blood mononuclear cells (PBMCs) from a blood sample obtained from a subject, comprises: (a) producing a T-cell enriched population of cells from a population of immune cells isolated from a subject; (b) transforming the T-cell enriched population of cells with a vector encoding a chimeric antigen receptor (CAR); and (c) culturing the cells obtained in step b) in media comprising beta-hydroxybutyrate (BHB), a BHB salt, a BHB precursor, a ketone diester, or a combination thereof.

[0155] In one aspect, the present invention provides a ketogenic composition exploiting the metabolic and physiological advantages of sustained ketosis (e.g. keto-adaptation) which utilizes ketones as an alternative fuel for proliferation and expansion of T-cells. The resulting T-cells constitute an improved source of T-cells for CAR-T cell therapy and CAR-T cell manufacturing.

[0156] A ketogenic composition comprises one or more ketone bodies or ketogenic ingredients, such as beta-hydroxybutyrate (BHB). The disclosed compositions can comprise a ketogenic composition or supplement in a pharmaceutically acceptable carrier. Ketone bodies are produced from fat and are an alternative caloric source to glucose, particularly when glucose is not available. During periods of fasting, extreme exercise, and / or low carbohydrate consumption, glucose and glycogen stores in the body are rapidly used and can become quickly depleted. Attorney Docket No: 046483-7485W01(04066)

[0157] Failure to replenish glucose stores as they become depleted causes the body to metabolically shift to using ketone bodies as primary caloric energy. This metabolic state is called “ketosis”. Exogenous ketone supplementation can cause a rapid and sustained elevation of BHB and reduced blood glucose levels.

[0158] In one embodiment, the ketogenic composition comprises beta-hydroxybutyrate (BHB), a BHB salt, a BHB precursor, a ketone diester, or a combination thereof. In another embodiment, the ketogenic composition comprises D-beta-hydroxybutyric (D-BHB) acid, L-beta- hydroxybutyric (L-BHB) acid, racemic beta-hydroxy butyric acid or a salt, monoester, polyester, or mixture thereof.

[0159] In another embodiment, the ketogenic composition comprises a BHB salt or BHB ester. In certain embodiments, the ketogenic composition comprises a BHB mineral salt, a BHB organic salt, or a combination thereof.

[0160] In one embodiment, the ketogenic composition comprises a BHB mineral salt selected from the group consisting of sodium beta-hydroxybutyrate, potassium beta-hydroxybutyrate, calcium beta-hydroxybutyrate, magnesium beta-hydroxybutyrate, lithium beta-hydroxybutyrate, and a mixture thereof.

[0161] In another embodiment, the ketogenic composition comprises a BHB organic salt selected from the group consisting of arginine beta-hydroxybutyrate, lysine beta- hydroxybutyrate, histidine beta-hydroxybutyrate, ornithine beta-hydroxybutyrate, creatine beta- hydroxybutyrate, agmatine beta-hydroxybutyrate, citrulline beta-hydroxybutyrate, and a mixture thereof.

[0162] In another embodiment, the ketogenic composition comprises a beta-hydroxy butyrate sodium salt, a beta-hydroxy butyrate potassium salt, a beta-hydroxy butyrate calcium salt, a betahydroxy butyrate magnesium salt, or a mixture thereof.

[0163] In another embodiment, the ketogenic composition comprises a BHB precursor selected from the group consisting of 1,3-butanediol (BDO), ethyl acetoacetate, ethyl beta- hydroxybutyrate, and mixture thereof. Attorney Docket No: 046483-7485W01(04066)

[0164] In another embodiment, the ketogenic composition comprises a ketone diester. Tn certain embodiments, the ketone diester is 7?,S l,3-butanediol di acetoacetate or R,S 1,3 butanediol acetoacetate diester.

[0165] In another embodiment, the ketogenic composition further comprises a medium chain fatty acid or ester thereof. In certain embodiments, the medium chain fatty acid or ester thereof is selected from the group consisting of coconut oil, coconut milk powder, fractionated coconut oil, palm oil, palm kernel oil, caprylic acid, isolated hexanoic acid, isolated octanoic acid, isolated decanoic acid, ester derivatives of the medium chain fatty acids ethoxylated triglyceride, enone triglyceride derivatives, aldehyde triglyceride derivatives, monoglyceride derivatives thereof, diglyceride derivatives thereof, triglyceride derivatives thereof, medium chain triglyceride salts, salt derivatives thereof, and mixtures thereof.

[0166] In some embodiments, pharmaceutical composition comprising a ketogenic composition, such as those provided for herein, are provided. The pharmaceutical compositions can be used in conjunction with CAR-T therapies to enhance or improve the CAR-T therapy. As provided for herein, the pharmaceutical composition comprising the ketogenic composition can be administered to the patient before, after, or during the administration of the CAR-T therapy. In some embodiments, the pharmaceutical composition is sterile and only comprises pharmaceutically grade compounds, such as pharmaceutical grade beta-hydroxybutyrate, or any other BHB type compound, such as those provided for herein.

[0167] In some embodiments, a pharmaceutical compositions comprising a ketogenic composition for use in conjunction with a CAR-T therapy are provided.

[0168] Chimeric Antigen Receptors (CARs)

[0169] A chimeric antigen receptor (CAR) refers to an artificial immune cell receptor that is engineered to recognize and bind to an antigen expressed by tumor cells. Generally, a CAR is designed for a T cell and is a chimera of a signaling domain of the T-cell receptor (TCR) complex and an antigen binding domain (e.g., an antibody single chain variable fragment (scFv) or other antigen binding fragment). A T cell that expresses a CAR is referred to as a CAR-T cell. CARs have the ability to redirect T-cell specificity and reactivity toward a selected target in a Attorney Docket No: 046483-7485W01(04066) non-MHC-restricted manner. The non-MHC-restricted antigen recognition gives T-cells expressing CARs the ability to recognize an antigen independent of antigen processing, thus bypassing a major mechanism of tumor escape.

[0170] In an embodiments, the CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular domain. In some embodiments, the intracellular domain further comprises a costimulatory signaling domain, and an intracellular signaling domain. The transmembrane domain connects the antigen binding domain of the CAR-To the intracellular domain and is defined by a region spanning the plasma membrane of a cell (e.g., T-cell).

[0171] In certain embodiments, the CAR comprises an antigen binding domain selected from the group consisting of a Fab, a single-chain variable fragment (scFv), or a nanobody. In some embodiments, the CAR comprises an antigen binding domain that specifically binds a B cell protein. In certain embodiments, the B cell protein is selected from the group consisting of CD10, CD19, CD20, CD22, CD79b, CD34, CD52, CD123, FLT-3, ROR1, CD179b, and CD79a. In one embodiment, the CAR comprises an anti -CD 19 antigen binding domain.

[0172] In some embodiments, the CAR comprises an antigen binding domain that specifically binds a tumor associated antigen. In certain embodiments, the tumor associated antigen is selected from the group consisting of alpha feto-protein (AFP) / HLA-A2, AXL, B7-H3, B7H4, BCMA, CA-IX, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD30, CD33, CD38, CD44v6, CD70, CD79a, CD79b, CD80, CD86, CD117, CD 123, CD 133, CD 147, CD171, CD276, CEA, CEACAM5, claudin 18.2, c-Met, DLL3, DR5, EGFR, EGFRvIII, EpCAM, EphA2, FAP, folate receptor alpha (FRa) / folate binding protein (FBP), GD-2, Glycolipid F77, glypican-2 (GPC2), glypican-3 (GPC3), HER2, HLA-A2, ICAM1, IL3Ra, IL13Ra2, LAGE-1, Lewis Y, LMP1 (EBV), MAGE-A1, MAGE-A3, MAGE-A4, Melan A, mesothelin, MG7 (glycosylated CEA), MMP, MUC1, Nectin4 / FAP, NKG2D-Ligands (MIC-A, MIC-B, and the ULBPs 1 to 6), NY-ESO-1, P16, PD-L1, PSCA, PSMA, ROR1, ROR2, TIM-3, TM4SF1, TnMucl, VEGFR2, and any combination thereof. In one embodiment, the tumor associated antigen is mesothelin.

[0173] In an embodiment, the CAR comprises a transmembrane domain from a protein selected from the group consisting of alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, Attorney Docket No: 046483-7485W01(04066)

[0174] CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154. In certain embodiments, the CAR comprises a transmembrane domain selected from the group consisting of CD8, CD28, ICOS, and 0X40.

[0175] The intracellular domain of the CAR is responsible for activation of at least one of the effector functions of the cell in which the CAR is expressed (e.g., T-cell). The intracellular domain transduces the effector function signal and directs the cell (e.g., immune cell) to perform its specialized function, e.g., harming and / or destroying a target cell. Examples of an intracellular domain for use in the invention include, but are not limited to, the cytoplasmic portion of a surface receptor, co-stimulatory molecule, and any molecule that acts in concert to initiate signal transduction in the T cell, as well as any derivative or variant of these elements and any synthetic sequence that has the same functional capability.

[0176] In certain embodiments, the intracellular domain comprises a costimulatory domain and an intracellular signaling domain. In certain embodiments, the costimulatory domain is from a protein selected from the group consisting of CD28, 4-1BB (CD137), ICOS (CD278), 0X40, CD5, CD27, LFA-1 (CD1 la / CD18), ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAL, ITGAM, CDl lb, ITGAX, CDl lc, ITGB1, CD29, ITGB2, CD 18, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP- 76, PAG / Cbp, BTLA, an MHC claim I molecule, and a ligand that specifically binds with CD8. In certain embodiments, the costimulatory domain is derived from 4-1BB, CD28, ICOS, or 0X40.

[0177] In an embodiment, the intracellular signaling domain comprises an immunoreceptor tyrosine-based activation motif. In some embodiments, the intracellular signaling domain is from a protein selected from the group consisting of CD3 zeta, common FcR gamma, FcyRIII Fc gamma Rlla, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, Attorney Docket No: 046483-7485W01(04066)

[0178] CD66d, DAP10, and DAP12. Tn one embodiment, the intracellular signaling domain is from CD3 zeta.

[0179] In an embodiment, the antigen-binding domain is connected to the transmembrane domain by a hinge region, which is optional. The hinge region of the CAR is a hydrophilic region which is located between the antigen binding domain and the transmembrane domain. In some embodiments, this domain facilitates proper protein folding for the CAR. In some embodiments, the transmembrane domain is interposed between a hinge region and the intracellular domain. In certain embodiments, the hinge region is from a protein selected from the group consisting of an Fc fragment of an antibody, a hinge region of an antibody, a CH2 region of an antibody, a CH3 region of an antibody, an artificial hinge domain, a hinge comprising an amino acid sequence of CD8, or any combination thereof. In one embodiment, the hinge region is from CD8 or CD28.

[0180] In an embodiment, the CAR comprises a leader sequence encoding an N-terminal signal peptide. In one embodiment, the leader sequence encodes a CD8a signal peptide.

[0181] CAR-T Cells

[0182] In one aspect, the present invention provides a population of CAR-T cells expressing a CAR having specificity to an antigen of interest (e.g., a cancer or tumor antigen). CAR-T-cells are useful for the treatment, prevention and / or amelioration of a disease or disorder. For example, the CAR-T-cells of the present invention are useful for the treatment of cancer, an infection, or an inflammatory disease.

[0183] A CAR-T cell may be envisioned to comprise any antigen binding domain, any hinge, any transmembrane domain, any intracellular costimulatory domain, and any intracellular signaling domain described herein or known to those skilled in the art, and can readily be understood and made by a person of skill in the art in view of the disclosure herein.

[0184] In some embodiments, the CAR-T cell is an autologous cell. In some embodiments, the CAR-T is an allogeneic cell.

[0185] In some embodiments, a CAR-T cell of the present invention is genetically edited to disrupt the expression of one or more endogenously expressed genes to reduce the risk of Attorney Docket No: 046483-7485W01(04066) autoreactivity and / or increase the efficacy of the CAR-T cells. Tn certain embodiments, the gene- edited CAR-T cells have a reduction, deletion, elimination, knockout, or disruption in expression of one or more endogenously expressed receptor. In one embodiment, the CAR-T cell is genetically edited to disrupt the expression of an endogenous TCR gene product (e.g., gene products of T Cell Receptor Alpha Constant (TRAC) and T Cell Receptor Beta Constant (TRBC)), Beta-2-microglobulin (B2M), and Class II Major Histocompatibility Complex Transactivator (CIITA).

[0186] In certain embodiments, the CAR-T cell of the present disclosure is genetically edited to disrupt the expression of endogenous PD-1 gene products. In certain embodiments, disrupting the expression of endogenous PD-1 may create “checkpoint” resistant CAR-T cells, resulting in increased tumor control. Checkpoint resistant CAR-T cells may also be created by disrupting the expression of, for example, without limitation, the Adenosine A2A receptor (A2AR), B7-H3 (CD276), B7-H4 (VTCN1), the B and T Lymphocyte Attenuator protein (BTLA / CD272), CD96, the Cytotoxic T-Lymphocyte Associated protein 4 (CTLA-4 / CD152), Indoleamine 2,3- dioxygenase (IDO), the Killer-cell Immunoglobulin-like Receptor (KIR), the Lymphocyte Activation Gene-3 (LAG3), the T cell immunoreceptor with Ig and ITIM domains (TIGIT), T- cell Immunoglobulin domain and Mucin domain 3 (TIM-3), or the V-domain Ig suppressor of T cell activation (VISTA).

[0187] In some embodiments, the metabolic activity of the CAR-T cells may be measured using a Seahorse® assay. The Seahorse® assay measures the extracellular flux of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). OCR reflects the rate at which cells consume oxygen during oxidative phosphorylation, a process that occurs in the mitochondria. ECAR measures the production of protons resulting from glycolysis, the metabolic pathway that generates energy from glucose. In some embodiments, CAR-T cells are added to specialized microplates with wells that contain sensors for detecting OCR and ECAR changes. The cells are exposed to experimental conditions, such as different concentrations of drugs or metabolic substrates, and the OCR and ECAR are measured at intervals. In some aspects, the Seahorse® assay is performed using 0.5 pM FCCP. In some aspects the Seahorse® assay is performed using 2 pM FCCP. Attorney Docket No: 046483-7485W01(04066)

[0188] In some embodiments, the CAR-T cells have an OCR above 100 pmol / min. In some embodiments, the CAR-T cells have an OCR above 40 pmol / min. In some embodiments, the CAR-T cells have an OCR above 150 pmol / min. In some embodiments, the CAR-T cells have an OCR from about 50 pmol / min to about 200 pmol / min. In some embodiments, the CAR-T cells have an ECAR above 30 mpH / min. In some embodiments, the CAR-T cells have an ECAR above 50 mpH / min. In some embodiments, the CAR-T cells have an ECAR above 30 mpH / min. In some embodiments, the CAR-T cells or TCR cells have an ECAR from about 30 mpH / min to about 60 mpH / min.

[0189] Methods for Generating CAR-T Cells

[0190] The present invention provides methods for producing / generating a CAR-T cell or precursor cell thereof. The CAR-T cells are engineered by introducing a vector or nucleic acid encoding a CAR into a T-cell. Methods of introducing nucleic acids or expression constructs into a cell include physical, biological, and chemical methods. Physical methods for introducing a polynucleotide, such as RNA, into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. RNA can be introduced into target cells using commercially available methods which include electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (Harvard Instruments, Boston, MA) or the Gene Pulser II (BioRad, Denver, CO), Multiporator (Eppendorf, Hamburg Germany). RNA can also be introduced into cells using cationic liposome mediated transfection using lipofection, using polymer encapsulation, using peptide mediated transfection, or using biolistic particle delivery systems such as “gene guns” (see, for example, Nishikawa, et al. Hum Gene Ther., 12(8): 861 -70 (2001).

[0191] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from lentivirus, poxviruses, herpesviruses, adenoviruses, and adeno- associated viruses, and the like. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362. Attorney Docket No: 046483-7485W01(04066)

[0192] In some embodiments, a nucleic acid encoding the CAR of the invention is introduced into a cell by an expression vector. Expression vectors comprising a nucleic acid encoding the CAR are provided herein. Suitable expression vectors include lentivirus vectors, gamma retrovirus vectors, foamy virus vectors, adeno associated virus (AAV) vectors, adenovirus vectors, engineered hybrid viruses, naked DNA, including but not limited to transposon mediated vectors, such as Sleeping Beauty, Piggyback, and Integrases such as Phi31. Other suitable expression vectors include Epstein-Barr virus (EBV), herpes simplex virus (HSV), and retrovirus expression vectors.

[0193] Adenovirus expression vectors are based on adenoviruses, which have a low capacity for integration into genomic DNA but a high efficiency for transfecting host cells. Adenovirus expression vectors contain adenovirus sequences sufficient to: (a) support packaging of the expression vector and (b) to ultimately express the CAR in the host cell. In some embodiments, the adenovirus genome is a 36 kb, linear, double stranded DNA, where a foreign DNA sequence (e.g., a nucleic acid encoding the CAR) may be inserted to substitute large pieces of adenoviral DNA in order to make the expression vector of the present invention (see, e.g., Danthinne and Imperiale, Gene Therapy (2000) 7(20): 1707-1714).

[0194] Another expression vector is based on an adeno associated virus, which takes advantage of the adenovirus coupled systems. This AAV expression vector has a high frequency of integration into the host genome. It can infect non-dividing cells, thus making it useful for delivery of genes into mammalian cells, for example, in tissue cultures or in vivo. The AAV vector has a broad host range for infectivity. Details concerning the generation and use of AAV vectors are described in U.S. Patent Nos. 5,139,941 and 4,797,368.

[0195] Retrovirus expression vectors are capable of integrating into the host genome, delivering a large amount of foreign genetic material, infecting a broad spectrum of species and cell types, and being packaged in special cell lines. The retrovirus vector is constructed by inserting a nucleic acid (e.g., a nucleic acid encoding the CAR) into the viral genome at certain locations to produce a virus that is replication defective. Though the retrovirus vectors are able to infect a broad variety of cell types, integration, and stable expression of the CAR, requires the division of host cells. Attorney Docket No: 046483-7485W01(04066)

[0196] Lentivirus vectors are derived from lentiviruses, which are complex retroviruses that, in addition to the common retroviral genes gag, pol, and env, contain other genes with regulatory or structural function (see, e.g., U.S. Patent Nos. 6,013,516 and 5,994, 136). Some examples of lentiviruses include the human immunodeficiency viruses (HIV-1, HIV-2) and the simian immunodeficiency virus (SIV). Lentivirus vectors have been generated by multiply attenuating the HIV virulence genes, for example, the genes env, vif, vpr, vpu and nef are deleted making the vector biologically safe. Lentivirus vectors are capable of infecting non-dividing cells and can be used for both in vivo and ex vivo gene transfer and expression, e.g., of a nucleic acid encoding the CAR (see, e.g., U.S. Patent No. 5,994,136).

[0197] Expression vectors including a nucleic acid of the present disclosure can be introduced into a host cell by any means known to persons skilled in the art. The expression vectors may include viral sequences for transfection, if desired. Alternatively, the expression vectors may be introduced by fusion, electroporation, biolistics, transfection, lipofection, or the like. The host cell may be grown and expanded in culture before introduction of the expression vectors, followed by the appropriate treatment for introduction and integration of the vectors. The host cells are then expanded and may be screened by virtue of a marker present in the vectors. Various markers that may be used are known in the art, and may include hprt, neomycin resistance, thymidine kinase, hygromycin resistance, etc. As used herein, the terms “cell,” “cell line,” and “cell culture” may be used interchangeably. In some embodiments, the host cell is an immune cell or precursor thereof, e.g., a T cell, an NK cell, or an NKT cell.

[0198] The present invention also provides genetically engineered cells which include and stably express the CAR of the present disclosure. In some embodiments, the genetically engineered cells are genetically engineered T-lymphocytes (T cells), regulatory T cells (Tregs), naive T cells (TN), memory T cells (for example, central memory T cells (TCM), effector memory cells (TEM)), natural killer cells (NK cells), natural killer T cells (NKT cells), gamma delta cells and macrophages capable of giving rise to therapeutically relevant progeny. In one embodiment, the genetically engineered cells are autologous cells.

[0199] Modified cells (e.g., comprising the CAR) may be produced by stably transfecting host cells with an expression vector including a nucleic acid of the present disclosure. Additional Attorney Docket No: 046483-7485W01(04066) methods to generate a CAR-T cell of the present disclosure include, without limitation, chemical transformation methods (e.g., using calcium phosphate, dendrimers, liposomes and / or cationic polymers), non-chemical transformation methods (e.g., electroporation, optical transformation, gene electrotransfer and / or hydrodynamic delivery) and / or particle-based methods (e.g., impalefection, using a gene gun and / or magnetofection). Transfected cells expressing the CAR may be expanded ex vivo.

[0200] Physical methods for introducing an expression vector into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells including vectors and / or exogenous nucleic acids are well- known in the art. See, e.g., Sambrook et al. (2001), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York.

[0201] Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0202] Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine (“DMPC”) can be obtained from Sigma, St. Louis, MO; dicetyl phosphate (“DCP”) can be obtained from K & K Laboratories (Plainview, NY); cholesterol (“Choi”) can be obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) and other lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at about -20°C. Chloroform is used as the only solvent since it is more readily evaporated than methanol. “Liposome” is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and Attorney Docket No: 046483-7485W01(04066) dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). However, compositions that have different structures in solution than the normal vesicular structure are also encompassed. For example, the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules. Also contemplated are lipofectamine- nucleic acid complexes.

[0203] Regardless of the method used to introduce exogenous nucleic acids into a host cell or otherwise expose a cell to the inhibitor of the present invention, in order to confirm the presence of the nucleic acids in the host cell, a variety of assays may be performed. Such assays include, for example, “molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; “biochemical” assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the invention.

[0204] Moreover, the nucleic acids may be introduced by any means, such as transducing the expanded T cells, transfecting the expanded T cells, and electroporating the expanded T cells. One nucleic acid may be introduced by one method and another nucleic acid may be introduced into the T cell by a different method.

[0205] As provided for herein, the CAR-T cells can be generated by ex vivo or in vivo methods. In some embodiments, the CAR-T cells are generated by an ex vivo method. In some embodiments, the CAR-T cells are generated by an in vivo method.

[0206] CAR-T Cell Therapy

[0207] CAR-T-cell therapy is a type of cellular therapy where immune cells (e.g., T-cells) are genetically modified ex vivo or in vivo to express a CAR and the CAR-expressing cell (e.g. a CAR-T-cell). If the cell is produce ex vivo, the cells can be infused to a recipient in need thereof. The infused cell is able to kill diseased cells expressing the target of the CAR in the recipient. Unlike antibody therapies, CAR-T cells are able to replicate in vivo resulting in longterm persistence that can lead to sustained tumor control. In various embodiments, the CAR-T- cells are administered to the patient, or their progeny, persist in the patient for at least four months, five months, six months, seven months, eight months, nine months, ten months, eleven Attorney Docket No: 046483-7485W01(04066) months, twelve months, thirteen months, fourteen month, fifteen months, sixteen months, seventeen months, eighteen months, nineteen months, twenty months, twenty-one months, twenty -two months, twenty -three months, two years, three years, four years, or five years after administration of the CAR-T-cell to the patient.

[0208] If the cells are produced in vivo, the cells are generated by administering a vector, such as a viral or non-viral vector, that targets the immune cell to produce the cell in vivo expressing the chimeric antigen receptor.

[0209] In one aspect, the present invention provides a method for treating a disease or disorder comprising administering to a subject in need thereof a CAR-T cell therapy or pharmaceutical composition thereof (as described herein), in an amount effective for treating a disease or disorder.

[0210] In some embodiments, the disease is cancer. In certain embodiments, the cancer comprises a solid tumor. In other embodiments, the cancer is a hematological malignancy. In some embodiments, the CAR-T therapy (cells or vector) described herein may be included in a pharmaceutical composition for immunotherapy or adoptive cell therapy. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0211] The CAR-T therapy of the present invention can be administered to an animal, preferably a mammal, even more preferably a human, to treat a cancer to produce an apoptotic response and / or cell-mediated immune response against tumor cells, where it is desirable to treat or alleviate the disease.

[0212] In an embodiment, the cancer to be treated is a solid tumor. In some embodiments, the cancer is melanoma, lung cancer, liver cancer, pancreatic cancer, stomach cancer, colon cancer, kidney cancer, brain cancer, head and neck cancer, breast cancer, skin cancer, rectal cancer, uterine cancer, cervical cancer, ovarian cancer, testicular cancer, skin cancer, esophageal cancer, and / or the cancer includes a sarcoma cell, a rhabdoid cancer cell, a neuroblastoma cell, retinoblastoma cell, or a medulloblastoma cell, and / or the cancer is uterine carcinosarcoma, brain lower grade glioma, thymoma, testicular germ cell tumors, glioblastoma multiforme, skin cutaneous melanoma, liver hepatocellular carcinoma, uveal melanoma, kidney chromophobe, thyroid cancer, kidney renal clear cell carcinoma, kidney renal papillary cell carcinoma, stomach Attorney Docket No: 046483-7485W01(04066) adenocarcinoma, cholangiocarcinoma, adenoid cystic carcinoma, prostate adenocarcinoma, pheochromocytoma and paraganglioma, lung adenocarcinoma, head-neck squamous cell carcinoma, pancreatic adenocarcinoma, breast cancer, mesothelioma, colon and rectal adenocarcinoma, rectum adenocarcinoma, esophageal carcinoma, ovarian cancer, lung squamous cell carcinoma, bladder urothelial carcinoma, sarcoma, or uterine corpus endometrial carcinoma .

[0213] In some embodiments, the solid tumor is selected from the group consisting of melanoma, breast cancer, liver cancer, lung cancer, ovarian cancer, cervical cancer, uterus cancer, head and neck cancer, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal carcinoma, kidney cancer, prostate cancer, gastric cancer, bronchus cancer, pancreatic cancer, urinary bladder cancer, and brain cancer.

[0214] In another embodiment, the cancer to be treated is a hematological cancer. In one embodiment, the cancer is a leukemia. In another embodiment, the cancer is a myeloma. In another embodiment, the cancer is a lymphoma. Exemplary hematological cancers be treated with the compositions of the present invention include those selected from the group consisting of chronic lymphocytic leukemia, mantle cell lymphoma, multiple myeloma, acute lymphoid leukemia, Hodgkin lymphoma, B-cell acute lymphoid leukemia, T-cell acute lymphoid leukemia, small lymphocytic leukemia, B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt lymphoma, diffuse large B cell lymphoma (DLBCL), DLBCL associated with chronic inflammation, follicular lymphoma, pediatric follicular lymphoma, hairy cell leukemia, small cell- or a large cell-follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma (extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue), Marginal zone lymphoma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom macroglobulinemia, splenic marginal zone lymphoma, splenic lymphoma / leukemia, splenic diffuse red pulp small B-cell lymphoma, hairy cell leukemia-variant, lymphoplasmacytic lymphoma, a heavy chain disease, plasma cell myeloma, solitary plasmocytoma of bone, extraosseous plasmocytoma, nodal marginal zone lymphoma, pediatric nodal marginal zone lymphoma, primary cutaneous follicle center lymphoma, lymphomatoid granulomatosis, primary mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, ALK+ large Attorney Docket No: 046483-7485W01(04066)

[0215] B-cell lymphoma, large B-cell lymphoma arising in HHV8-associated multi centric Castleman disease, primary effusion lymphoma, B-cell lymphoma, or unclassifiable lymphoma.

[0216] In certain exemplary embodiments, the CAR-T cells of the invention are used to treat a myeloma, or a condition related to myeloma. Examples of myeloma or conditions related thereto include, without limitation, light chain myeloma, non-secretory myeloma, monoclonal gamopathy of undetermined significance (MGUS), plasmacytoma (e.g., solitary, multiple solitary, extramedullary plasmacytoma), amyloidosis, and multiple myeloma. In one embodiment, a method of the present disclosure is used to treat multiple myeloma. In one embodiment, a method of the present disclosure is used to treat refractory myeloma. In one embodiment, a method of the present disclosure is used to treat relapsed myeloma.

[0217] In some embodiments, the subject has been treated with a therapeutic agent prior to administration of the CAR-T therapy. In some embodiments, the subject is refractory or non- responsive to the other therapeutic agent. In some embodiments, the subject has persistent or relapsed disease, e.g., following treatment with another therapeutic intervention, including chemotherapy, radiation, and / or hematopoietic stem cell transplantation (HSCT), e.g., allogenic HSCT, and / or other CAR-T therapy. In some embodiments, the administration effectively treats the subject despite the subject having become resistant to another therapy.

[0218] In some embodiments, the subject is responsive to the other therapeutic agent, and treatment with the CAR-T therapy reduces disease burden. In some embodiments, the subject is initially responsive to the other therapeutic agents, but exhibits a relapse of the disease or condition over time. In some embodiments, the subject has not relapsed. In some such embodiments, the subject is determined to be at risk for relapse, such as at a high risk of relapse, and thus the CAR-T therapy are administered prophylactically, e.g., to reduce the likelihood of or prevent relapse. In some aspects, the subject has not received prior treatment with another therapeutic agent.

[0219] In some embodiments, the subject has persistent or relapsed disease, e.g., following treatment with another therapeutic intervention, including chemotherapy, radiation, hematopoietic stem cell transplantation (HSCT), e.g., allogenic HSCT, and / or other CAR-T cell Attorney Docket No: 046483-7485W01(04066) therapies. In some embodiments, the administration effectively treats the subject despite the subject having become resistant to another therapy.

[0220] In any of the methods disclosed herein, the improved clinical outcome may comprise one or more of the following: (i) increased clinical or anti-tumor response to the CAR-T therapy in the subject; (ii) increased persistence of the engineered human CAR-T cells in the subject; (iii) reduced tumor size or tumor cell numbers in the subject; (iv) decreased cell lysis of the engineered human CAR-T cells in the subject; and (v) reduced natural killer (NK) cell activity in the subject.

[0221] Administration of the CAR-T therapy may be carried out in any convenient manner known to those of skill in the art. In some embodiments, the CAR-T therapy may be administered to a subject by injection or transfusion. In some embodiments, the CAR-T therapy described herein may be administered to a patient intratumorally, transarterially, subcutaneously, intradermally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In other instances, the CAR-T therapy of the invention are injected directly into a site of inflammation in the subject, a local disease site in the subject, a lymph node, an organ, a tumor, and the like.

[0222] For the prevention or treatment of disease, the appropriate dosage may depend on the type of disease to be treated, the type of targeting proteins or target proteins, the severity and course of the disease, whether the CAR-T therapy are administered for preventive or therapeutic purposes, previous therapy, the subject's clinical history and response to the CAR-T therapy, and / or the discretion of the attending physician. In some embodiments, the compositions in some embodiments are suitably administered to the subject at one time or over a series of treatments.

[0223] In some embodiments, the therapy is administered at a desired dosage, which in some aspects includes a desired dose or number of cells or cell type(s) and / or a desired ratio of cell types. Thus, the dosage of therapy in some embodiments is based on a total number of cells or amount of vector (or number per kg body weight) and, for example, for cells at a desired ratio of the individual populations or sub-types, such as the CD4+ to CD8+ ratio. In some embodiments, the dosage of cells is based on a desired total number (or number per kg of body weight) of cells Attorney Docket No: 046483-7485W01(04066) in the individual populations or of individual cell types. In some embodiments, the dosage is based on a combination of such features, such as a desired number of total cells, desired ratio, and desired total number of cells in the individual populations.

[0224] In certain embodiments, the cells, or individual populations of sub-types of cells, are administered to the subject at a range of about one million to about 100 billion cells, such as, e.g., 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), such as about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the foregoing values), and in some cases about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells) or any value in between these ranges.

[0225] In some embodiments, where a vector is used the dosage is measured by amounts or by infectious units, which can be determined by one of skill in the art.

[0226] In some embodiments, the dose of total cells and / or dose of individual sub-populations of cells is within a range of between at or about IxlO5cells / kg to about IxlO11cells / kg 104and at or about 1011cells / kilograms (kg) body weight, such as between 105and 106cells / kg body weight, for example, at or about 1 x 105cells / kg, 1.5 x 105cells / kg, 2 x 105cells / kg, or 1 x 106cells / kg body weight. For example, in some embodiments, the cells are administered at, or within a certain range of error of, between at or about 104and at or about 109T cells / kilograms (kg) body weight, such as between 10’ and 106T cells / kg body weight, for example, at or about 1 x 105T cells / kg, 1.5 x 105T cells / kg, 2 x 105T cells / kg, or 1 x 106T cells / kg body weight. In other exemplary embodiments, a suitable dosage range of CAR-T cells for use in a method of the present disclosure includes, without limitation, from about IxlO5cells / kg to about IxlO6cells / kg, from about IxlO6cells / kg to about IxlO7cells / kg, from about IxlO7cells / kg about Attorney Docket No: 046483-7485W01(04066)

[0227] Ixl O8cells / kg, from about IxlO8cells / kg about IxlO9cells / kg, from about IxlO9cells / kg about IxlO10cells / kg, from about IxlO10cells / kg about IxlO11cells / kg. In an exemplary embodiment, a suitable dosage for use in a method of the present disclosure is about IxlO8cells / kg. In an exemplary embodiment, a suitable dosage for use in a method of the present disclosure is about IxlO7cells / kg. In other embodiments, a suitable dosage is from about IxlO7total cells to about 5xl07total cells. In some embodiments, a suitable dosage is from about IxlO8total cells to about 5xl08total cells. In some embodiments, a suitable dosage is from about 1.4xl07total cells to about l.lxlO9total cells. In an exemplary embodiment, a suitable dosage for use in a method of the present disclosure is about 7xl09total cells.

[0228] In some embodiments, the cells are administered at or within a certain range of error of between at or about 104and at or about 109CD4+and / or CD8+cells / kilograms (kg) body weight, such as between 105and 106CD4+and / or CD8+cells / kg body weight, for example, at or about 1 x 105CD4+and / or CD8+cells / kg, 1.5 x 105CD4+and / or CD8+cells / kg, 2 x 105CD4+and / or CD81cells / kg, or 1 x 106CD41and / or CD8 cells / kg body weight. In some embodiments, the cells are administered at or within a certain range of error of, greater than, and / or at least about 1 x 106, about 2.5 x 106, about 5 x 106, about 7.5 x 106, or about 9 x 106CD4+cells, and / or at least about l x 106, about 2.5 x 106, about 5 x 106, about 7.5 x 106, or about 9 x 106CD8+ cells, and / or at least about 1 x 106, about 2.5 x 106, about 5 x 106, about 7.5 x 106, or about 9 x 106T cells. In some embodiments, the cells are administered at or within a certain range of error of between about 108and 1012or between about IO10and 1011T cells, between about 108and 1012or between about IO10and 1011CD4+cells, and / or between about 108and 1012or between about 1010and 1011CD8+cells.

[0229] In some embodiments, the cells are administered at or within a tolerated range of a desired output ratio of multiple cell populations or sub-types, such as CD4+ and CD8+ cells or sub-types. In some aspects, the desired ratio can be a specific ratio or can be a range of ratios, for example, in some embodiments, the desired ratio (e.g., ratio of CD4+to CD8+cells) is between at or about 5: 1 and at or about 5: 1 (or greater than about 1:5 and less than about 5: 1), or between at or about 1 :3 and at or about 3 : 1 (or greater than about 1 :3 and less than about 3: 1), such as between at or about 2: 1 and at or about 1 :5 (or greater than about 1 :5 and less than about 2: 1, Attorney Docket No: 046483-7485W01(04066) such as at or about 5: 1, 4.5: 1, 4: 1, 3.5: 1 , 3: 1, 2.5: 1 , 2: 1, 1.9: 1, 1.8: 1 , 1.7: 1, 1.6: 1, 1.5: 1, 1.4: 1, 1.3: 1, 1.2: 1, 1.1: 1, 1 : 1, 1 : 1.1, 1 : 1.2, 1: 1.3, 1: 1.4, 1 : 1.5, 1 : 1.6, 1: 1.7, 1 : 1.8, 1 : 1.9: 1 :2, 1 :2.5, 1 :3, 1 :3.5, 1 :4, 1 :4.5, or 1 :5. In some aspects, the tolerated difference is within about 1%, about 2%, about 3%, about 4% about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50% of the desired ratio, including any value in between these ranges.

[0230] In some embodiments, a dose of CAR-T therapy is administered to a subject in need thereof, in a single dose or multiple doses. In some embodiments, a dose of CAR-T therapy is administered in multiple doses, e.g., once a week or every 7 days, once every 2 weeks or every 14 days, once every 3 weeks or every 21 days, once every 4 weeks or every 28 days. In an exemplary embodiment, a single dose of CAR-T therapy is administered to a subject in need thereof. In an exemplary embodiment, a single dose of CAR-T therapy is administered to a subject in need thereof by rapid intravenous infusion. The therapy can also be administered by injection.

[0231] For the prevention or treatment of disease, the appropriate dosage may depend on the type of disease to be treated, the type of cells or recombinant receptors, the severity and course of the disease, whether the cells are administered for preventive or therapeutic purposes, previous therapy, the subject's clinical history and response to the cells, and the discretion of the attending physician. The compositions and cells are in some embodiments suitably administered to the subject at one time or over a series of treatments.

[0232] In some embodiments, the therapy is administered as part of a combination treatment, such as simultaneously with or sequentially with, in any order, another therapeutic intervention, such as an antibody or engineered cell or receptor or agent, such as a cytotoxic or therapeutic agent. The therapy in some embodiments is co-administered with one or more additional therapeutic agents or in connection with another therapeutic intervention, either simultaneously or sequentially in any order. In some contexts, the therapy is co-administered with another therapy sufficiently close in time such that the therapy enhances the effect of one or more additional therapeutic agents, or vice versa. In some embodiments, the therapy is administered prior to the one or more additional therapeutic agents. In some embodiments, the CAR-T therapy Attorney Docket No: 046483-7485W01(04066) is administered after the one or more additional therapeutic agents. In some embodiments, the one or more additional agents includes a cytokine, such as IL-2, for example, to enhance persistence. In some embodiments, the methods comprise administration of a chemotherapeutic agent.

[0233] In certain embodiments, the CAR-T therapy of the invention may be administered to a subject in combination with an immune checkpoint antibody (e.g., an anti-PDl, anti-CTLA-4, or anti-PDLl antibody). For example, the CAR-T therapy may be administered in combination with an antibody or antibody fragment targeting, for example, PD-1 (programmed death 1 protein). Examples of anti -PD-1 antibodies include, but are not limited to, pembrolizumab (KEYTRUDA®, formerly lambrolizumab, also known as MK-3475), and nivolumab (BMS- 936558, MDX-1106, ONO-4538, OPDIVA®) or an antigen-binding fragment thereof. In certain embodiments, the CAR-T cell may be administered in combination with an anti-PD-Ll antibody or antigen-binding fragment thereof. Examples of anti-PD-Ll antibodies include, but are not limited to, BMS-936559, MPDL3280A (TECENTRIQ®, Atezolizumab), and MEDI4736 (Durvalumab, Imfinzi). In certain embodiments, the CAR-T cell may be administered in combination with an anti-CTLA-4 antibody or antigen-binding fragment thereof. An example of an anti- CTLA-4 antibody includes, but is not limited to, Ipilimumab (trade name Yervoy). Other types of immune checkpoint modulators may also be used including, but not limited to, small molecules, siRNA, miRNA, and CRISPR systems. Immune checkpoint modulators may be administered before, after, or concurrently with the CAR-T cells. In certain embodiments, combination treatment comprising an immune checkpoint modulator may increase the therapeutic efficacy of a therapy comprising a CAR-T cell of the present invention.

[0234] Following administration of the therapy, the biological activity of the engineered cell populations in some embodiments can be measured, e.g., by any of a number of known methods. Parameters to assess include specific binding of an engineered or natural T cell or other immune cell to antigen, in vivo, e.g., by imaging, or ex vivo, e.g., by ELISA or flow cytometry. In certain embodiments, the ability of the engineered cells to destroy target cells can be measured using any suitable method known in the art, such as cytotoxicity assays described in, for example, Kochenderfer et al., J. Immunotherapy, 32(7): 689-702 (2009), and Herman et al. J. Attorney Docket No: 046483-7485W01(04066)

[0235] Immunological Methods, 285(1): 25-40 (2004). Tn certain embodiments, the biological activity of the cells is measured by assaying expression and / or secretion of one or more cytokines, such as CD 107a, IFNy, IL-2, and TNF. In some aspects the biological activity is measured by assessing clinical outcome, such as reduction in tumor burden or load.

[0236] In certain embodiments, the subject is provided a secondary treatment. Secondary treatments include but are not limited to chemotherapy, radiation, surgery, and medications.

[0237] In some embodiments, the subject can be administered a conditioning therapy prior to CAR-T therapy. In some embodiments, the conditioning therapy comprises administering an effective amount of cyclophosphamide to the subject. In some embodiments, the conditioning therapy comprises administering an effective amount of fludarabine to the subject. In preferred embodiments, the conditioning therapy comprises administering an effective amount of a combination of cyclophosphamide and fludarabine to the subject. Administration of a conditioning therapy prior to CAR-T therapy may increase the efficacy of the CAR-T therapy. Methods of conditioning patients for CAR-T therapy are described in U.S. Patent No. 9,855,298, which is incorporated herein by reference in its entirety.

[0238] In some embodiments, a specific dosage regimen of the present disclosure includes a lymphodepletion step prior to the administration of the CAR-T therapy. In an exemplary embodiment, the lymphodepletion step includes administration of cyclophosphamide and / or fludarabine. In some embodiments, no lymphodepletion step prior to the administration of the CAR-T therapy is performed, such as, but not limited to, when the therapy is a vector that is used to deliver the nucleic acid encoding the CAR to the immune cell in vivo.

[0239] In some embodiments, the lymphodepletion step includes administration of cyclophosphamide at a dose of between about 200 mg / m2 / day and about 2000 mg / m2 / day (e.g., 200 mg / m2 / day, 300 mg / m2 / day, or 500 mg / m2 / day). In an exemplary embodiment, the dose of cyclophosphamide is about 300 mg / m2 / day. In some embodiments, the lymphodepletion step includes administration of fludarabine at a dose of between about 20 mg / m2 / day and about 900 mg / m2 / day (e.g., 20 mg / m2 / day, 25 mg / m2 / day, 30 mg / m2 / day, or 60 mg / m2 / day). In an exemplary embodiment, the dose of fludarabine is about 30 mg / m2 / day. Attorney Docket No: 046483-7485W01(04066)

[0240] In some embodiment, the lymphodepletion step includes administration of cyclophosphamide at a dose of between about 200 mg / m2 / day and about 2000 mg / m2 / day (e.g., 200 mg / m2 / day, 300 mg / m2 / day, or 500 mg / m2 / day), and fludarabine at a dose of between about 20 mg / m2 / day and about 900 mg / m2 / day e.g., 20 mg / m2 / day, 25 mg / m2 / day, 30 mg / m2 / day, or 60 mg / m2 / day). In an exemplary embodiment, the lymphodepletion step includes administration of cyclophosphamide at a dose of about 300 mg / m2 / day, and fludarabine at a dose of about 30 mg / m2 / day.

[0241] In an exemplary embodiment, the dosing of cyclophosphamide is 300 mg / m2 / day over three days, and the dosing of fludarabine is 30 mg / m2 / day over three days.

[0242] Dosing of lymphodepletion chemotherapy may be scheduled on Days -6 to -4 (with a -1- day window, i.e., dosing on Days -7 to -5) relative to CAR-T cell infusion on Day 0.

[0243] In an exemplary embodiment, for a subject having cancer, the subject receives lymphodepleting chemotherapy including 300 mg / m2of cyclophosphamide by intravenous infusion 3 days prior to administration of the CAR-T cells. In an exemplary embodiment, for a subject having cancer, the subject receives lymphodepleting chemotherapy including 300 mg / m2of cyclophosphamide by intravenous infusion for 3 days prior to administration of the CAR-T cells.

[0244] In an exemplary embodiment, for a subject having cancer, the subject receives lymphodepleting chemotherapy including fludarabine at a dose of between about 20 mg / m2 / day and about 900 mg / m2 / day (e.g., 20 mg / m2 / day, 25 mg / m2 / day, 30 mg / m2 / day, or 60 mg / m2 / day). In an exemplary embodiment, for a subject having cancer, the subject receives lymphodepleting chemotherapy including fludarabine at a dose of 30 mg / m2for 3 days.

[0245] In an exemplary embodiment, for a subject having cancer, the subject receives lymphodepleting chemotherapy including cyclophosphamide at a dose of between about 200 mg / m2 / day and about 2000 mg / m2 / day (e.g., 200 mg / m2 / day, 300 mg / m2 / day, or 500 mg / m2 / day), and fludarabine at a dose of between about 20 mg / m2 / day and about 900 mg / m2 / day (e.g., 20 mg / m2 / day, 25 mg / m2 / day, 30 mg / m2 / day, or 60 mg / m2 / day). In an exemplary embodiment, for a subject having cancer, the subject receives lymphodepleting chemotherapy Attorney Docket No: 046483-7485W01(04066) including cyclophosphamide at a dose of about 300 mg / m2 / day, and fludarabine at a dose of 30 mg / m2for 3 days.

[0246] Therapies of the invention can be administered in dosages and routes and at times to be determined in appropriate pre-clinical and clinical experimentation and trials. Therapy compositions may be administered multiple times at dosages within these ranges. Administration of the therapies of the invention may be combined with other methods useful to treat the desired disease or condition as determined by those of skill in the art.

[0247] It is known in the art that one of the adverse effects following infusion of CAR-T cells is the onset of immune activation, known as cytokine release syndrome (CRS). CRS is immune activation resulting in elevated inflammatory cytokines. CRS is a known on-target toxicity, development of which likely correlates with efficacy. Clinical and laboratory measures range from mild CRS (constitutional symptoms and / or grade-2 organ toxicity) to severe CRS (sCRS; grade >3 organ toxicity, aggressive clinical intervention, and / or potentially life threatening). Clinical features include high fever, malaise, fatigue, myalgia, nausea, anorexia, tachycardia / hypotension, capillary leak, cardiac dysfunction, renal impairment, hepatic failure, and disseminated intravascular coagulation. Dramatic elevations of cytokines including interferon-gamma, granulocyte macrophage colony-stimulating factor, IL- 10, and IL-6 have been shown following CAR-T-cell infusion. One CRS signature is elevation of cytokines including IL-6 (severe elevation), IFN-gamma, TNF-alpha (moderate), and IL -2 (mild). Elevations in clinically available markers of inflammation including ferritin and C-reactive protein (CRP) have also been observed to correlate with the CRS syndrome. The presence of CRS generally correlates with expansion and progressive immune activation of adoptively transferred cells. It has been demonstrated that the degree of CRS severity is dictated by disease burden at the time of infusion as patients with high tumor burden experience a more sCRS.

[0248] Accordingly, the invention provides for, following the diagnosis of CRS, appropriate CRS management strategies to mitigate the physiological symptoms of uncontrolled inflammation without dampening the antitumor efficacy of the engineered cells (e.g, CAR-T cells). CRS management strategies are known in the art. For example, systemic corticosteroids Attorney Docket No: 046483-7485W01(04066) may be administered to rapidly reverse symptoms of sCRS (e.g., grade 3 CRS) without compromising initial antitumor response.

[0249] In some embodiments, an anti-IL-6R antibody may be administered. An example of an anti-IL-6R antibody is the Food and Drug Administration-approved monoclonal antibody tocilizumab, also known as atlizumab (marketed as Actemra, or RoActemra). Tocilizumab is a humanized monoclonal antibody against the interleukin-6 receptor (IL-6R). Administration of tocilizumab has demonstrated near-immediate reversal of CRS.

[0250] CRS is generally managed based on the severity of the observed syndrome and interventions are tailored as such. CRS management decisions may be based upon clinical signs and symptoms and response to interventions, not solely on laboratory values alone.

[0251] Mild to moderate cases generally are treated with symptom management with fluid therapy, non-steroidal anti-inflammatory drug (NSAID) and antihistamines as needed for adequate symptom relief. More severe cases include patients with any degree of hemodynamic instability; with any hemodynamic instability, the administration of tocilizumab is recommended. The first-line management of CRS may be tocilizumab, in some embodiments, at the labeled dose of 8 mg / kg IV over 60 minutes (not to exceed 800 mg / dose); tocilizumab can be repeated Q8 hours. If suboptimal response to the first dose of tocilizumab, additional doses of tocilizumab may be considered. Tocilizumab can be administered alone or in combination with corticosteroid therapy. Patients with continued or progressive CRS symptoms, inadequate clinical improvement in 12-18 hours or poor response to tocilizumab, may be treated with high- dose corticosteroid therapy, generally hydrocortisone 100 mg IV or methylprednisolone 1-2 mg / kg. In patients with more severe hemodynamic instability or more severe respiratory symptoms, patients may be administered high-dose corticosteroid therapy early in the course of the CRS. CRS management guidance may be based on published standards (Lee et al. (2019) Biol Blood Marrow Transplant, doi.org / 10.1016 / j.bbmt.2018.12.758; Neelapu et al. (2018) Nat Rev Clin Oncology, 15:47; Teachey et al. (2016) Cancer Di scov, 6(6):664-679).

[0252] Features consistent with Macrophage Activation Syndrome (MAS) or Hemophagocytic lymphohistiocytosis (HLH) have been observed in patients treated with CAR-T therapy (Henter, 2007), coincident with clinical manifestations of the CRS. MAS appears to be a reaction to Attorney Docket No: 046483-7485W01(04066) immune activation that occurs from the CRS, and should therefore be considered a manifestation of CRS. MAS is similar to HLH (also a reaction to immune stimulation). The clinical syndrome of MAS is characterized by high grade non-remitting fever, cytopenias affecting at least two of three lineages, and hepatosplenomegaly. It is associated with high serum ferritin, soluble interleukin-2 receptor, and triglycerides, and a decrease of circulating natural killer (NK) activity.

[0253] Pharmaceutical compositions and formulations

[0254] Also provided are populations of T cells of the invention, compositions containing cells and / or enriched for such cells, such as in which cells expressing a CAR make up at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more of the total cells in the composition or cells of a certain type such as T cells or CD8+ or CD4+ cells. Among the compositions are pharmaceutical compositions and formulations for administration, such as for adoptive cell therapy. Also provided are therapeutic methods for administering the cells and compositions to subjects, e.g., patients.

[0255] Also provided are compositions including the cells for administration, including pharmaceutical compositions and formulations, such as unit dose form compositions including the number of cells for administration in a given dose or fraction thereof. The pharmaceutical compositions and formulations generally include one or more optional pharmaceutically acceptable carrier or excipient. In some embodiments, the composition includes at least one additional therapeutic agent. The term "pharmaceutical formulation" refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative. In some aspects, the choice of carrier is determined in part by the particular cell and / or by the method of administration. Accordingly, there are a variety of suitable formulations. For example, the pharmaceutical composition can contain Attorney Docket No: 046483-7485W01(04066) preservatives. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition. Carriers are described, e.g., by Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG).

[0256] Buffering agents in some aspects are included in the compositions. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some aspects, a mixture of two or more buffering agents is used. The buffering agent or mixtures thereof are typically present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail in, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).

[0257] The formulations can include aqueous solutions. The formulation or composition may also contain more than one active ingredient useful for the particular indication, disease, or condition being treated with the cells, preferably those with activities complementary to the cells, Attorney Docket No: 046483-7485W01(04066) where the respective activities do not adversely affect one another. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended. Thus, in some embodiments, the pharmaceutical composition further includes other pharmaceutically active agents or drugs, such as chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, and / or vincristine. The pharmaceutical composition in some embodiments contains the cells in amounts effective to treat or prevent the disease or condition, such as a therapeutically effective or prophylactically effective amount. Therapeutic or prophylactic efficacy in some embodiments is monitored by periodic assessment of treated subjects. The desired dosage can be delivered by a single bolus administration of the cells, by multiple bolus administrations of the cells, or by continuous infusion administration of the cells.

[0258] Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some embodiments, the cell populations are administered parenterally. The term "parenteral," as used herein, includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. In some embodiments, the cells are administered to the subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection. Compositions in some embodiments are provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may in some aspects be buffered to a selected pH. Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues. Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol) and suitable mixtures thereof. Attorney Docket No: 046483-7485W01(04066)

[0259] Sterile injectable solutions can be prepared by incorporating the cells in a solvent, such as in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like. The compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, and / or colors, depending upon the route of administration and the preparation desired. Standard texts may in some aspects be consulted to prepare suitable preparations.

[0260] Various additives which enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, and sorbic acid. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0261] The formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by fdtration through sterile filtration membranes.

[0262] Sources of cells for ex-vivo production of CAR-T cells

[0263] Prior to expansion, and possible genetic modification or other modification, a cell population of T-cells is obtained from a subject for ex vivo manipulation. Sources of T-cells for ex vivo manipulation may also include, e.g., autologous or heterologous donor blood, cord blood, peripheral blood mononuclear cells, lymph tissue, lymphoid organs, thymus tissue, bone marrow, ascites, pleural effusion, and spleen tissue. Other exemplary cells include stem cells, such as multipot ent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs). In certain aspects, the cells are human cells. With reference to the subject to be treated, the cells may be allogeneic and / or autologous. The cells typically are primary cells, such as those isolated directly from a subject and / or isolated from a subject and frozen.

[0264] In some embodiments, the above sources of T-cells may be from the subject to be treated with the CAR-T cells of the invention. Non-limiting examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. In certain exemplary embodiments, the subject is Attorney Docket No: 046483-7485W01(04066) a human. With reference to the subject to be treated, the cells may be allogeneic and / or autologous. The cells typically are primary cells, such as those isolated directly from a subject and / or isolated from a subject and frozen.

[0265] In certain embodiments, the T cell is a CD8+ T cell (e.g., a CD8+ naive T cell, central memory T cell, or effector memory T cell), a CD4+ T cell, a natural killer T cell (NKT cells), a gamma-delta T cell, a regulatory T cell (Treg), a stem cell memory T cell, a lymphoid progenitor cell, a hematopoietic stem cell, a natural killer cell (NK cell), a natural killer T cell (NKT cell) or a dendritic cell. In some embodiments, the cells are monocytes or granulocytes, e.g., myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and / or basophils. In an embodiment, the target cell is an induced pluripotent stem (iPS) cell or a cell derived from an iPS cell, e.g., an iPS cell generated from a subject, manipulated to alter (e.g., induce a mutation in) or manipulate the expression of one or more target genes, and differentiated into, e.g., a T cell, e.g., a CD8+ T cell (e.g., a CD8+ naive T cell, central memory T cell, or effector memory T cell), a CD4+ T cell, a stem cell memory T cell, a lymphoid progenitor cell or a hematopoietic stem cell.

[0266] In some embodiments, the cells include one or more subsets of T cells or other cell types, such as whole T cell populations, CD4+ cells, CD8+ cells, and subpopulations thereof, such as those defined by function, activation state, maturity, potential for differentiation, expansion, recirculation, localization, and / or persistence capacities, antigen- specificity, type of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and / or degree of differentiation. Among the sub-types and subpopulations of T cells and / or of CD4+ and / or of CD8+ T cells are naive T (TN) cells, effector T cells (TEFF), memory T cells and sub-types thereof, such as stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM), or terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa- associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha / beta T cells, and gamma / delta T cells. In certain embodiments, any number of T cell lines available in the art, may be used. Attorney Docket No: 046483-7485W01(04066)

[0267] In one embodiment, the population of T cells is comprised within cells such as peripheral blood mononuclear cells, cord blood cells, a purified population of T cells, or a T cell line. In another embodiment, peripheral blood mononuclear cells comprise the population of T cells. In yet another embodiment, purified T cells comprise the population of T cells.

[0268] In some embodiments, the methods include isolating immune cells from the subject, preparing, processing, culturing, and / or engineering them. In some embodiments, preparation of the engineered cells includes one or more culture and / or preparation steps. The cells for engineering as described may be isolated from a sample, such as a biological sample, e.g., one obtained from or derived from a subject. In some embodiments, the subject from which the cell is isolated is one having the disease or condition or in need of a cell therapy or to which cell therapy will be administered. The subject in some embodiments is a human in need of a particular therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated, processed, and / or engineered. Accordingly, the cells in some embodiments are primary cells, e.g., primary human cells. The samples include tissue, fluid, and other samples taken directly from the subject, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic engineering (e.g., transduction with viral vector), washing, and / or incubation. The biological sample can be a sample obtained directly from a biological source or a sample that is processed. Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom.

[0269] In certain aspects, the sample from which the cells are derived or isolated is blood or a blood-derived sample, or is or is derived from an apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsil, or other organ, and / or cells derived therefrom. Samples include, in the context of cell therapy, e.g., adoptive cell therapy, samples from autologous and allogeneic sources. Attorney Docket No: 046483-7485W01(04066)

[0270] In some embodiments, the cells are derived from cell lines, e.g., T cell lines. The cells in some embodiments are obtained from a xenogeneic source, for example, from mouse, rat, nonhuman primate, and pig. In some embodiments, isolation of the cells includes one or more preparation and / or non-affinity-based cell separation steps. In some examples, cells are washed, centrifuged, and / or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich for desired components, and lyse or remove cells sensitive to particular reagents. In some examples, cells are separated based on one or more property, such as density, adherent properties, size, sensitivity and / or resistance to particular components.

[0271] In some examples, cells from the circulating blood of a subject are obtained, e.g., by apheresis or leukapheresis. The samples, in certain aspects, contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and / or platelets, and in certain aspects contains cells other than red blood cells and platelets. In some embodiments, the blood cells collected from the subject are washed, e.g., to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments , a washing step is accomplished by tangential flow filtration (TFF) according to the manufacturer's instructions. In certain embodiments, the cells are resuspended in a variety of biocompatible buffers after washing. In certain embodiments, components of a blood cell sample are removed, and the cells directly resuspended in culture media. In some embodiments, the methods include density-based cell separation methods, such as the preparation of white blood cells from peripheral blood by lysing the red blood cells and centrifugation through a Percoll or Ficoll gradient.

[0272] In one embodiment, immune cells are obtained from the circulating blood of an individual are obtained by apheresis or leukapheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. The cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media, such as phosphate buffered saline (PBS) or wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations, for subsequent processing steps. As those of ordinary skill in the Attorney Docket No: 046483-7485W01(04066) art would readily appreciate a washing step may be accomplished by methods known to those in the art, such as by using a semi-automated “flow-through” centrifuge (for example, the Cobe 2991 cell processor, the Baxter CytoMate, or the Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca2+-free, Mg2+-free PBS, PlasmaLyte A, or another saline solution with or without buffer. In some embodiments, the undesirable components of the apheresis sample may be removed, and the cells directly resuspended in culture media.

[0273] In some embodiments, the isolation methods include the separation of different cell types based on the expression or presence in the cell of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acid. In some embodiments, any known method for separation based on such markers may be used. In some embodiments, the separation is affinity- or immunoaffmity-based separation. For example, the isolation in certain aspects includes separation of cells and cell populations based on the cells' expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, followed generally by washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner. Such separation steps can be based on positive selection, in which the cells having bound the reagents are retained for further use, and / or negative selection, in which the cells having not bound to the antibody or binding partner are retained. In some examples, both fractions are retained for further use. In certain aspects, negative selection can be particularly useful where no antibody is available that specifically identifies a cell type in a heterogeneous population, such that separation is best carried out based on markers expressed by cells other than the desired population. The separation need not result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection of or enrichment for cells of a particular type, such as those expressing a marker, refers to increasing the number or percentage of such cells, but need not result in a complete absence of cells not expressing the marker. Likewise, negative selection, removal, or depletion of cells of a particular type, such as those expressing a Attorney Docket No: 046483-7485W01(04066) marker, refers to decreasing the number or percentage of such cells, but need not result in a complete removal of all such cells.

[0274] In certain exemplary embodiments, multiple rounds of separation steps are carried out, where the positively or negatively selected fraction from one step is subjected to another separation step, such as a subsequent positive or negative selection. In certain exemplary embodiments, a single separation step can deplete cells expressing multiple markers simultaneously, such as by incubating cells with a plurality of antibodies or binding partners, each specific for a marker targeted for negative selection. Likewise, multiple cell types can simultaneously be positively selected by incubating cells with a plurality of antibodies or binding partners expressed on the various cell types.

[0275] In some embodiments, one or more of the T cell populations is enriched for or depleted of cells that are positive for (marker+) or express high levels (marker111811) of one or more particular markers, such as surface markers, or that are negative for (marker ) or express relatively low levels (marker'0") of one or more markers. For example, in certain aspects, specific subpopulations of T cells, such as cells positive or expressing high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+ T cells, are isolated by positive or negative selection techniques. In some cases, such markers are those that are absent or expressed at relatively low levels on certain populations of T cells (such as non-memory cells) but are present or expressed at relatively higher levels on certain other populations of T cells (such as memory cells). In one embodiment, the cells (such as the CD8+ cells or the T cells, e.g., CD3+ cells) are enriched for (i.e., positively selected for) cells that are positive or expressing high surface levels of CD45RO, CCR7, CD28, CD27, CD44, CD 127, and / or CD62L and / or depleted of (e.g., negatively selected for) cells that are positive for or express high surface levels of CD45RA. In some embodiments, cells are enriched for or depleted of cells positive or expressing high surface levels of CD122, CD95, CD25, CD27, and / or IL7-Ra (CD127). In certain exemplary embodiments, CD8+ T cells are enriched for cells positive for CD45RO (or negative for CD45RA) and for CD62L. For example, CD3+, CD28+ T cells can be positively selected using CD3 / CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3 / CD28 T Cell Expander). Attorney Docket No: 046483-7485W01(04066)

[0276] In some embodiments, T cells are separated from a PBMC sample by negative selection of markers expressed on non-T cells, such as B cells, monocytes, or other white blood cells, such as CD 14. In certain aspects, a CD4+ or CD8+ selection step is used to separate CD4+ helper and CD8+ cytotoxic T cells. Such CD4+ and CD8+ populations can be further sorted into subpopulations by positive or negative selection for markers expressed or expressed to a relatively higher degree on one or more naive, memory, and / or effector T cell subpopulations. In some embodiments, CD8+ cells are further enriched for or depleted of naive, central memory, effector memory, and / or central memory stem cells, such as by positive or negative selection based on surface antigens associated with the respective subpopulation. In some embodiments, enrichment for central memory T (TCM) cells is carried out to increase efficacy, such as to improve longterm survival, expansion, and / or engraftment following administration, which in certain aspects is particularly robust in such sub-populations. In some embodiments, combining TCM-enriched CD8+ T cells and CD4+ T cells further enhances efficacy.

[0277] In some embodiments, memory T cells are present in both CD62L+ and CD62L- subsets of CD8+ peripheral blood lymphocytes. PBMC can be enriched for or depleted of CD62L-CD8+ and / or CD62L+CD8+ fractions, such as using anti-CD8 and anti-CD62L antibodies. In some embodiments, a CD4+ T cell population and / or a CD8+ T population is enriched for central memory (TCM) cells. In some embodiments, the enrichment for central memory T (TCM) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and / or CD 127; in certain aspects, it is based on negative selection for cells expressing or highly expressing CD45RA and / or granzyme B. In certain aspects, isolation of a CD8+ population enriched for TCM cells is carried out by depletion of cells expressing CD4, CD 14, CD45RA, and positive selection or enrichment for cells expressing CD62L. In one aspect, enrichment for central memory T (TCM) cells is carried out starting with a negative fraction of cells selected based on CD4 expression, which is subjected to a negative selection based on expression of CD 14 and CD45RA, and a positive selection based on CD62L. Such selections in certain aspects are carried out simultaneously and in other aspects are carried out sequentially, in either order. In some embodiments, the same CD4 expression-based selection step used in preparing the CD8+ cell population or subpopulation, also is used to generate the CD4+ cell population or sub- Attorney Docket No: 046483-7485W01(04066) population, such that both the positive and negative fractions from the CD4-based separation are retained and used in subsequent steps of the methods, optionally following one or more further positive or negative selection steps.

[0278] CD4+ T helper cells are sorted into naive, central memory, and effector cells by identifying cell populations that have cell surface antigens. CD4+ lymphocytes can be obtained by standard methods. In some embodiments, naive CD4+ T lymphocytes are CD45RO-, CD45RA+, CD62L+, CD4+ T cells. In some embodiments, central memory CD4+ cells are CD62L+ and CD45RO+. In some embodiments, effector CD4+ cells are CD62L- and CD45RO. In one example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD1 lb, CD16, HLA-DR, and CD8. In some embodiments, the antibody or binding partner is bound to a solid support or matrix, such as a magnetic bead or paramagnetic bead, to allow for separation of cells for positive and / or negative selection.

[0279] In some embodiments, the methods include selection of a specific subpopulation of T- cells that are a T regulatory (Treg) cell-depleted population. A CD25+ depleted cell population, for example, can be obtained using, e.g., a negative selection technique, e.g., described herein. Preferably, the population of T regulatory depleted cells contains less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% of CD25+ cells. In some embodiments, ART inhibitors and / or an inhibitors of a PH domain protein may be used to reduce Treg cells during culturing.

[0280] In some embodiments, the manufacturing methods comprise reducing the number of (e g., depleting) Treg cells prior to manufacturing of the CAR-expressing cell. Methods of depleting TREG cells are known in the art. Methods of decreasing Treg cells include, but are not limited to, cyclophosphamide, anti-GITR antibody (an anti-GITR antibody described herein), CD25- depletion, and combinations thereof. For example, manufacturing methods comprise contacting the sample, e.g., the apheresis sample, with an anti-GITR antibody and / or an anti- CD25 antibody (or fragment thereof, or a CD25-binding ligand), e.g., to deplete Treg cells prior to manufacturing of the CAR-expressing cell (e.g., T-cell, NK cell) product.

[0281] In one embodiment, Treg cells, e.g., CD25+ T-cells, are removed from the population using an anti-CD25 antibody, or fragment thereof, or a CD25 -binding ligand, IL- 2. In another Attorney Docket No: 046483-7485W01(04066) embodiment, the anti-CD25 antibody, or fragment thereof, or CD25-binding ligand is conjugated to a substrate, e.g., a bead, or is otherwise coated on a substrate, e.g., a bead. In one embodiment, the anti-CD25 antibody, or fragment thereof, is conjugated to a substrate as described herein.

[0282] In some embodiments, the cells are incubated and / or cultured prior to or in connection with genetic engineering. The incubation steps can include culture, cultivation, stimulation, activation, and / or propagation. In some embodiments, the compositions or cells are incubated in the presence of stimulating conditions or a stimulatory agent. Such conditions include those designed to induce proliferation, expansion, activation, and / or survival of cells in the population, to mimic antigen exposure, and / or to prime the cells for genetic engineering, such as for the introduction of a recombinant antigen receptor. The conditions can include one or more of particular media, temperature, oxygen content, carbon dioxide content, time, agents, e.g., nutrients, amino acids, antibiotics, ions, and / or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cells. In some embodiments, the stimulating conditions or agents include one or more agent, e.g., ligand, which is capable of activating an intracellular signaling domain of a TCR complex. In certain aspects, the agent turns on or initiates TCR / CD3 intracellular signaling cascade in a T cell. Such agents can include antibodies, such as those specific for a TCR component and / or costimulatory receptor, e.g., anti-CD3, anti-CD28, for example, bound to solid support such as a bead, and / or one or more cytokines. Optionally, the expansion method may further comprise the step of adding anti-CD3 and / or anti CD28 antibody to the culture media (e.g., at a concentration of at least about 0.5 ng / ml). In some embodiments, the stimulating agents include IL-2 and / or IL-15, for example, an IL-2 concentration of at least about 10 units / mL.

[0283] In another embodiment, T cells are isolated from peripheral blood by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL™ gradient. Alternatively, T cells can be isolated from an umbilical cord. In any event, a specific subpopulation of T cells can be further isolated by positive or negative selection techniques.

[0284] The cord blood mononuclear cells so isolated can be depleted of cells expressing certain antigens, including, but not limited to, CD34, CD8, CD14, CD19, and CD56. Depletion of these Attorney Docket No: 046483-7485W01(04066) cells can be accomplished using an isolated antibody, a biological sample comprising an antibody, such as ascites, an antibody bound to a physical support, and a cell bound antibody.

[0285] Enrichment of a T cell population by negative selection can be accomplished using a combination of antibodies directed to surface markers unique to the negatively selected cells. An exemplary method is cell sorting and / or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected. For example, to enrich for CD4+cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD1 lb, CD 16, HLA-DR, and CD8.

[0286] For isolation of a desired population of cells by positive or negative selection, the concentration of cells and surface (e.g., particles such as beads) can be varied. In certain embodiments, it may be desirable to significantly decrease the volume in which beads and cells are mixed together (i.e., increase the concentration of cells), to ensure maximum contact of cells and beads. For example, in one embodiment, a concentration of 2 billion cells / ml is used. In one embodiment, a concentration of 1 billion cells / ml is used. In a further embodiment, greater than 100 million cells / ml is used. In a further embodiment, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / ml is used. In yet another embodiment, a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells / ml is used. In further embodiments, concentrations of 125 or 150 million cells / ml can be used. Using high concentrations can result in increased cell yield, cell activation, and cell expansion.

[0287] T cells can also be frozen after the washing step, which does not require the monocyteremoval step. While not wishing to be bound by theory, the freeze and subsequent thaw step provides a more uniform product by removing granulocytes and to some extent monocytes in the cell population. After the washing step that removes plasma and platelets, the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and will be useful in this context, in a non-limiting example, one method involves using PBS containing 20% DMSO and 8% human serum albumin, or culture media containing 10% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin and 7.5% DMSO, or 31.25% Plasmalyte-A, 31.25% Dextrose 5%, 0.45% NaCl, 10% Dextran 40 and 5% Dextrose, 20% Human Serum Attorney Docket No: 046483-7485W01(04066)

[0288] Albumin, and 7.5% DMSO or other suitable cell freezing media containing, for example, Hespan and PlasmaLyte A. The cells are then frozen to -80°C at a rate of 1°C per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing may be used as well as uncontrolled freezing immediately at -20°C or in liquid nitrogen.

[0289] In certain embodiments, cryopreserved cells are thawed and washed as described herein and allowed to rest for one hour at room temperature prior to activation using the methods of the present invention.

[0290] Also contemplated in the context of the invention is the collection of blood samples or apheresis product from a subject at a time period prior to when the expanded cells as described herein might be needed. As such, the source of the cells to be expanded can be collected at any time point necessary, and desired cells, such as T-cells, isolated and frozen for later use in immune cell therapy for any number of diseases or conditions that would benefit from immune cell therapy, such as those described herein. In one embodiment a blood sample or an apheresis is taken from a generally healthy subject. In certain embodiments, a blood sample or an apheresis is taken from a generally healthy subject who is at risk of developing a disease, but who has not yet developed a disease, and the cells of interest are isolated and frozen for later use. In certain embodiments, the T-cells may be expanded, frozen, and used at a later time. In certain embodiments, samples are collected from a patient shortly after diagnosis of a particular disease as described herein but prior to any treatments. In a further embodiment, the cells are isolated from a blood sample or an apheresis from a subject prior to any number of relevant treatment modalities, including but not limited to treatment with agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies, Cytoxan, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, and irradiation. ex-vivo

[0291] Whether prior to or after modification of cells to express the CAR, the cells can be activated and expanded in number using methods as described, for example, in U.S. Patent Nos. Attorney Docket No: 046483-7485W01(04066)

[0292] 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681 ; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Publication No. 20060121005.

[0293] For example, the T cells of the invention may be expanded by contact with a surface having attached thereto an agent that stimulates a CD3 / TCR complex associated signal and a ligand that stimulates a co-stimulatory molecule on the surface of the T cells. In particular, immune cell populations may be stimulated by contact with an anti-CD3 antibody, or an antigenbinding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore. For costimulation of an accessory molecule on the surface of the T cells, a ligand that binds the accessory molecule is used. For example, T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody, under conditions appropriate for stimulating proliferation of the T cells. Examples of an anti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France) and these can be used in the invention, as can other methods and reagents known in the art (see, e.g., ten Berge et al., Transplant Proc. (1998) 30(8): 3975-3977; Haanen et al., J. Exp. Med. (1999) 190(9): 1319-1328; and Garland et al., J. Immunol. Methods (1999) 227(1-2): 53- 63).

[0294] Expanding the T cells by the methods disclosed herein can be multiplied by about 10- fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700 fold, 800-fold, 900-fold, 1000-fold, 2000-fold, 3000- fold, 4000-fold, 5000-fold, 6000-fold, 7000-fold, 8000-fold, 9000-fold, 10,000-fold, 100,000- fold, 1,000,000-fold, 10,000,000-fold, or greater, and any and all whole or partial integers therebetween. In one embodiment, the T cells expand in the range of about 20-fold to about 50- fold.

[0295] Following culturing, the T cells can be incubated in cell media in a culture apparatus for a period of time or until the cells reach confluency or high cell density for optimal passage before passing the cells to another culture apparatus. The culturing apparatus can be of any culture apparatus commonly used for culturing cells in vitro. In certain exemplary embodiments, the level of confluence is 70% or greater before passing the cells to another culture apparatus. In Attorney Docket No: 046483-7485W01(04066) particularly exemplary embodiments, the level of confluence is 90% or greater. A period of time can be any time suitable for the culture of cells in vitro. The immune cell media may be replaced during the culture of the T cells at any time. In certain exemplary embodiments, the immune cell media is replaced about every 2 to 3 days. The T cells are then harvested from the culture apparatus whereupon the T cells can be used immediately or cryopreserved to be stored for use at a later time. In one embodiment, the invention includes cryopreserving the expanded T cells. The cryopreserved T cells are thawed prior to introducing nucleic acids into the immune cell.

[0296] In another embodiment, the method comprises isolating T cells and expanding the T cells. In another embodiment, the invention further comprises cryopreserving the T cells prior to expansion. In yet another embodiment, the cryopreserved T cells are thawed for electroporation with the RNA encoding the chimeric membrane protein.

[0297] Another procedure for ex vivo expansion cells is described in U.S. Pat. No. 5,199,942 (incorporated herein by reference). Expansion, such as described in U.S. Pat. No. 5,199,942 can be an alternative or in addition to other methods of expansion described herein. Briefly, ex vivo culture and expansion of T cells comprises the addition to the cellular growth factors, such as those described in U.S. Pat. No. 5,199,942, or other factors, such as Flt3-L, IL-1, IL-3, and c-kit ligand. In one embodiment, expanding the T cells comprises culturing the T cells with a factor selected from the group consisting of Flt3-L, IL-1, IL-3, and c-kit ligand.

[0298] The culturing step as described herein (contact with agents as described herein or after electroporation) can be very short, for example less than 24 hours such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours. The culturing step as described further herein (contact with agents as described herein) can be longer, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more days.

[0299] Various terms are used to describe cells in culture. Cell culture refers generally to cells taken from a living organism and grown under controlled condition. A primary cell culture is a culture of cells, tissues or organs taken directly from an organism and before the first subculture. Cells are expanded in culture when they are placed in a growth medium under conditions that facilitate cell growth and / or division, resulting in a larger population of the cells. When cells are Attorney Docket No: 046483-7485W01(04066) expanded in culture, the rate of cell proliferation is typically measured by the amount of time required for the cells to double in number, otherwise known as the doubling time.

[0300] Each round of subculturing is referred to as a passage. When cells are subcultured, they are referred to as having been passaged. A specific population of cells, or a cell line, is sometimes referred to or characterized by the number of times it has been passaged. For example, a cultured cell population that has been passaged ten times may be referred to as a PIO culture. The primary culture, i.e., the first culture following the isolation of cells from tissue, is designated P0. Following the first subculture, the cells are described as a secondary culture (Pl or passage 1). After the second subculture, the cells become a tertiary culture (P2 or passage 2), and so on. It will be understood by those of skill in the art that there may be many population doublings during the period of passaging. Therefore, the number of population doublings of a culture is greater than the passage number. The expansion of cells (i.e., the number of population doublings) during the period between passaging depends on many factors, including but is not limited to the seeding density, substrate, medium, and time between passaging.

[0301] In one embodiment, the cells may be cultured for several hours (about 3 hours) to about 14 days or any hourly integer value in between. Conditions appropriate for immune cell culture include an appropriate media (e.g., Minimal Essential Media or RPMI Media 1640 or, X-vivo 15, (Lonza)) that may contain factors necessary for proliferation and viability, including serum (e. ., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-gamma, IL-4, IL-7, GM- CSF, IL-10, IL-12, IL-15, TGF-beta, and TNF-a or any other additives for the growth of cells known to the skilled artisan. Other additives for the growth of cells include, but are not limited to, surfactant, plasmanate, and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol. Media can include RPMI 1640, AIM-V, DMEM, MEM, a-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, with added amino acids, sodium pyruvate, and vitamins, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and / or an amount of cytokine(s) sufficient for the growth and expansion of T cells. Antibiotics, e.g., penicillin and streptomycin, are included only in experimental cultures, not in cultures of cells that are to be infused into a subject. The target cells are maintained under conditions Attorney Docket No: 046483-7485W01(04066) necessary to support growth, for example, an appropriate temperature e.g., 31° C) and atmosphere (e.g., air plus 5% CO2).

[0302] The media used to culture the T cells may include an agent that can co-stimulate the T cells. For example, an agent that can stimulate CD3 is an antibody to CD3, and an agent that can stimulate CD28 is an antibody to CD28. This is because, as demonstrated by the data disclosed herein, a cell isolated by the methods disclosed herein can be expanded approximately 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300- fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, 6000-fold, 7000-fold, 8000-fold, 9000-fold, 10,000-fold, 100,000-fold, 1,000,000-fold, 10,000,000-fold, or greater. In one embodiment, the T cells expand in the range of about 2-fold to about 50-fold, or more by culturing the electroporated population. In one embodiment, human T regulatory cells are expanded via anti-CD3 antibody coated KT64.86 artificial antigen presenting cells (aAPCs). Methods for expanding and activating T cells can be found in U.S. Patent Numbers 7,754,482, 8,722,400, and 9,555,105, the contents of which are incorporated herein in their entirety.

[0303] In certain embodiments, it may be desired to administer activated T-cells to a subject and then subsequently re-draw blood (or have an apheresis performed), activate and expand the immune cells therefrom, and reinfuse the patient with these activated and expanded cells. This process can be carried out multiple times every few weeks. In certain embodiments, Immune cells can be activated from blood draws of from 10 cc to 400 cc. In certain embodiments, immune cells are activated from blood draws of 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc, or 100 cc. Using this multiple blood draw / multiple reinfusion protocol may serve to select out certain populations of immune cells.

[0304] In one embodiment, the method of expanding the T cells can further comprise isolating the expanded T cells for further applications. In another embodiment, the method of expanding can further comprise a subsequent electroporation of the expanded T cells followed by culturing. The subsequent electroporation may include introducing a nucleic acid encoding an agent, such as a transducing the expanded T cells, transfecting the expanded T cells, or electroporating the expanded T cells with a nucleic acid, into the expanded population of T cells, wherein the agent Attorney Docket No: 046483-7485W01(04066) further stimulates the immune cell. The agent may stimulate the T cells, such as by stimulating further expansion, effector function, or another immune cell function.

[0305] The contents of the articles, patents, and patent applications, and all other documents and electronically available information mentioned or cited herein, are hereby incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to physically incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other physical and electronic documents.

[0306] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made, and equivalents may be substituted without departing from the true spirit and scope of the invention. It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods described herein may be made using suitable equivalents without departing from the scope of the embodiments disclosed herein. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit, and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto. Having now described certain embodiments in detail, the same will be more clearly understood by reference to the following examples, which are included for purposes of illustration only and are not intended to be limiting.

[0307] EXPERIMENTAL EXAMPLES

[0308] The invention is now described with reference to the following Examples. These Examples are provided for the purpose of illustration only, and the invention is not limited to these Examples, but rather encompasses all variations that are evident as a result of the teachings provided herein.

[0309] 1: Ketogenic diet enhances CAR T cell antitumor function via Attorney Docket No: 046483-7485W01(04066)

[0310] To compare the effect of different established diets in CAR T cell immunotherapy, a syngeneic mouse B-cell lymphoma model (Balb / c, CD19+A20) was used in which mice were fed one of six diets, inoculated with tumors and subsequently infused with CD19-directed murine CAR T cells (mCART19) (FIGs. 1A, 2A). Each diet had a distinct nutrient profile representative of common human dietary patterns: high fiber, high protein, Western, high fat, and ketogenic (KD) (FIGs. IB, 2B and Table 1). Among all dietary interventions. CAR T-cell-treated mice fed a KD were found to experience the greatest improvement in tumor control and overall survival (FIGs. 2C, 2D, 9C, 9D, 9F, and 9G). Moreover, a higher proportion of tumor-infiltrating CAR T cells in mice under KD was observed compared to the control diet, suggesting increased intratumoral activity in response to KD (FIG. 9E). Notably, the body weights of mice on each of these diets increased over the course of intervention and did not differ significantly from the control diet, suggesting that the reduced tumor volume observed with the KD was not due to caloric restriction (FIG. 8B) or weight fluctuation (FIG. 9H). Importantly, mice fed a KD did not experience improved tumor control when infused with untransduced (UTD) control T cells, suggesting that a combination of antigen-mediated CAR stimulation and dietary intervention was required for maximal beneficial effects of the KD (FIGs. 2E and 91).

[0311] Table 1. Composition of diets in FIG. 1.

[0312] Attorney Docket No: 046483-7485W01(04066)

[0313] To investigate how KD might improve CAR T cell-mediated tumor control in an in vitro experiment, it was observed that serum from KD-fed mice could augment CAR T cell proliferation against tumor cells compared to serum from control diet-fed mice. This finding suggested that a circulating factor was sufficient to confer the beneficial effects of a KD on CAR T cells. Since diet- derived metabolites in the circulation can drive T cell effector functions (5), untargeted metabolomics was performed on the serum of mice in each diet group (seven days post-infusion) to examine how KD could influence and improve CAR T cell function (FIGs. 1A-1B). Hie serum metabolic profile of KD-fed mice clustered distinctly from that of all other diet groups (FIGs. 2G-2H). Expectedly, amongst KD-enriched metabolites, elevated levels of BHB were observed relative to all other diets. These results were further confirmed using a standard ketone monitor (FIG. 9J). In addition to BHB, docosanoic acid, cc-linolenic acid, linoleic acid, erucic acid, and tetradecanedioic acid were among the metabolites differentially enriched in KD serum (FIG. 2L). To determine specifically which of these KD-derived metabolites could support CAR T cell function, the top six KD-enriched metabolites (BHB, docosanoic acid, a -linoleic Attorney Docket No: 046483-7485W01(04066) acid, linoleic acid, erucic acid, tetradecanedioic acid) were tested for antigen-mediated CAR T cell activation in vitro. Of these metabolites, BHB was the only one that improved CAR T cell proliferation (FIG. 2 J and 81).

[0314] A ketogenic diet can be challenging to implement in the clinic due to strict macronutrient requirements, risk of nutrient deficiencies, and possible challenges related to cancer-associated appetiteloss (6). To test whether BHB alone could provide a functional benefit to CAR T cells equivalent to that of KD, To first measure the effect of BHB on CAR T cell function in vitro, BHB was added to culture medium during coculture of mouse CART19 cells and A20 lymphoma cells and found that it enhanced CART19 cytotoxicity and proliferation (FIGs. 12A, 12B). Importantly, BHB did not alter stimulate A20 growth in vitro (FIG. 12C). Next, to validate these observations in vivo, A20 tumor-bearing mice were placed on either BHB-supplemented water or KD (FIGs. 1J, 12D). Following CART19 infusion, both BHB and KD led to comparable improvements in tumor control relative to the standard diet (FIGs. 12E- 12F). Notably, when infusing untransduced control (UTD) T cells into mice under each diet intervention, no differences were observed in A20 tumor growth or the overall survival of these mice (FIGs. 1J, 12G) suggesting that enhanced tumor control with BHB or KD requires the CAR-antigen interaction. Of note, all mice gained weight over the course of treatment, ruling out body weight as a confounding factor (FIG. 12H).

[0315] To investigate whether BHB and KD impact CAR T cell function within the tumor bed, the phenotype of A20-tumor infdtrating CART 19 cells was analyzed seven days post-infusion in mice receiving either BHB or KD. Both BHB and KD increased CAR T cell cytokine secretion (IL-2+IFNy+cells) and reduced CAR T cell exhaustion (PD1+TIM3+) within the tumor (FIGs. IK, 121). Additionally, neither the tumor monocyte fraction nor antigen expression (MHCI / II) was affected by BHB or KD (FIG. 8G). Moreover, BHB or KD alone did not provide intrinsic tumor suppression relative to control diet (FIGs. 8H-8I). These findings suggest that KD enhances CAR T cell efficacy, and may do so through production of BHB that supports active CAR T cells rather than direct modification of the tumor bed and that enhanced tumor control with BHB or KD requires the CAR-antigen interaction but also that BHB does not directly affect the growth of A20 cancer cells.

[0316] While these results emphasize the combinatorial effects of CAR T cell treatment and dietary intervention, to see whether BHB or KD could additionally stimulate the endogenous T cell and immune response, and whether this contributes to the improved tumor control observed with CART19 in the A20 model, A20 tumors were implanted and infused with a very low dose of CART19 cells (CD45.1+) that was subtherapeutic to better observe endogenous T cell infiltration (CD45.2+) at high proportions in Attorney Docket No: 046483-7485W01(04066) tumors (FIG. 12 J). In mice fed a control diet, endogenous T cells were more exhausted (PD1+LAG3+) than in KD- or BHB-fed mice, and in KD-fed mice, endogenous T cells exhibited greater effector cytokine secretion (IL2+TNFa ) following ex vivo stimulation (FIGs. 12K-12M). Whether BHB or KD can affect components of the tumor microenvironment was investigated, but the results of this analysis found that neither the tumor myeloid cell fraction nor antigen expression (MHC-I / II) was affected by BHB or KD (FIG. 12N). These findings suggest that, in the A20 mouse lymphoma model, KD enhances CAR T cell efficacy and may additionally stimulate the endogenous T cell response against tumors, but does not seem to modify antigen presentation in the tumor bed.

[0317] Example 2: BHB supplementation enhances CAR T cell antitumor function in multiple xenograft models of cancer

[0318] Next, it was of interest to investigate how the duration of BHB administration affects CART19- mediated tumor control. It was initially observed that while BHB does improve in vitro CART19 killing against A20 cells when in culture medium, pre-exposing A20 cells to BHB did not sensitize them to CART19 killing (FIGs. 10A-10B). This suggests that BHB acts primarily on CAR T cells during activation and killing. To further explore this concept, tumor-bearing mice were supplemented with BHB according to one of three intervention protocols: (1) administration of BHB from tumor implantation to CAR T cell infusion only (“early stop”), (2) administration after CAR T cell infusion only (“late start”), or (3) continuous administration throughout the entire course of the experiment (“continuous treatment”) (FIG. 3A, 13A). Elevated blood ketone levels in cohorts placed on BHB prior to infusion was confirmed (FIG. 10C, 13B) However, significant improvement in overall survival and tumor control was only- observed when BHB was administered continuously (FIGs. 3B-3C, 13C-13D). Finally, the impact of providing BHB during ex vivo human CAR T cell manufacturing was evaluated to assess whether preexposing CAR T cells to BHB was sufficient to enhance antitumor function, including the potency of the final CAR T product (FIG. 10D). First, no differences in the distribution of T cell memory subsets were observed after expanding human CAR T cells with or without BHB (FIGs. 10E, 13E). Ex vivo BHB- treated human CART19 cells were found to have no functional benefit when infused into CD19+tumorbearing immunodeficient mice (FIGs. 10F-10G, 13F). Importantly, ex vivo BHB-treated human CART19 cells had no improvement in tumor control as compared to control CART 19 when infused into CD19+tumor-bearing NOD scid gamma chain-deficient (NSG) mice (FIGs. 13G-13H). Overall, these results suggest that BHB must be present in the circulation to confer a functional benefit to CAR T cells in vivo. Attorney Docket No: 046483-7485W01(04066)

[0319] Next, clinically relevant human xenografts of liquid and solid cancers were employed to validate the foregoing findings. First, the effect of BHB administration in the OCI-Lyl8 CD19+diffuse large B cell lymphoma (DLBCL) model was tested using human CART19. To this end, NOD scid gamma chain deficient (NSG) mice were subcutaneously inoculated with OCI-Lyl8 cells, while being supplemented daily with BHB via oral gavage (FIGs. 3D and 10H). After tumor engraftment, mice were randomized to receive either UTD cells or CAR T cells, with or without continuous BHB supplementation. While BHB had no effect on mice receiving UTD cells, BHB significantly enhanced CART 19 tumor control with 6 out of 7 mice showing complete response (FIGs. 3E-3F). BHB also boosted both CAR T cell expansion in peripheral blood and serum levels of IFNy (FIGs. 3H-3I). Overall, mice treated with both CART19 and BHB experienced dramatically improved overall survival (FIG. 3J). To investigate whether the effect of BHB could be reproduced in other B-cell malignancies, the established Nahn6 CD19+B cell acute lymphoblastic leukemia (B-ALL) model was used. Nalm6-bearing mice were placed on BHB ad libitum and infused with CART19 (FIG. 3K). Here, mice treated with both CART19 and BHB experienced a substantial improvement in tumor control and overall survival relative to controls, while BHB showed no effect on mice that received UTD cells (FIGs. 3L-3N).

[0320] Solid malignancies have historically been unresponsive to CAR T therapies (7). Thus, it was of interest to investigate whether BHB could provide a benefit for CAR T cells targeting solid tumors. Hie AsPC-1 pancreatic cancer xenograft model was utilized in this study in which tumor-bearing NSG mice were treated with clinically relevant anti-mesothelin CAR T cells (CARTmeso, NCT03054298) (FIG. 3P). Consistently, in this model, BHB was found to enhance tumor control and improve the overall survival of mice treated with CAR T cells compared to controls (FIGs. 3Q-3R). Interestingly, in this model, delayed tumor growth was observed in mice infused with UTD cells under BHB relative to vehicle. Consistent with these findings, BHB suppressed AsPC-1 cell growth in vitro at 5 mM of BHB (FIG. 130), possibly suggesting differential direct effect of BHB on solid versus liquid cancers in addition to its effect on CAR T cells (<*?). Taken together, these data show that BHB enhances human CAR T cell function in multiple xenograft models of liquid and solid cancers.

[0321] Example 3; BHB fuels CAR T cell metabolism

[0322] Next, the mechanisms driving the observed benefits of BHB on CAR T cell effector function was investigated. As a metabolite of fatty acid oxidation in the liver, BHB enters peripheral tissues, bypasses traditional glycolysis, and undergoes ketolysis to form acetyl-CoA that initiates the citric acid cycle. Therefore, BHB may be more readily converted into acetyl- Attorney Docket No: 046483-7485W01(04066)

[0323] CoA in CAR T cells compared to glucose and more efficiently drive the citric acid cycle. It was hypothesized that the enhanced CAR T cell function observed in several cancer models following BHB administration may be attributed to improved oxidative phosphorylation in CAR T cells. Indeed, increased mitochondrial function, as measured by higher VCh max (i.e., maximum rate of oxygen consumption) and spare respiratory capacity (SRC), in engineered T- cell therapies is associated with robust therapeutic responses (9-17). To test this hypothesis, antigen-activated CAR T cells were grown in a cell culture medium containing either relabelled glucose ([U-13Ce]- glucose) with unlabeled BHB or13C-labelled BHB ([U-13C4]-BHB) with unlabeled glucose. After mass spectrometry, it was found that while carbon from glucose preferentially formed lactate, labelled carbon from BHB was highly enriched in citric acid cycle intermediates, such as fumaric acid, a-ketoglutarate, and succinate (FIGs. 4A-4B and 14).

[0324] To characterize the kinetics of BHB and glucose uptake in CAR T cells, antigen-activated CAR T cells were cultured in media containing [l,6-13Ce]-glucose and [U-13C4]-BHB, permitting simultaneous detection of glucose and BHB-derived intermediates (FIG. 4J). Over a time course of 5 hours, CAR T cells from the culture were analyzed byt mass spectrometry at each interval. The results of this analysis showed that both glucose and BHB were rapidly taken up by activated CAR T cells, with no observable differences in their uptake kinetics, and that isotopic steady state was achieved (FIG. 4K). Key TCA intermediates, such as citric acid and glutamate, were readily labeled from BHB (m+2) but not from glucose (m+1) over time (FIGs. 4M and 4N). Overall, simultaneous tracing further validated that BHB preferentially enriches the TCA cycle over glycolytic intermediates (FIG. 4L). These findings provide evidence that BHB is a principal anaplerotic substrate supporting TCA cycle function in activated CAR T cells.

[0325] Given that BHB carbon is preferentially incorporated into TCA components of CAR T cells fueling CAR T cell oxygen-dependent metabolism (i.e., electron-transport chain), the effect of BHB on overall CAR T cell bioenergetics was tested using the Seahorse assay. The results of this analysis show that murine CAR T cells treated with BHB during manufacturing process experienced a substantial improvement in OCR relative to no BHB in antigen-activated human CAR T cells (FIG. 4Q). Taken together, these data suggest that BHB can serve as a more efficient fuel source for mitochondrial energy production in CAR T cells compared to glucose. Attorney Docket No: 046483-7485W01(04066)

[0326] Since increased Acetyl-CoA from BHB metabolism has been shown to acetylate histones of active T cells (5, 12), BHB-mediated modulation of CAR T cells was examined at both transcriptional and epigenetic levels. Both RNA sequencing and an assay for transposase- accessible chromatin with sequencing ATAC-sequencing on antigen-activated CAR T cells was performed. BHB treatment was found to increase transcriptional activation of IFNy and decrease accessibility of dysfunction markers (i.e., CTLA4, BCL6, and IL-2Ra) (FIGs. 13A-13F). Additionally, ex vivo activated murine CART19 cells displayed mildly enhanced chromatin opening of effector T cell regions (e.g., FOXO1, TCF7) and closing of suppressive chromatin regions (TOX, TGF0) suggesting an increase of histone acetylation (FIG. 4D).

[0327] To confirm the mechanism of enhanced TCA cycle activation as described above and that BHB metabolism via ketolysis is necessary to observe the functional benefits of BHB on CAR T cell function, CRISPR-Cas9 was used in CAR T cells to knock out 3 -hydroxybutyrate dehydrogenase 1 (BDH1), the enzyme responsible for converting BHB to acetoacetate (in the process of ketolysis) that precedes acetyl-CoA (FIG. 4E). Next, the anti-tumor function of BD / TT-deficient CAR T cells in A20 tumor-bearing mice was tested (FIG. 4F). BDH1 knockout CAR T cells displayed reduced tumor control capability compared to control CAR T cells when administered BHB but not when vehicle was administered (FIG. 4G). Further, it was of interest to examine how BHB influences CAR T cells under hypoxic conditions in which mitochondrial respiration is reduced. The results of this analysis show that the levels of IFNY increased in a dose-dependent manner with BHB under normoxic conditions, while no increase was observed under hypoxia (FIG. 4H). Collectively, these findings suggest that BHB metabolism enhances CAR T cell function by fueling the citric acid cycle.

[0328] Example 4; BHB promotes the metabolic fitness of T cells in cancer patients and healthy volunteers

[0329] Encouraged by the results from in vitro and in mouse models, an observational study was performed to investigate whether administration of BHB to healthy volunteers could impact T cell metabolism. To this end, peripheral blood was isolated from healthy volunteers (w=10) who ingested BHB (105 mL Ketone-IQ) after an overnight fast (FIG. 6A). Circulating BHB levels rose overtire following 90 minutes while glucose levels remained unchanged (FIG. 6B). After 90 minutes, peripheral blood was drawn again. T cells were Attorney Docket No: 046483-7485W01(04066) isolated from each volunteer’s blood pre- and post-BHB ingestion, and oxygen consumption was measured immediately upon isolation. Serum metabolomics revealed a specific enrichment of BHB at 90 minutes, along with detectable interconversion to other ketone bodies (e.g., acetone), and decrease of several long- chain fatty acids (e.g., linoleic acid and oleic acid) as well as prostaglandin Ez (PGE2) (FIG. 6C), consistent with sequencing data showing induction of the PGEz-degrading enzyme HPGD (FIG. 15K). Moreover, BHB supplementation increased serum levels of BHB-ASN, BHB-DOPA, and BHB-MET, in line with a recently described BHB shunt pathway linking ketone metabolism to energy balance (FIG. 6C)(75). Following BHB ingestion, an overall increase in peripheral T cell spare respirator} capacity, VO2max, and ATP production was observed (FIGs. 6D-6G and 17A). The conversion of BHB to acetoacetate is known to generate NADH, which can be subsequently used to generate the mitochondrial proton gradient and establish membrane potential (A m). Following BHB administration, in a subset of volunteers (w=4) significantly increased intracellular NADH (3 of 4 volunteers) and mitochondrial membrane potential was found (4 of 4 volunteers) (FIGs. 6H-6J and 17B). Notably, an increase in effector cytokine production did not occur at 90 minutes, suggesting that the changes in T cell function following this single BHB dose are primarily metabolic in nature during this short interval (FIG. 17C).

[0330] To investigate whether BHB could enhance patient-derived T cell expansion, 4-lBB^ anti-CD19 CAR T cells were generated using T cells isolated from the apheresis products of three lymphoma patients (who subsequently received tisagenlecleucel), and the cells were cultured with or without BHB supplementation. The addition of BHB during manufacturing was found to promote both proliferation and mitochondrial function of patient-derived T cells (FIGs. 17D-17G). Further, to gain clinical insights into the significance of BHB, serum samples were retrospectively collected and analyzed. The serum samples were collected seven days post-infusion from CART 19 patients (n=17) enrolled in the first single-center study of CART19 (CTL019, now tisagenlecleucel) for relapsed / refractory (r / r) DLBCL and follicular lymphoma (NCT02030834), and analyzed by mass spectrometry (FIG. 18A)(76). Higher serum BHB concentrations were associated with greater peripheral CART 19 expansion, suggesting that variability in BHB levels may contribute to differences in CAR T cell proliferation in patients (FIG. 18B).

[0331] FIG. 18C shows the association between serum BHB levels and incidence of CRS. CRS grading was carried out at day 7 post CART19 infusion, split by BHB levels of n=39 CART19 patients. CRS was categorized as no CRS (white), low-grade CRS (grade 1-2. blue), or high-grade CRS (grade >3, red). Importantly, BHB levels did not correlate meaningfully with the incidence of cytokine release syndrome (CRS) (FIG. 18C), suggesting that BHB variability may not impact adverse event rates following CART 19 infusion. The graphical observation of increased high-grade CRS (G3+) in the high BHB group in FIG. Attorney Docket No: 046483-7485W01(04066)

[0332] 18C is driven by a single patient who had a Grade 4 CRS. Since a small patient pool was utilized for analysis, it was not possible to perform meaningful statistics within the high-grade CRS group.

[0333] Table 2 shows the raw values and breakdown of CRS for tire 39 patients from which serum metabolomics and CFRS analyses was perfonned:

[0334] Table 2

[0335] As shown in FIG. 18C, the data has been stratified by Low7CRS (G1+G2) and High CRS (G3+), as well as show ing the chi-squarcd statistics, w hich confirm the absence of a trend tow ards CRS in tire high BHB High Group (FIG. 18C).

[0336] To explore potential clinical safety concerns, a complete metabolic panel (CMP) was performed. Specifically, healthy donors’ serum during both short-interval (90 min) and long-term (14 day) BHB consumption was tested. No abnormal deviation of lab values were found that would suggest tissue damage or toxicity (FIGs. 18D and 18E). Overall, these results demonstrate that exogenous BHB administration can safely enhance circulating T cell metabolic function in humans and have justified the implementation of a proof-of-concept trial in patients receiving commercial CART19 therapies (FIG. 18F, NCT06610344).

[0337] To test the translational relevance of the foregoing findings, CART19 patient serum (w=17) w as retrospectively analyzed. CART 19 patient serum was taken seven days post-infusion from the first singlecenter study of CART19 (CTL019, now tisagenlecleucel) for replased / refractory DLBCL and follicular lymphoma (UPCC13413) trial using mass spectrometry (FIG. 7A). In line with the hypothesis that BHB can support CAR T cell proliferation, BHB serum concentration was found to positively correlate with CART 19 peripheral expansion (p=0.0164) with no observed increase in toxicity (FIGs. 7B-7C).

[0338] To examine whether BHB could, indeed, support patient CAR T cell growth, CAR T cells were generated and expanded using T cells isolated from the apheresis products of three patients (who later received tisagelecleucel) with or without BHB in the cell culture medium. Supplementing BHB during manufacturing boosted proliferation and mitochondrial function (FIGs. 7D-7F).

[0339] Finally, to investigate whether BHB could provide transient benefit to peripheral T cells shortly following ingestion when blood ketone levels were elevated, peripheral blood was isolated from healthy Attorney Docket No: 046483-7485W01(04066) donors administered BHB following an overnight fast (105mL Ketone-IQ) (FIG. 7G). Circulating ketone levels rose over the next 90 minutes while glucose levels remained unchanged (FIG. 7H). After 90 minutes, peripheral blood was drawn once more. T cells pre- and post-BHB ingestion were isolated from each donor’s blood, and oxygen consumption was measured immediately upon isolation. Further, an increase in peripheral naive T cell oxygen consumption and ATP production was observed following BHB ingestion (FIGs. 71- 7K). Importantly, these increases were proportional to each donor's respective increase in circulating ketone levels. Overall, these results demonstrate that exogenous BHB administration can safely enhance circulating T cell metabolic function in humans.

[0340] Example 5: BHB induces transcriptional and epigenetic changes in CAR T cells that promote effector function

[0341] To identify key regulatory genes and pathways associated with the improved performance of BHB-treated CAR T cells, CD19+OCI-Lyl8 lymphoma tumors were subcutaneously implanted into NSG mice and infused with curative doses of CAR T cells to induce robust in vivo proliferation without driving overt exhaustion (FIG. 5A). On day 17 postinfusion, circulating T cells were isolated and single-cell RNA sequencing was performed. Analysis of circulating CAR T cells revealed six major T cell subsets, including activated CD4+T cells, GZMB+CD8+T cells, MK167+CD47CD8+T cells, CD4+ / CD8+T central memory cells, CD8+T effector memory cells, and gamma-delta (yS) T cells (FIGs. 5B and 5C). BHB-treated CART19 cells comprised a higher proportion of GZMB+CD8+T cells and MKI67+CD4+ / CD8+T cells (FIGs. 5D and 5E), suggesting a shift towards a more cytotoxic state. Next, a targeted analysis of genes involved in mitochondrial respiration across each cluster was performed. Genes encoding components of complex III (UQCR10), ubiquinone and complex I (NDUFB10 and NDUFBF), cytochrome c (MT-CO2, COX8A, COX5A and COX4H), and ATP synthase (ATP5MG, ATP5ME, ATP5MC3, ATP5F1E, ATP5F1D and ATP5F1B) were upregulated BHB- treated activated CD4+T cells, GZMB+CD8+T cells, and MKI67+CD4+and CD8+populations which represent functionally active CART 19 cells (FIG. 5F). In contrast, these same genes shared similar expression levels between the BHB- and vehicle-treated groups across clusters of CD4+ / CD8+T central memory cells, CD8+T effector memory cells, and y5 T cells (FIG. 15A). Overall, global differential gene expression analysis across all CART19 clusters showed that Attorney Docket No: 046483-7485W01(04066)

[0342] BHB treatment downregulated exhaustion-associated genes such as RGS16, TGFB1 and upregulated genes related to oxidative phosphorylation (ATP5MC3) and cytolytic function (GZMB, GZMK) (FIG. 5G). Unbiased pathway analysis and gene set enrichment analysis (GSEA) validated that BHB induced CAR T cell signatures corresponding to enhanced oxidative phosphorylation (FIG. 5H). Overall, these data suggest that BHB helps to shift CAR T cell transcriptional programming towards enhanced oxidative phosphorylation during active in vivo tumor control.

[0343] Increased acetyl-CoA levels from BHB metabolism have been shown to acetylate histones of active T cells (8, 12). Therefore, the effect of BHB on CAR T cell epigenetics was examined. Using ATAC-sequencing, in vitro anti-CD3 / CD28-stimulated CAR T cells treated with BHB were found to exhibit enhanced chromatin opening of effector T cell regions (e. ., F0X01, TCF7) and closing of suppressive chromatin regions (e.g., TOX, TGFfl) (FIG. 15B). Next, to examine joint transcriptional and epigenetic effects of BHB during active CAR T cell killing, bulk RNA- and ATAC-sequencing was performed on in vitro antigen-activated CART 19 cells. Bulk RNA sequencing of CD19-stimulated CAR T cells treated with BHB revealed robust transcriptional reprogramming, in particular upregulation of inflammatory markers (e.g., IL-22, ADGRG1, CXCL8) and downregulation of interferon-stimulated genes (e.g., 1SG15, OAS1, IF16) (FIG. 15C). To explore the potential metabolomic correlations of these transcriptional changes mediated by BHB, Human Metabolome Database (HMDB) enrichment analysis based on upregulated genes revealed predicted enrichment in lipid metabolism (glycerophospholipid and Lys phosphatidylglycerol species), and amino-acid metabolism (L-arginine) (FIGs. 15C and 15D). These findings were accompanied by activation of GPCR downstream signaling and the urea cycle, consistent with enhanced metabolic and signaling reprogramming in BHB-treated CAR T cells (FIG. 15E). ATAC-seq of antigen-stimulated CAR T cells revealed BHB-induced accessibility at loci encoding effector molecules critical for CAR T memory and persistence and function (e.g., CCR7, EOMES, ICOS, CARD11, BCL2), as well as genes regulating ATP synthesis and hydrolysis (ATP2C1, ATP6AOV2) (FIGs. 5I-5K) (13, 14). Furthermore, profiling of H3K27 acetylation via CUT&RUN- sequencing revealed increased acetylation in several mitochondrial transport-related genes (e.g., SLC25A42, TOMM40, AFG3L2, GRPELl) (FIGs. Attorney Docket No: 046483-7485W01(04066)

[0344] 15F-15H). Overall, BHB was found to induce broad epigenetic changes in activated CAR T cells that supported T cell signaling and metabolism (FIGs. 151 and 15J). Finally, integration of ATAC-seq, RNA-seq, and CUT&RUN data identified HPGD, a NAD+-dependent prostaglandindegrading enzyme, as uniquely upregulated, with concomitant increases in chromatin accessibility and intergenic H3K27 acetylation (FIG. 15K). These data indicate that BHB may modulate transcriptional and epigenetic programs that promote a more metabolically resilient state, supporting enhanced effector CAR T cell function.

[0345] Example 6; Discussion

[0346] CAR T cell metabolic fitness, persistence, and resistance to exhaustion are critical determinants of therapeutic success. Current strategies to enhance CAR T cell function often involve approaches like CRISPR-mediated knockouts of exhaustion-related checkpoints or overexpression of memory-associated transcription factors (9, 11, 17-21'). These methods, however, are expensive and time-consuming as they require the modification of the cellular product.

[0347] In this study, the effect of diet - a modifiable patient lifestyle factor - on the anti-tumor efficacy of CAR T cell therapy was assessed. Among six different diets representative of distinct nutrient patterns, we found that a ketogenic diet significantly improved CAR T cell in vivo antitumor function relative to a control diet. Serum profiling of KD-fed mice revealed elevated BHB levels relative to other diets. Since a major goal of T cell immunotherapies has been to improve metabolic fitness in the tumor bed, it was hypothesized that BHB could provide an alternative fuel for active CAR T cells. Although the reported studies here are primarily focused on metabolic enhancement via increased oxidative phosphorylation, the full impact of BHB on CAR T cell function through epigenetics, particularly through histone acetylation and more recently P-hydroxybutyrylation, remains to be fully elucidated. The present findings underscore the role of ketone-driven metabolic and epigenetic remodeling in enhancing T cell function and demonstrate that BHB administration enhances CAR T cell function in several clinically relevant models of cancer. Most critically, a prospective observational study was conducted in healthy human volunteers, which revealed that oral BHB supplementation significantly improved the Attorney Docket No: 046483-7485W01(04066) metabolic function of peripheral T cells. This represents a rare and essential step in translational immunometabolism, demonstrating in real time and in human beings that a defined metabolite can enhance T cell function outside of a disease model. These findings not only validate the mechanistic underpinnings observed in mice but also provide a compelling rationale for clinical translation of BHB-based metabolic interventions in T cell-mediated cancer immunotherapies (39).

[0348] The results described herein have several important implications. In cancer patients, metabolite supplementation may be a more feasible and targeted approach to enhancing T-cell function than dietary interventions, offering better patient compliance and circumventing metabolic side effects of strict dietary patterns. Retrospective work has shown that cancer patients undergoing immune checkpoint blockade had improved responses when consuming a high-fiber diet, likely due to the effect of resultant metabolites (i.e., short-chain fatty acids) on effector T cells (2). In addition, other recent reports have supported the use of metabolites like trans-vaccenic acid to boost T cell effector functions (7). Of note, BHB is a unique exogenous carbon source that can produce the high-energy electron carrier NADH in a single enzymatic step, making it a particularly efficient energy substrate to be administered externally (-7). In addition, the multi-omics studies described herein converged on HPGD enrichment following BHB addition, and human serum metabolomics after BHB ingestion showed downregulation of PGE2 (22). Since HPGD inactivates prostaglandin E2, and PGE2 suppresses IL-2 signaling and effector T-cell expansion, induction of HPGD may enable BHB to counteract prostaglandin- mediated immunosuppression and thereby promote T-cell differentiation. Overall, based on these studies, it is conceivable that a series of metabolites may present highly efficient interventions which not only enhances CAR T cell effector functions but also integrates seamlessly into existing treatment regimens without adverse effects. Thus, the present study encourages the clinical evaluation of metabolite supplementation to improve the efficacy of T cell-mediated immunotherapies. istic effects of BHB on solid tumor cell killi Attorney Docket No: 046483-7485W01(04066)

[0349] In contrast to the lymphoma and leukemia models, BHB was found to exert its effects on PDAC growth even in the absence of CAR T cells. Specifically, untransduced T cell (UTD) controls combined with BHB suppressed AsPC-1 tumor growth. Previously, it was demonstrated that BHB can suppress epithelial cell-derived colorectal cancer through the upregulation of the tumor suppressor genes and inhibition of epithelial cell proliferation. To further explore this finding in vitro experiments were performed. Specifically, in vitro killing assays of AsPC-1 PDAC cells (FIG. 19A) and B 16 melanoma cells (FIG. 19B) cells following 60-hour treatment with vehicle, 2 mM, or 5 mM BHB were carried. Additionally, in vitro killing assays were carried out for SKOV3 ovarian cancer cells (FIG. 19C) and 4T1 breast cancer cells (FIG. 19D) following 60-hour treatment with vehicle, 1 mM, 2 mM, 5 mM or 10 mM BHB. Statistics were determined at 60 h by determined by one-way ANOVA with post hoc Tukey tests (FIGs. 19A- 19D).The results of this analysis showed that BHB (5mM) significantly slowed the growth rate of AsPC-1 cells in culture (FIGs. 130 and 19A). In addition, BHB (lOmM) was found to slow the growth rate of two additional solid tumor cell lines: B 16 melanoma and SKOV-3 ovarian cancer (FIGs. 19B and 19C).

[0350] Example 8: BDH1 knockdown abolishes the survival benefit of BHB

[0351] A potential biological limitation of CRISPR concerns low knockout efficiency (e.g., reduced targetability of the Bdhl locus by gRNA). Thus, an alternative approach was employed to knock out BDH1 in T cells. Specifically, a lentiviral short-hairpin RNA (shRNA) system (Santa Cruz Biotechnology) targeting human BDH1 was utilized in which the shRNA was integrated into a lentiviral vector and introduced into CAR T cells during manufacturing (FIG. 20A). Because this vector carries a puromycin-resi stance gene, CAR T cells were cultured in puromycin-supplemented media (5 pg / mL) until the end of the manufacturing to ensure a pure BDHl-shRNA+ CAR T cell population.

[0352] Briefly, human T cells (CD4:CD8 at 1: 1) were activated with anti-CD3 / CD28, transduced with anti-CD19 CAR and a BDH1 shRNA lentiviral vector, selected with puromycin (5 pg / mL), expanded, and cryopreserved. Attorney Docket No: 046483-7485W01(04066)

[0353] Next, a model of human leukemia (CD19+Nalm6) was utilized in which CD19+Nalm6 cells were engrafted into NSG mice and supplemented their drinking water with either vehicle or BHB (FIG. 20B). NSG mice bearing CD19+Nalm6 leukemia received BHB supplementation or vehicle beginning before tumor CART19 infusion. BDHl-shRNA CART19 cells were infused intravenously on day 0. Tumor burden was monitored by bioluminescence imaging (BLI) and survival was recorded (FIG. 20C). Following infusion of BDH1 -knockdown CAR T cells, BHB supplementation had no statistically significant effect on tumor control or survival (FIGs. 20C- 20D).

[0354] Overall, these results support the conclusion that BDH1 is necessary and relevant for BHB-mediated enhancement of CAR T-cell function.

[0355] Example 9; Acetoacetate (AcAc) fails to rescue metabolic function in BDHl-deficient CAR T cells

[0356] In an attempt to rescue mitochondrial function in BDHl-deficient CART19 cells, an experimental arm was set up in which activated Cre+ Bdhlfl / fl CART19 were supplemented with acetoacetate (i.e., AcAc), the metabolite downstream of BDH1. In this case, SeahorseXF was performed on BDHl-deficient CAR T cells after 24 h of culture with either vehicle, BHB, or AcAc. Despite AcAc being the product of BDH1 dehydrogenation of BHB, however, no improvement in OCR upon addition of AcAc was observed (FIG. 21B). This finding suggests a specific role for BDH1 and supports the hypothesis that the full holistic effect of BHB on CAR T cells may also include its additional anaplerotic function: upon BDH1 -mediated conversion of BHB to AcAc, NADH is generated, which can directly fuel the electron transport chain (FIG. 21A).

[0357] Example 10: The role of Hcar2 and Hopx in BHB-regulated CAR T cell function

[0358] HCAR2 has been previously implicated as a receptor mediating a portion of the signaling effects of BHB 1, and in a previous study BHB was found to suppress colorectal cancer proliferation through the HCAR2-HOPX pathway. To examine whether the HCAR2 receptor is appreciably expressed in T cells, as it is in epithelial cells that use it to mediate HOPX Attorney Docket No: 046483-7485W01(04066) expression, Human Protein Atlas expression of HCAR2 across cell types was examined. The results of this analysis showed that epithelial cells exhibit high HCAR2 expression, whereas T cells showed little to none (FIG. 22A). This suggests that the low expression of HCAR2 in T cells makes it unlikely to be a major mediator of BHB’s effects in these cells.

[0359] To directly test a possible role for HCAR2 activity in T cells, CD19-expressing B16 melanoma cells were generated, seeded onto xCELLigence E-plates, and co-cultured with CD 19- directed CAR T cells derived from wild-type (FIG. 22B), Hcar2 / (FIG. 22C), or Hopx / (FIG. 22D) at an effector-to-target (E:T) ratio of 1:2 in the presence of 0, 2, or 5 mM BHB in order to assay for cytotoxicity. Real-time changes in impedance (cell index) were recorded using the xCELLigence system. Data are presented as mean ± SEM of technical replicates. The results of this analysis showed that in both Hcar2 / and Hopx / genetic backgrounds CART cells with 5 mM BHB still significantly enhanced CAR T cell-mediated killing, compared to vehicle supplemented Hcar2 / orHopx- / " CART19 cells (FIGs. 22B-22D). Overall, these findings demonstrate that, unlike in colorectal cancer epithelial cells, the beneficial effect of BHB on CAR T function is independent of HCAR2 or HOPX signaling.

[0360] Example 11: BHB enhances proliferation without altering mTORCl signaling

[0361] To test whether BHB might act through mTOR modulation, antigen-mediated proliferation in the presence of rapamycin, a selective mTORCl inhibitor, was assessed by examining phosphorylation of S6 kinase (S6K), a canonical mTORCl downstream target. Here, anti-CD19 human CAR T cells were co-cultured for 96 h with an irradiated CD19+ lymphoma cell line (OCI-Lyl8; effectortarget = 0.25, n=3 technical replicates) in the presence or absence of 2 mM BHB and increasing rapamycin concentrations (FIG. 23A). Rapamycin alone was found to have little effect on CART 19 proliferation (black line), but abolished the proliferative advantage obtained from 2 mM BHB as rapamycin dose increased (red line) (FIG. 23 A). However, immunoblot analysis of phosphorylated S6 kinase (p-S6K), total S6K, and 0-actin in human CART 19 cells stimulated on human CD19-coated wells with 5 mM BHB and / or 50 nM rapamycin for 1 h or 24 h of antigen-stimulated CART 19 cells treated for 1 h or 24 h showed that BHB did not alter p-S6K levels, whereas rapamycin robustly suppressed this signal (FIG. Attorney Docket No: 046483-7485W01(04066)

[0362] 23B) Together, these findings suggest that while BHB’s proliferative benefit in CAR T cells may require intact mTORCl activity, BHB does not exert its effects through direct modulation of mTORCl signaling in CAR T cells.

[0363] 12: Materials and Methods

[0364] Cancer cell lines, primary cells, and general cell culture

[0365] Mouse CD 19+ B-cell malignant cell line was used (murine large B cell lymphoma: A20). Two human CD 19+ B-cell malignant cell lines used include (B-ALL Nalm6; DLBCL: OCI- Lyl8). All human suspension cell lines were grown and cultured in standard culture mediium “RIO” (RPMI 1640 + 10% FBS, 1% Penicillin / Streptomycin, 1% HEPES, 1% GlutaMAX) at 37°C in 5% ambient CO2 and maintained at a concentration of 0.3-1 x 106cells / mL. All human suspension cell lines were transduced with a lentiviral vector containing a 2A-self cleaving peptide-linked transgene encoding both click-beetle-green (CBG) luciferase and GFP. The A20 model was cultured in RIO medium supplemented with 0.05 mM 2-mercaptoethanol under standard conditions. One human mesothelin+ pancreatic cancer cell line was used (AsPC-1), and grown and cultured in standard culture medium R10 at 37°C in 5% ambient CO2 and maintained at a confluency of -60-70%. One murine melanoma transduced with murine CD 19 was used (B16-muCD19), cultured in “DMEM10” (Dulbecco's Modified Eagle Medium (DMEM) + 10% FBS, 1% Penicillin / Streptomycin, 1% HEPES, 1% GlutaMAX), and maintained at a confluency of -60-70% under standard conditions. All suspension and adherent cell lines were originally procured from ATCC or DSMZ, authenticated by the University of Arizona Genetics Core, and ensured to be free of Mycoplasma contamination (Lonza).

[0366] Primary human T cells (for standard CAR T cell production, and subsequent in vitro / in vivo experiments), were obtained through the University of Pennsylvania’s Human Immunology Core. Primary murine T cells were isolated from freshly resected spleens of 6 to 8-week-old Balb / c or C57BL / 6 mice. Spleens were then harvested, grinded, and passed through a 70 pm mesh filter. Splenocytes were pelleted, resuspended in 2 m of IX RBC Lysis Buffer (BioLegend) per spleen, incubated for 2-3 minutes before washing with 10 mL R10 + 0.05 mM 2-mercpatoethanol per spleen. Bulk murine T cells were then purified from splenocytes using the Attorney Docket No: 046483-7485W01(04066)

[0367] Miltenyi Biotec Pan-T cell Isolation Kit II (negative selection) according to the manufacturer’s instructions.

[0368] Lentivirus production and human CAR T cell manufacturing

[0369] Replication-defective, third-generation lentiviral vectors were produced using HEK293T cells (ATCC ACS-4500). Briefly, 8 x io6cells were seeded per T175 culture flask in standard RIO media and incubated overnight at 37°C. After 24 hr, the HEK193T cells (-70-80% confluency) were transfected using a mixture of Lipofectamine 2000 (116 pL, Invitrogen), pMDG.l (7 pg), pRSV.rev (18 pg), pMDLg / p.RRE (18 pg) packaging plasmids and 15 pg of transfer / expression plasmid (e. ., 4-lBBz CAR (23, 27), per flask. Lipofectamine and plasmid DNA were diluted in 4.6 mb Opti-MEM Reduced Serum media (Thermo-Fisher) prior to transfer into lentiviral production flasks. At 24 hr and 48 hr following transfection, the culture media from each flask was collected, filtered (0.45 pm), and concentrated using high-speed ultracentrifugation (8,000 x g for 16 hr or 25,000 x g for 2.5 hr). Lentiviral concentrates were divided into aliquots, snap-frozen, and stored in -80°C.

[0370] To generate human CAR T cells, T cells were first obtained from the University of Pennsylvania Fluman Immunology Core. CD4+ and CD8+ subsets were mixed at a 1 : 1 ratio and stimulated with CD3 / CD28 Dynabeads (Thermo Fisher) at a bead-to-cell ratio of 2: 1, followed by overnight incubation at 37 °C. After 24 hours, CAR lentiviral vectors were introduced into the stimulated cultures at a multiplicity of infection (MOI) ranging from 1.5 to 2.5. On day 5, stimulation beads were removed, and cells were subsequently counted every other day until growth kinetics and cell size indicated recovery from stimulation (average cell volume -350 fL). Unless otherwise explicitly specified in the Figure legend, all experiments employed CAR19 encoding the CTL019 construct, which incorporates the FMC63 scFv, 4- IBB, and CD3(^ signaling domains.

[0371] Retrovirus production and mouse CAR T cell manufacturing

[0372] One MSGV (Murine stem cell virus-based splice-gag vector) encoding murine CD19-directed 4- 1BB co-stimulated CD3^ CAR construct (muCARl 9-MSGV) was used. One MSGV encoding murine mesothelin-directed 4- IBB CD3^ CAR-T2A-GFP construct (mmesoCAR-T2A-GFP-MSGV) was used. Murine gamma retrovirus from MSGV vectors were generated from Platinum-E (Plat-E) cells. Plat-E Attorney Docket No: 046483-7485W01(04066) cells were cultured in DMEM10 + 1 pg / mL puromycin + 10 pg / m blasticidin. 24 hrprior to transfection, 8 x 106cells Plat-E cells were seeded per T175 culture flask, and the medium was exchanged with DMEM10 (without puromycin or blasticidin). On the day of Plat-E transfection (~70- 80% confluency), a mixture of Lipofectamine 2000 (60 pL, Invitrogen) and muCAR19-MSGV retroviral construct (15 pg) was diluted in 3 mL Opti-MEM Reduced Serum medium (Thermo-Fisher) per retroviral production flask and transferred to said flasks. At both 48- and 72-hr post-transfection, media containing virus was collected, and filtered (0.45 pm). The viral supernatant from 48- and 72- hr were combined, flash-frozen, and stored in -80°C.

[0373] Mouse CAR T cells were generated using a 5 to 6-day protocol. T cells were isolated from Balb / c or C57BL / 6 mice and activated with anti-mouse CD3 / CD28 Dynabeads (Invitrogen) at a 2:1 bead-to-cell ratio. In parallel, 24-well plates were coated with RetroNectin (Takara Bio, 1 : 50 in PBS) overnight at 4 °C. The following day, wells were blocked with 2% BSA for 30 min, after which muCAR19-MSGV retrovirus was added, centrifuged at 2,000g for 1 h at 4 °C, and incubated at 37 °C for 30 min. Activated T cells were then transferred to the retrovirus-coated wells (0.5-1 x io6cells per well).

[0374] Cells were cultured for 5 to 6 days in R10 supplemented with 0.05 mM 2- mercaptoethanol and 10 ng / ml mouse or human IL-2 and mouse IL-7, with fresh cytokines added daily. On day 5 or 6, Dynabeads were removed, and CAR expression was assessed by flow cytometry using an anti-G4S stain (Cell Signaling Technology). Expanded mouse CAR T cells were resuspended in either fresh R10 media for in vitro experiments or lx PBS for infusion into mice.

[0375] Electroporation of Cas9 + BDH1 sgRNA ribonucleoprotein (RNP) of mouse T cells Mouse T cells were electroporated using the Lonza 4D-Nucleofector System as previously described (25). In order to form a Cas9 + BDH1 sgRNA Ribonucleoprotein (RNP) complex, 10 pg of TrueCut v2 Cas9 (Thermo-Fisher) and 5 pg single-guide RNA (IDT) were combined and incubated for 10 min at room temperature. Primary murine T cells were collected, pelleted at 300 x g for 5 min, and resuspended at 1-5 x io6per 100 pL of Lonza Buffer P3 (P3 Primary Cell Solution: Supplement 1 = 4.55: 1 vol / vol). 100 pL of T cells in Buffer P3 were gently pipette-mixed with pre-incubated RNP (or Cas9 alone for “mock” electroporation conditions). The T cell / RNP mixture was transferred to a Lonza 100 pL Nucleocuvette and Attorney Docket No: 046483-7485W01(04066) electroporated. For murine T cell editing, pulse code DN-100 was used. Following electroporation, T cells were transferred from cuvettes and recovered in warm culture medium at ~5 x io6per mL. RIO recovery medium for murine T cells was supplemented with 10 ng / mL murine IL-2, 10 ng / mL of murine IL-7, and 0.05 mM 2-mercaptoethanol.

[0376] CellTraceViolet (CTV) Proliferation Assays

[0377] T cells were resuspended in PBS with cell trace violet (CTV. Thermo-Fisher) stain (1 : 1000 dilution), at a concentration of 1 x 106per mL, and incubated for 15 min at 37°C. T cells were washed with 10 mL R10 medium, resuspended, and plated in coculture with cancer cells. For the experiment involving diluted mouse serum (FIG. 2F), serum was obtained from mice fed either control or ketogenic diet. Samples were collected, frozen at -80 °C until use, thawed once, and passed through a 0.22 pm fdter to remove debris. For proliferation assays, serum was added to cell cultures at a final 1 :10 dilution. CAR T cell proliferation was assessed by flow cytometric analysis of CTV dilution after 96 h of co-culture.

[0378] Cytotoxicity Assays

[0379] Cytotoxicity of human CART 19 cells were measured by bioluminescence-based cytotoxicity assays. Briefly, OCI-Lyl8 cells were engineered via a CBG-2A-GFP lentiviral vector construct to express CBG and GFP. CART19 cells and cancer cells were co-cultured at the indicated effector :target ratios with PBS or 5 mM of BHB. Following 72 h incubation, d- luciferin potassium salt (PerkinElmer) was added to cultures at a final concentration of 15 pg mL1and incubated at 37 °C for 10 min. Bioluminescence intensity was measured using a BioTek Synergy H4 Imager and analyzed with the corresponding BioTek Gen5 software. The percentage of tumor killing was calculated relative to a control of cancer / target cells alone without effector T cells. Flow-based cytotoxicity assays were used to assess the cytotoxicity of murine CART 19 cells. Murine CART 19 cells and A20 cells were seeded at specified effectortarget ratios, with or without 5 mM of BHB. after 48 h of coculturing, cytotoxicity was evaluated by determining the absolute counts of T cells and cancer cells using flow cytometry using Flow-Count Fluorospheres (Beckman Coulter).

[0380] Flow Cytometry Attorney Docket No: 046483-7485W01(04066)

[0381] Cells were resuspended in FACS staining buffer (PBS + 2% FBS) using the following antibodies: human CD3 (clone OKT3, BioLegend), human CD45 (clone 2D1 BioLegend), or mouse CD45 (clone 30-F11, BioLegend). CAR expression was detected using a PE-conjugated anti-G4S linker antibody. To monitor cell proliferation, cells were stained with CellTraceViolet according to manufacturer's instructions (Invitrogen). For quantification of absolute cell numbers (tumor or T cells) acquired during flow cytometry, FlowCount FluoroSpheres were used (BeckmanCoulter). Cell viability was established using Propidium iodide (PI, 1 :200 dilution), Live / Dead Violet Fixable stain (Thermo-Fisher, 1:400 dilution), or ViaKrome 808 Fixable Viability Dye (Beckman Coulter, 1 :400 dilution). Unless otherwise specified, all gating strategies began in the following order: SSC-A vs. FSC-A (Lymphocytes), FSC-A vs. FSC-H (Singlets), FSC-A vs. Live / Dead (Viability). All data were acquired on Beckman Coulter CytoFLEX S Flow Cytometer. All data analysis was performed using FlowJo version 10.8.1 (FlowJo, LLC).

[0382] Immunodeficient mice and xenograft models

[0383] Six- to 8-week-old NSG (NOD SCID gamma) mice were obtained from the Stem Cell and Xenograft Core at the University of Pennsylvania. NSG mice were housed in pathogen-free conditions. Briefly, the OCI-Lyl8 subcutaneous model was established as previously described (37). To engraft OCI-Lyl8 tumors, 5 x 106OCI-Lyl8 cells were prepared in a 100 pL solution (50% PBS, 50% Matrigel, Corning) and implanted under the skin of the right flank of NSG mice. Tumor size was monitored by digital caliper according to the following equation: Tumor volume (mm3) = % (L x W2), where L (mm) is the major axis of the tumor and ffl (mm) is the minor axis perpendicular to L. On day eight, daily oral gavage of vehicle (i.e., water) or BHB (280 pL of Ketone-IQ HVMN) began. Eleven (11) days after implantation, mice were randomized on the basis of bioluminesenence (IVIS, mice imaged left lateral recumbent) and infused with 3 x 106CAR19+T cells were infused via tail vein. Tumor size was monitored l-2x per week. Animals were continuously monitored for signs of disease progression and overt toxicity, such as xeno- Graft-Versus-Host-Disease (GVHD), as evidenced by >20% loss in body weight, excessive fur loss, diarrhea, conjunctivitis, disease-induced hind limb paralysis. Animals were also continuously monitored for signs of distress affecting gait or normal posture. NIH Guidelines Attorney Docket No: 046483-7485W01(04066) were followed for animal care and use. All experimental protocols and endpoints, including tumor volume thresholds of up to 4.2 cm3for the OCI-Lyl8 model, were approved by the University of Pennsylvania Animal Care and Use Committee (IACUC).

[0384] Syngeneic mouse models

[0385] Six- to 8-week-old Balb / c mice were purchased from The Jackson Laboratory and contained in conventional housing (BSL-1). To establish the A20 murine lymphoma subcutaneous model, 2 x 106A20 cells were prepared in a 100 pL solution (50% PBS, 50% Matrigel, Coming) and implanted under the skin of the right flank in Balb / c mice. Fourteen (14) days post-implantation, one day prior to adoptive cell transfer, mice were administered 100 mg / kg cyclophosphamide via intraperitoneal injection. On day 15, mice were randomized (subcutaneous tumors had reached -100-200 mm3) and infused with 0.35 x 106CAR+T cells. Tumors were monitored twice per week and euthanized according to IACUC standards as described above.

[0386] Diets and BHB administration

[0387] Micro- and macronutrient-matched diets, as shown in Table 1, were ordered from Inotive. All diets were irradiated and stored at 4°C or -20°C until use. For BHB added as a supplement in the drinking water, a solution of 200 mL of Ketone-IQ (HVMN) per IL water was prepared. Mice were given diets or supplemented drinking water ad libitum. Alternatively, 80 mg BHB was orally gavaged daily per mouse. BHB levels were measured in mouse blood obtained via a tail nick and the Nova Max® PLUS™ Glucose and Ketone monitor.

[0388] Healthy Human Volunteers Trial

[0389] Ten healthy adult volunteers (w=10, 4 females and 6 males; age range, 25-42 years) participated in this study following overnight fasting. An initial blood sample was collected via venipuncture prior to ingestion of a 30g dose of R-1,3 butanediol. (Ketone-IQ, HVMN), which is converted to BHB. Following ingestion, circulating BHB levels were monitored through finger pricks at 0, 15, 30, and 90 minutes. At 90 min, another blood sample was collected. The study protocol was approved by the University of Pennsylvania Institutional Review Board (IRB) under protocol number 855923. Attorney Docket No: 046483-7485W01(04066)

[0390] Stable Isotope Labeling and Metabolomics

[0391] For stable isotope tracing, human CART19 cells were first co-cultured with irradiated OCI-Lyl8 cells (100 Gy) at an effector: target ratio of 0.25 for 72 h. Cell viability and complete OCI-Lyl8 clearance were confirmed by flow cytometry, and cells were washed 2-3 times with PBS to remove dead cells and debris. For the short-term labeling experiment (FIGs. 3A, 41), antigen-stimulated CAR T cells were cultured in medium containing either 5 mM [U-13C4]-BHB and 10 mM unlabeled glucose, or 5 mM unlabeled BHB and 10 mM [U-13Cs]-glucose. Cells were maintained at a density of 1x106cells / mL for 5 h. 3x106cells per condition were harvested at 5 h. washed with ice-cold saline, snap-frozen on dry ice, and stored at -80 °C until extraction.

[0392] For the kinetic labeling experiment (FIGs. 4J-4N), CAR T cells were first incubated for 1 h in medium containing 0.5 mM unlabeled BHB and 1 mM unlabeled D-glucose. Isotopelabeled substrates were then introduced to achieve a final concentration of 5 mM U-,3C-BHB and 10 mM [l,6-13Ce]-glucose. Cells (5 x 106) and corresponding media were collected at the following time points: -10 min, 0 min, 10 min, 70 min, 150 min, and 5 h.

[0393] For metabolite extraction, harvested cell pellets were washed once with ice-cold saline, centrifuged (800 g, 5 min, 0 °C), and resuspended in -80 °C acetonitrile / methanol / water (2:2: 1, v / v) containing 0.1% formic acid. Samples were vortexed for 10 s, incubated on dry ice for 3 min, and supplemented with 69 pL of 15% ammonium bicarbonate. Extracts were stored at -80 °C until further processing. Prior to LC-MS analysis, samples were pulse-sonicated on ice, centrifuged at 21,000 g for 15 min at 0 °C, and clarified supernatants were dried under nitrogen gas and reconstituted in LC-MS grade acetonitrile / water (1 : 1, v / v).

[0394] Samples were analyzed by hydrophilic interaction chromatography (HILIC) coupled to high-resolution LC-MS.

[0395] A TA C-Sequencing

[0396] ATAC-seq libraries were prepared using the Active Motif ATAC-Seq kit following the manufacturer’s protocol. Nuclei from 50,000 sorted CART19 cells were isolated and were subjected to the transposition reaction using Tn5 transposase (Illumina) for 30 minutes at 37°C with lOOOrp mixing. The transposed DNA was purified using DNA Clean and Concentrator Attorney Docket No: 046483-7485W01(04066)

[0397] (Zymo) and fragments were barcoded with unique i5 and / or i7 indexed primers. Final libraries were double size selected using AMPure beads prior to sequencing. Sequencing was performed on an Illumina NextSeq 2000 instrument to produce 75-bp paired-end reads.

[0398] To analyze mouse ATAC-seq data, adapters and stretches of low-quality sequences were removed from raw reads using Trim Galore (v0.6.10) and Cutadapt (v4.6) with the parameter — stringency 5. Reads were then mapped to the mm 10 mouse reference genome using Bowtie2 (v2.5.2) with the following alignment options: —very-sensitive-local —dovetail -no-discordant — no-mixed. Reads aligning to the mitochondrial genome were identified with Samtools (vl.19) idxstats and removed. Samtools was also used to remove PCR duplicates (rmdup) and unpaired or low-quality alignments (view -f 2 -q 20). Reads overlapping blacklisted genomic regions were excluded using Bedtools (v2.31.0) intersect and the mouse ATAC-seq blacklist (github.com / Boyle-Lab / Blacklist / blob / master / lists / mmlO-blacklist.v2. bed. gz). The resulting filtered alignment files were analyzed with ChrAccR (vO.9.21) in R (v4.4) to quantify reads mapped to regions 1500 bp upstream and 500 bp downstream of transcription start sites (promoters) across the mm 10 genome. Regions represented in fewer than 75% of samples or located on sex chromosomes were excluded. Quantile-normalized counts were then used to test for differential chromatin accessibility between BHB-treated and control samples using the built- in ChrAccR: :getDiffAcc function. Significance was determined using a Benjamini-Hochberg adjusted p-value cutoff of 0.05 and a log2 fold change threshold of ±0.1. Data were organized with dplyr (vl.1.4) and visualized using Compl exHeatmap (v2.20.0) and ggplot2 (v3.5.1).

[0399] To analyze human ATAC-seq data, reads were first assessed for quality and adaptertrimmed using FastQC. Clean reads were then aligned to the GRCh38 / hg38 reference genome with Bowtie2 (26). Genrich was used in ATAC-seq mode to call peaks, applying a q-value cutoff of <0.01 for high-confidence peaks. For visualization, normalized fragment signal bigWig files were generated using DeepTools2 (27) with BPM (Bins Per Million) normalization, 10 bp bin size, and read centering by fragment length. Normalized signals ±2 kb around ATAC-seq peak centers were further used for unsupervised k-means clustering via DeepTools.A consensus peak set was defined using DiffBind (28), and differential enrichment was determined with DESeq2 (Love et al., 2014). Functional annotation was performed using GREAT (29) with the Attorney Docket No: 046483-7485W01(04066)

[0400] GRCh38 / hg38 genome as background. Transcription factor footprints within ATAC-seq peaks were identified using HINT (vO.13.0) (30), while motif enrichment was determined with the HOMER de novo algorithm (v4.11)(37) based on the cumulative binomial distribution. Peaks were annotated using HOMER’s annotatePeaks function, assigning sites to genomic features such as transcription start sites (TSS), transcription termination sites (TTS), coding exons, 5' and 3' UTR exons, introns, and intergenic regions.

[0401] RNA-sequencing and analysis

[0402] Total RNA was extracted using the Qiagen RNAeasy mini kit as per the manufacturer’s protocol. Libraries were prepared using the Illumina stranded mRNA kit with Illumina DNA / RNA unique dual indices according to the manufacturer’s instructions. Concentration of libraries was measured using Qubit 4 and an Agilent 4200 TapeStation was used to perform quality control of the libraries. Libraries were sequenced on an Illumina NextSeq 2000 instrument to produce 118-bp single-end reads with an average sequencing depth of 10 million reads per sample.

[0403] To analyze the data, raw reads were pseudo-mapped to the hg38 human reference transcriptome using kallisto vO.46.1 (32) and the outputs were converted into raw count matrices with tximport vl.32.0 (33) in R. Kallisto abundance values were rounded to the nearest integer prior to testing for differential expression with DESeq2 (vl.44.0)(34), which assumes integer count data. Prior to testing, any genes with duplicate names were removed as well as those whose cumulative counts across samples were in the lowest 20% of genes. Then, DESeq2 was used to test for differential expression of the remaining genes between BHB-treated and control samples using a p-value (Benjamini -Hochberg adjusted) cutoff of 0.05 and log2 fold change cutoff of ±0.1. As above, in R, dplyr was used for data reshaping followed by ComplexHeatmap and ggplot2 for generating plots.

[0404] Cleavage Under Targets and Release Using Nuclease (CUT&RUN)-Sequencing and Analysis

[0405] A total of 10,000 CD19-stimulated CART19 cells were processed using the ChIC / CUT&RUN Assay Kit (Active Motif, #53180). DNA libraries were prepared with the Illumina DNA Library Prep Kit (Cat. #53220) and indexed using NEBNext Multiplex Oligos for Attorney Docket No: 046483-7485W01(04066)

[0406] Illumina (New England Biolabs, #E7335S). The antibody against Acetyl-Histone 143 (Lys27) was obtained from Cell Signaling Technology (#8173T). Library quality and size distribution were assessed with the Bioanalyzer High Sensitivity DNA Analysis Kit (Agilent, #5067-4626) according to the manufacturer’s instructions. Libraries were sequenced as paired-end 100 bp reads on a NextSeq 2000 (Illumina, P2 100-cycle flow cell).

[0407] FASTQ fdes were processed through quality control, trimming, mapping, and filtering. H3K27ac peaks were called using MACS2 (v2.0.1)(35) against matched input controls with a p- value cutoff of 1x109, and peaks overlapping hg38 ENCODE blacklisted regions were excluded. A consensus peak set was generated with DiffBind (v2.0.1). Accessible enhancers overlapping H3K27ac peaks were defined as active enhancers. Differential enrichment of chromatin marks was assessed using DESeq2 (v3.28.1), with significance defined as p < 0.05 and |log2FC| > 0.3 (gain or loss) under inflammatory conditions. Gene ontology enrichment was performed with GREAT (Genomic Regions Enrichment of Annotations Tool)(29).

[0408] Single-cell RNA sequencing and analysis

[0409] Diffuse large B-cell lymphoma (DLBCL) tumors (CD19+OCI-Lyl8, 5x l06cells) were implanted subcutaneously into immunodeficient NOD SCID gamma (NSG) mice. Mice received daily oral BHB or vehicle. Ten days post-implantation, a curative dose of CART 19 (5x 106CAR+cells) was infused intravenously. Peripheral blood was collected 17 days after CAR T cell infusion, and red blood cell lysis was performed.

[0410] Human cells were enriched using the EasySep Mouse / Human Chimera Isolation Kit (StemCell Technologies, cat. no. 19849) according to the manufacturer’s protocol. To further purify T cells, samples were stained with anti-human CD3, CD5, and CD2 antibodies and sorted on a BD FACSMelody cell sorter. Sorted cells were washed twice with 0.04% BSA in PBS and loaded onto the lOx Chromium Controller for partitioning. Libraries were prepared using the lOx Genomics Chromium Single Cell 3' Reagent Kit v2.

[0411] Libraries were sequenced on an Illumina NextSeql000 / 2000 platform. Raw sequencing data were processed using the Cell Ranger pipeline (lOx Genomics). Data were fdtered in Seurat with the following parameters before clustering: 200 < number of RNA features < 5,500; percent mitochondrial genes <10. Clustering was guided by a predefined set of canonical T-cell markers, Attorney Docket No: 046483-7485W01(04066) as described by Szabo et al. 36). Differential expression and pathway enrichment analyses were conducted using clusterProfder (version 4.0)(37).

[0412] Metabolomics

[0413] Global metabolomics was performed as previously described with some modifications (38) with modifications as noted. Polar metabolites were extracted from 15 pl of plasma with 150 pl of LC-MS grade ice-cold 80:20 methanol / water containing 1.5 pM internal standards (Cambridge Isotope Laboratories, MSK-A2-1.2). A quality control (QC) sample was generated by pooling equal volumes of all extracts. LC-MS analysis was performed on a Thermo Scientific Q Exactive Plus mass spectrometer with HESI II probe and Vanquish Horizon UHPLC system. Hydrophilic interaction liquid chromatography (HILIC) was performed at a flow rate of 0.2 ml / min on a ZIC-pHILIC column (150 x 2.1 mm, 5 pm particle size, EMD Millipore, 1504600001) with aZIC-pHILIC guard column (20 x 2.1 mm, EMD Millipore, 1504380001) at 45 °C. Solvent A was 20 mM ammonium carbonate, 5 pM medronic acid, 0.1% ammonium hydroxide, pH 9.2, and solvent B was acetonitrile. The chromatographic gradient was 85% B for 2 min, 85% B to 20% B over 15 min, 20% B to 85% B over 0. 1 min, and 85% B for 8.9 min. The autosampler was held at 4 °C, and 4 pl of each sample was injected. The following parameters were used for the MS analysis: sheath gas flow rate, 30; auxiliary gas flow rate, 5: sweep gas flow rate, 0; auxiliary gas heater temperature, 200 °C; spray voltage, 3.6 kV for both positive and negative modes; capillary temperature, 325 °C; and funnel RF leve 1(65). All samples were analyzed by full MS with polarity switching. The QC sample was analyzed at the start of the sample sequence and after every 10 or 11 injections. Tire QC sample was also analyzed by data-dependent MS / MS with separate runs for positive and negative ion modes. Full MS scans were acquired with 2 microscans at 70,000 resolution with an automatic gain control (AGC) target of le6. maximum injection time (IT) of 100 ms, and scan range of 65-975 m / z. Data-dependent MS / MS scans were acquired for the top 10 highest intensity ions at 17,500 resolution with an AGC target of 5e4, maximum IT of 50 ms, isolation width of 1.0 m / z, and stepped normalized collision energy (NCE) of 20, 40, 60. Data were analyzed using Compound Discoverer 3.3 SP3 (Thermo Scientific). Metabolites were annotated based on: 1) accurate mass and retention time by using an in-house database generated from pure standards or 2) accurate mass and MS / MS spectra by querying the mzCloud spectral database (mzCloud.org) and requiring a match score > 50. Periodic analysis of the QC sample was used to correct metabolite quantifications over the course of the batch analysis and further normalized based on the total signal of annotated metabolites in each sample. Attorney Docket No: 046483-7485W01(04066)

[0414] Mitochondrial respiration (Agilent Seahorse XF)

[0415] Mitochondrial function was evaluated using the Agilent / Seahorse Bioscience extracellular flux analyzer. Individual wells of an XF96 cell culture microplate were coated with Cell-Tak following the manufacturer’s protocol. The matrix was adsorbed overnight at 37°C, then aspirated, air-dried, and stored at 4°C until use. The XF assay medium (non-buffered RPMI 1640) contained 10 mM glucose, 2 mM L-glutamine, and 5 mM HEPES was prepared fresh prior to plating the experiments. For mitochondrial functional assays, human or murine T cells were centrifuged at 500 * g for 5 minutes and washed in lx PBS. The cell pellets were resuspended in XF assay medium and seeded at a density of 1 x 105cells per well. During instrument calibration, the microplate was centrifuged at 1,000 x g for 2 min and incubated in a C Ch-free environment at 37°C for 15 min. Oligomycin, BAM15, and Rotenone were used as the inhibitors for this assay. Cellular oxygen consumption rates (OCRs) and extracellular acidification rates (ECARs) were recorded under basal conditions and after treatment with 1.5 pM oligomycin and 2.5 pM BAM15, 0.5 m rotenone, with 0.5 M antimycin A (Seahorse XF T Cell Metabolic Profiling Kit, Agilent). Dynabead-activated murine T cells, or human T cells (from patient CART 19 expansions or healthy volunteers trial) were resuspended in XF assay medium supplemented with 10 mM Carnosine and 30 mM glycyl sarcosine (Gly-Sar) from Sigma. In all instances, the pH was adjusted to 7.4 before the assay. mitoATP production is the rate of ATP production (expressed in pmol ATP / min) specific to the mitochondrial oxidative phosphorylation. glycoATP production is the rate of ATP production (expressed in pmol ATP / min) correlated with the glycolytic pathway.

[0416] Mitochondrial membrane potential (Live Cell Imaging, MitoSOX™ Red)

[0417] A total of 100,000 T cells were washed twice with 4 mL of 1 x phosphate-buffered saline (PBS), then resuspended in 150 pL of Live Cell Imaging Solution (Invitrogen™, Cat. # A59688DJ) supplemented with 11 mM glucose and 2 mM glutamine (hereafter referred to as LC1S+). The cell suspension was subsequently seeded onto a glass-bottom dish (D35-I0-I .5-N, Cellvis) and incubated at 37 °C for 20 minutes to allow for cell immobilization. Following incubation, 1.8 mL of LCIS+ was gently added to each dish. Attorney Docket No: 046483-7485W01(04066)

[0418] To assess mitochondrial membrane potential, tetramethylrhodamine ethyl ester (TMRE; final concentration 200 nM) was added to the dishes after treatment with ORI. Cells were then incubated at 37 °C in the dark for 30 minutes (TMRE). After incubation, the fluorescence probes were removed by washing with PBS, and the cells were replenished with fresh LCIS+ prior to imaging.

[0419] For each experimental condition, images were acquired from 15 randomly selected fields of view (FOVs) per dish using a Zeiss Observer 7 wide-field fluorescence microscope equipped with a Plan-Apochromat 20x / 0.8 M27 objective lens, maintained at 37 °C. Each image had a spatial resolution of 0.293 pm x 0.293 pm per pixel, with a total FOV size of 562.56 pm x 356.29 pm. Detailed imaging parameters are provided in Table 3.

[0420] Table 3 ncsihatio (min)

[0421] NADH 378-400 414-450 25% 2 !

[0422] TMRE 540-570 580-610 40% 0.05 i 30

[0423] During imaging, transmitted light was employed for locating and focusing on regions of interest to minimize photo-bleaching, and vignette correction was applied. Image processing was performed using a custom MATLAB® script (Version 2019a, The MathWorks, Inc., Natick, MA, USA). Background subtraction was conducted by selecting a cell-free region of interest (ROI), and pixel intensity thresholds were set using a signal-to-noise ratio of 5 to quantify NADH signals. For each FOV, mean values of NADH and TMRE signals were calculated.

[0424] Enumerated Embodiments

[0425] The following enumerated embodiments are provided, the numbering of which is not to be construed as designating levels of importance.

[0426] Embodiment 1 provides a method for treating a patient with chimeric antigen receptor (CAR)-T therapy, the method comprising administering to a subject in need thereof a CAR-T therapy and a ketogenic composition comprising beta-hydroxy butyrate (BHB), a BHB salt, a BUB precursor, a ketone diester, or a combination thereof. Attorney Docket No: 046483-7485W01(04066)

[0427] Embodiment 2 provides a method for improving chimeric antigen receptor (CAR)-T therapy, the method comprising administering to a subject in need thereof a CAR-T therapy and a ketogenic composition comprising beta-hydroxybutyrate (BHB), a BHB salt, a BHB precursor, a ketone diester, or a combination thereof.

[0428] Embodiment 3 provides the method of embodiment 1 or 2, wherein administering the CAR-T therapy to the patient comprises administering a therapeutically effective amount of CAR-T cells.

[0429] Embodiment 4 provides the method of embodiment 1 or 2, wherein administering the CAR-T therapy comprises administering a vector to the patient, wherein the vector comprises a nucleic acid molecule encoding a chimeric antigen receptor.

[0430] Embodiment 5 provides the method of embodiment 4, wherein the vector is a viral vector.

[0431] Embodiment 6 provides the method of embodiment 5, wherein the viral vector is a lentivirus, such as a pseudotyped lentivirus.

[0432] Embodiment 7 provides the method of embodiment 4, wherein the vector is a lipid nanoparticle (LNP) comprising the nucleic acid molecule encoding the chimeric antigen receptor.

[0433] Embodiment 8 provides the method of claim 1 or 2, wherein the ketogenic composition comprises D-beta-hydroxybutyric (D-BHB) acid, L-beta-hydroxybutyric (L-BHB) acid, racemic beta-hydroxybutyric acid or a salt, monoester, polyester, or mixture thereof.

[0434] Embodiment 9 provides the method of claim 1 or 2, wherein the ketogenic composition comprises a BHB salt or BHB ester.

[0435] Embodiment 10 provides the method of embodiment 9, wherein the ketogenic composition comprises a BHB mineral salt, a BHB organic salt, or a combination thereof.

[0436] Embodiment 11 provides the method of embodiment 9 or 10, wherein the ketogenic composition comprises a BHB mineral salt selected from the group consisting of sodium beta- hydroxybutyrate, potassium beta-hydroxybutyrate, calcium beta-hydroxybutyrate, magnesium beta-hydroxybutyrate, lithium beta-hydroxybutyrate, and a mixture thereof. Attorney Docket No: 046483-7485W01(04066)

[0437] Embodiment 12 provides the method of embodiment 9 or 10, wherein the ketogenic composition comprises a BHB organic salt selected from the group consisting of arginine betahydroxybutyrate, lysine beta-hydroxybutyrate, histidine beta-hydroxybutyrate, ornithine betahydroxybutyrate, creatine beta-hydroxybutyrate, agmatine beta-hydroxybutyrate, citrulline beta- hydroxybutyrate, and a mixture thereof.

[0438] Embodiment 13 provides the method of embodiment 9 or 10, wherein the ketogenic composition comprises a beta-hydroxy butyrate sodium salt, a beta-hydroxy butyrate potassium salt, a beta-hydroxy butyrate calcium salt, a beta-hydroxy butyrate magnesium salt, or a mixture thereof.

[0439] Embodiment 14 provides the method of claim 1 or 2, wherein the ketogenic composition comprises a BHB precursor selected from the group consisting of 1,3 -butanediol (BDO), ethyl acetoacetate, ethyl beta-hydroxybutyrate, and mixture thereof.

[0440] Embodiment 15 provides the method of claim 1 or 2, wherein the ketogenic composition comprises a ketone diester.

[0441] Embodiment 16 provides the method of embodiment 15, wherein the ketone diester is R, S 1,3-butanediol di acetoacetate or R, S 1,3 butanediol acetoacetate diester.

[0442] Embodiment 17 provides the method of any one of embodiments 1-16, wherein the ketogenic composition further comprises a medium chain fatty acid ester thereof, or derivative thereof.

[0443] Embodiment 18 provides the method of embodiment 17, wherein the medium chain fatty acid, ester thereof, or derivative thereof is selected from the group consisting of coconut oil, coconut milk powder, fractionated coconut oil, palm oil, palm kernel oil, caprylic acid, isolated hexanoic acid, isolated octanoic acid, isolated decanoic acid, ethoxylated triglyceride or derivative thereof, enone triglyceride or derivative thereof, aldehyde triglyceride or derivative thereof, monoglyceride or derivative thereof, diglyceride or derivative thereof, triglyceride or derivative thereof, medium chain triglyceride salts, salt derivatives thereof, and mixtures thereof.

[0444] Embodiment 19 provides the method of any one of embodiments 1-18, wherein the ketogenic composition is administered to the subject before the CAR-T therapy is administered to the patient. Attorney Docket No: 046483-7485W01(04066)

[0445] Embodiment 20 provides the method of embodiment 19, wherein the ketogenic composition is administered to the subject during the course of CAR-T therapy.

[0446] Embodiment 21 provides the method of any one of embodiments 1-18, wherein the ketogenic composition is administered to the subject after the CAR-T therapy is administered to the patient.

[0447] Embodiment 22 provides the method of any one of embodiments 19-21, wherein the ketogenic composition is administered to the subject once per day, twice per day, or three times per day for a period of at least one week.

[0448] Embodiment 23 provides the method of embodiment 22, wherein the ketogenic composition is administered to the subject once per day, twice per day, or three times per day for a period of at least two weeks.

[0449] Embodiment 24 provides the method of embodiment 23, wherein the ketogenic composition is administered to the subject once per day, twice per day, or three times per day for a period of at least one month.

[0450] Embodiment 25 provides the method of any one of embodiments 1-24, wherein the betahydroxybutyrate (BHB), BHB salt, BHB precursor, or ketone diester is administered to the subject in an amount between 0.01-50 g / kg / day.

[0451] Embodiment 26 provides the method of embodiment 25, wherein the BHB, BHB salt, BHB precursor, or ketone diester is administered to the subject in an amount between 1-10 g / kg / day.

[0452] Embodiment 27 provides a method of producing a cell population comprising T-cells, the method comprising culturing T cells in cell culture media comprising BHB, a BHB salt, a BHB precursor, a ketone diester, or a combination thereof.

[0453] Embodiment 28. The method of embodiment 27, wherein the cell culture media comprises BHB in an amount between about 0.5-5 mM.

[0454] Embodiment 29 provides the method of embodiment 27 or 28, wherein the cell population comprises an oxygen consumption rate (OCR) between about 1-200 pmol / min.

[0455] Embodiment 30 provides the method of any one of embodiments 27-29, wherein the cell population comprises an extracellular acidification rate (ECAR) of about 1-150 pmol / min.

[0456] I l l Attorney Docket No: 046483-7485W01(04066)

[0457] Embodiment 31 provides the method of any one of embodiments 27-30, wherein the T- cells comprise a chimeric antigen receptor.

[0458] Embodiment 32 provides a method of manufacturing chimeric antigen receptor (CAR)- expressing T cells from peripheral blood mononuclear cells (PBMCs) from a blood sample obtained from a subject, comprising a) producing a T-cell enriched population of cells from a population of immune cells isolated from a subject; b) transforming the T-cell enriched population of T-cells with a vector encoding a chimeric antigen receptor (CAR); and c) culturing the cells obtained in step b) in cell culture media comprising betahydroxybutyrate (BHB), a BHB salt, a BHB precursor, a ketone diester, or a combination thereof.

[0459] Embodiment 33 provides the method of embodiment 32, wherein the media comprises D- b eta-hydroxy butyric (D-BHB) acid, L-beta-hydroxybutyric (L-BHB) acid, racemic betahydroxybutyric acid or a salt, monoester, polyester, or mixture thereof.

[0460] Embodiment 34 provides the method of embodiment 32 or 33, wherein the media comprises a BHB salt or BHB ester.

[0461] Embodiment 35 provides the method of embodiment 34, wherein the media comprises a BHB mineral salt, a BHB organic salt, or a combination thereof.

[0462] Embodiment 36 provides the method of embodiment 35, wherein the media comprises a BHB mineral salt selected from the group consisting of sodium beta-hydroxybutyrate, potassium beta-hydroxybutyrate, calcium beta-hydroxybutyrate, magnesium beta-hydroxybutyrate, lithium beta-hydroxybutyrate, and a mixture thereof.

[0463] Embodiment 37 provides the method of embodiment 35, wherein the media comprises a BHB organic salt selected from the group consisting of arginine beta-hydroxybutyrate, lysine beta-hydroxybutyrate, histidine beta-hydroxybutyrate, ornithine beta-hydroxybutyrate, creatine beta-hydroxybutyrate, agmatine beta-hydroxybutyrate, citrulline beta-hydroxybutyrate, and a mixture thereof. Attorney Docket No: 046483-7485W01(04066)

[0464] Embodiment 38 provides the method of embodiment 35, wherein the media comprises beta-hydroxy butyrate sodium salt, beta-hydroxy butyrate potassium salt, beta-hydroxy butyrate calcium salt, beta-hydroxy butyrate magnesium salt, or a mixture thereof.

[0465] Embodiment 39 provides the method of embodiment 32, wherein the media comprises a BHB precursor selected from the group consisting of 1,3 -butanediol (BDO), ethyl acetoacetate, ethyl beta-hydroxybutyrate, and mixture thereof.

[0466] Embodiment 40 provides the method of embodiment 32, wherein the media comprises a ketone diester.

[0467] Embodiment 41 provides the method of embodiment 40, wherein the ketone diester is R, S 1,3-butanediol di acetoacetate or R,S 1,3 butanediol acetoacetate diester.

[0468] Embodiment 42 provides the method of any one of embodiments 32-41, wherein the media further comprises a medium chain fatty acid, ester thereof, or derivative thereof.

[0469] Embodiment 43 provides the method of embodiment 42, wherein the medium chain fatty acid, ester thereof, or derivative thereof is selected from the group consisting of coconut oil, coconut milk powder, fractionated coconut oil, palm oil, palm kernel oil, caprylic acid, isolated hexanoic acid, isolated octanoic acid, isolated decanoic acid, ethoxylated triglyceride or derivative thereof, enone triglyceride or derivative thereof, aldehyde triglyceride or derivative thereof, monoglyceride or derivative thereof, diglyceride or derivative thereof, triglyceride or derivative thereof, medium chain triglyceride salts, salt derivatives thereof, and mixtures thereof.

[0470] Embodiment 44 provides a population of T-cells, wherein the population of T-cells comprises an oxygen consumption rate (OCR) between about 1-200 pmol / min and / or an extracellular acidification rate (ECAR) of about 1-150 pmol / min.

[0471] Embodiment 45 provides the population of T-cells of embodiment 44, wherein the population of T-cells comprises CAR-T cells.

[0472] Embodiment 46 provides a method for improving chimeric antigen receptor (CAR)-T therapy, comprising administering to a subject in need thereof a therapeutically effective amount of a CAR-T therapy, wherein the subject is in a state of ketosis before and / or after administration of the therapeutically effective amount of the CAR-T therapy, wherein the subject is in the state Attorney Docket No: 046483-7485W01(04066) of ketosis when the subject’s blood ketone levels are within a range of about 0.5 mmol / L and about 16 mmol / L.

[0473] Embodiment 47 provides the method of embodiment 46, wherein the subject is in the state of ketosis before and after administration of the therapeutically effective amount of the CAR-T therapy or a ketogenic composition a ketogenic composition comprising betahydroxybutyrate (BHB), a BHB salt, a BHB precursor, a ketone diester, or a combination thereof.

[0474] Embodiment 48 provides the method of embodiment 47, wherein the subject is in the state of ketosis for at least about 1-2 weeks before administration of the CAR-T therapy or the ketogenic composition.

[0475] Embodiment 49 provides the method of any one of embodiments 46-48, wherein the subject is in the state of ketosis for at least about 1 week following administration of the CAR-T therapy or the ketogenic composition.

[0476] Embodiment 50 provides the method of any one of embodiments 46-48, wherein the subject is in the state of ketosis for at least about 4 weeks following administration of the CAR-T therapy or the ketogenic composition.

[0477] Embodiment 51 provides the method of any one of embodiments 46-50, wherein the subject is in the state of ketosis following administration of the ketogenic composition.

[0478] Embodiment 52 provides the method of any one of embodiments 46-50, wherein the subject is in the state of ketosis by being on a ketogenic diet.

[0479] Embodiment 53 provides the method of any one of embodiments 46-52, wherein the subject in the state of ketosis has increased serum level(s) of BHB, acetoacetate, and / or acetone compared to a subject that is not in a state of ketosis.

[0480] Embodiment 54 provides the method of any one of embodiments 46-52, wherein the subject in the state of ketosis has increased serum level(s) of one or more metabolites compared to a subject that is not in a state of ketosis, wherein the one or more metabolites are selected from the group consisting of BHB, docosanoic acid, a-linolenic acid, linoleic acid, erucic acid, tetradecanedioic acid, 10-hydroxy decanoic acid, stearic acid, 1-norleucine, arachidic acid, 1- Attorney Docket No: 046483-7485W01(04066) norleucine, nicotinamide 1 -oxide, d-saccharic acid, n-acetyl-d-alloisoleucine, uric acid, methylcysteine, oleamide, palmitoylcarnitine, dl- -leucine, oleic acid, and combinations thereof.

[0481] Embodiment 55 provides the method of embodiment 53 or 54, wherein one or more of the serum level(s) are increased by at least 50% compared to a subject that is not in a state of ketosis.

[0482] Embodiment 56 provides the method of embodiment 53 or 54, wherein one or more of the serum level(s) are increased by at least 2-fold compared to a subject that is not in a state of ketosis.

[0483] Embodiment 57 provides the method of embodiment 53 or 54, wherein one or more of the serum level(s) are increased by at least about 5-fold to 20-fold compared to a subject that is not in a state of ketosis.

[0484] Embodiment 58 provides the method of any one of embodiments 53 or 57, wherein the subject that is not in a state of ketosis is the subject being administered the CAR-T therapy.

[0485] Embodiment 59 provides the method of any one of embodiments 46-58, wherein the CAR-T therapy is a therapeutically effective amount of CAR-T cells.

[0486] Embodiment 60 provides the method of any one of embodiments 46-58, wherein the CAR-T therapy is a vector comprising a nucleic acid molecule encoding a chimeric antigen receptor.

[0487] Embodiment 61 provides the method of embodiment 60, wherein the vector is a viral vector.

[0488] Embodiment 62 provides the method of embodiment 61, wherein the viral vector is a lentivirus, such as a pseudotyped lentivirus.

[0489] Embodiment 63 provides the method of embodiment 60, wherein the vector is a lipid nanoparticle (LNP) comprising the nucleic acid molecule encoding the chimeric antigen receptor.

[0490] Other Embodiments

[0491] The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of Attorney Docket No: 046483-7485W01(04066) listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

[0492] The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this disclosure has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this disclosure may be devised by others skilled in the art without departing from the true spirit and scope of the disclosure. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

[0493] Attorney Docket No: 046483-7485W01(04066)

[0494] REFERENCES

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Claims

Attorney Docket No: 046483-7485W01(04066)CLAIMSWhat is claimed:

1. A method for treating a patient with chimeric antigen receptor (C AR)-T therapy, the method comprising administering to a subject in need thereof a CAR-T therapy and a ketogenic composition comprising beta-hydroxybutyrate (BHB), a BHB salt, a BHB precursor, a ketone diester, or a combination thereof.

2. A method for improving chimeric antigen receptor (CAR)-T therapy, the method comprising administering to a subject in need thereof a CAR-T therapy and a ketogenic composition comprising beta-hydroxybutyrate (BHB), a BHB salt, a BHB precursor, a ketone diester, or a combination thereof.

3. The method of claim 1 or 2, wherein administering the CAR-T therapy to the patient comprises administering a therapeutically effective amount of CAR-T cells.

4. The method of claim 1 or 2, wherein administering the CAR-T therapy comprises administering a vector to the patient, wherein the vector comprises a nucleic acid molecule encoding a chimeric antigen receptor.

5. The method of claim 4, wherein the vector is a viral vector.

6. The method of claim 5, wherein the viral vector is a lentivirus, such as a pseudotyped lenti virus.

7. The method of claim 4, wherein the vector is a lipid nanoparticle (LNP) comprising the nucleic acid molecule encoding the chimeric antigen receptor.

8. The method of claim 1 or 2, wherein the ketogenic composition comprises D-beta- hydroxybutyric (D-BHB) acid, L-beta-hydroxybutyric (L-BHB) acid, racemic betahydroxybutyric acid or a salt, monoester, polyester, or mixture thereof.Attorney Docket No: 046483-7485W01(04066)9. The method of claim 1 or 2, wherein the ketogenic composition comprises a BHB salt or BHB ester.

10. The method of claim 9, wherein the ketogenic composition comprises a BHB mineral salt, a BHB organic salt, or a combination thereof.

11. The method of claim 9 or 10, wherein the ketogenic composition comprises a BHB mineral salt selected from the group consisting of sodium beta-hydroxybutyrate, potassium betahydroxybutyrate, calcium beta-hydroxybutyrate, magnesium beta-hydroxybutyrate, lithium beta- hydroxybutyrate, and a mixture thereof.

12. The method of claim 9 or 10, wherein the ketogenic composition comprises a BHB organic salt selected from the group consisting of arginine beta-hydroxybutyrate, lysine beta- hydroxybutyrate, histidine beta-hydroxybutyrate, ornithine beta-hydroxybutyrate, creatine beta- hydroxybutyrate, agmatine beta-hydroxybutyrate, citrulline beta-hydroxybutyrate, and a mixture thereof.

13. The method of claim 9 or 10, wherein the ketogenic composition comprises a beta-hydroxy butyrate sodium salt, a beta-hydroxy butyrate potassium salt, a beta-hydroxy butyrate calcium salt, a beta-hydroxy butyrate magnesium salt, or a mixture thereof.

14. The method of claim 1 or 2, wherein the ketogenic composition comprises a BHB precursor selected from the group consisting of 1,3-butanediol (BDO), ethyl acetoacetate, ethyl beta- hydroxybutyrate, and mixture thereof.

15. The method of claim 1 or 2, wherein the ketogenic composition comprises a ketone diester.

16. The method of claim 15, wherein the ketone diester is R, S 1,3-butanediol di acetoacetate or R, S 1,3 butanediol acetoacetate diester.

17. The method of any one of claims 1-16, wherein the ketogenic composition further comprises a medium chain fatty acid ester thereof, or derivative thereof.Attorney Docket No: 046483-7485W01(04066)18. The method of claim 17, wherein the medium chain fatty acid, ester thereof, or derivative thereof is selected from the group consisting of coconut oil, coconut milk powder, fractionated coconut oil, palm oil, palm kernel oil, caprylic acid, isolated hexanoic acid, isolated octanoic acid, isolated decanoic acid, ethoxylated triglyceride or derivative thereof, enone triglyceride or derivative thereof, aldehyde triglyceride or derivative thereof, monoglyceride or derivative thereof, diglyceride or derivative thereof, triglyceride or derivative thereof, medium chain triglyceride salts, salt derivatives thereof, and mixtures thereof.

19. The method of any one of claims 1-18, wherein the ketogenic composition is administered to the subject before the CAR-T therapy is administered to the patient.

20. The method of claim 19, wherein the ketogenic composition is administered to the subject during the course of CAR-T therapy.

21. The method of any one of claims 1-18, wherein the ketogenic composition is administered to the subject after the CAR-T therapy is administered to the patient.

22. The method of any one of claims 19-21, wherein the ketogenic composition is administered to the subject once per day, twice per day, or three times per day for a period of at least one week.

23. The method of claim 22, wherein the ketogenic composition is administered to the subject once per day, twice per day, or three times per day for a period of at least two weeks.

24. The method of claim 23, wherein the ketogenic composition is administered to the subject once per day, twice per day, or three times per day for a period of at least one month.

25. The method of any one of claims 1-24, wherein the beta-hydroxybutyrate (BHB), BHB salt, BHB precursor, or ketone diester is administered to the subject in an amount between 0.01-50 g / kg / day.

26. The method of claim 25, wherein the BHB, BHB salt, BHB precursor, or ketone diester is administered to the subject in an amount between 1-10 g / kg / day.Attorney Docket No: 046483-7485W01(04066)27. A method of producing a cell population comprising T-cells, the method comprising culturing T cells in cell culture media comprising BHB, a BHB salt, a BHB precursor, a ketone diester, or a combination thereof.

28. The method of claim 27, wherein the cell culture media comprises BHB in an amount between about 0.5-5 mM.

29. The method of claim 27 or 28, wherein the cell population comprises an oxygen consumption rate (OCR) between about 1-200 pmol / min.

30. The method of any one of claims 27-29, wherein the cell population comprises an extracellular acidification rate (ECAR) of about 1-150 pmol / min.

31. The method of any one of claims 27-30, wherein the T-cells comprise a chimeric antigen receptor.

32. A method of manufacturing chimeric antigen receptor (CAR)-expressing T cells from peripheral blood mononuclear cells (PBMCs) from a blood sample obtained from a subject, comprising a) producing a T-cell enriched population of cells from a population of immune cells isolated from a subject; b) transforming the T-cell enriched population of T-cells with a vector encoding a chimeric antigen receptor (CAR); and c) culturing the cells obtained in step b) in cell culture media comprising betahydroxybutyrate (BHB), a BHB salt, a BHB precursor, a ketone diester, or a combination thereof.

33. The method of claim 32, wherein the media comprises D-beta-hydroxybutyric (D-BHB) acid, L-beta-hydroxybutyric (L-BHB) acid, racemic beta-hydroxybutyric acid or a salt, monoester, polyester, or mixture thereof.

34. The method of claim 32 or 33, wherein the media comprises a BHB salt or BHB ester.Attorney Docket No: 046483-7485W01(04066)35. The method of claim 34, wherein the media comprises a BHB mineral salt, a BHB organic salt, or a combination thereof.

36. The method of claim 35, wherein the media comprises a BHB mineral salt selected from the group consisting of sodium beta-hydroxybutyrate, potassium beta-hydroxybutyrate, calcium beta-hydroxybutyrate, magnesium beta-hydroxybutyrate, lithium beta-hydroxybutyrate, and a mixture thereof.

37. The method of claim 35, wherein the media comprises a BHB organic salt selected from the group consisting of arginine beta-hydroxybutyrate, lysine beta-hydroxybutyrate, histidine beta- hydroxybutyrate, ornithine beta-hydroxybutyrate, creatine beta-hydroxybutyrate, agmatine beta- hydroxybutyrate, citrulline beta-hydroxybutyrate, and a mixture thereof.

38. The method of claim 35, wherein the media comprises beta-hydroxy butyrate sodium salt, beta-hydroxy butyrate potassium salt, beta-hydroxy butyrate calcium salt, beta-hydroxy butyrate magnesium salt, or a mixture thereof.

39. The method of claim 32, wherein the media comprises a BHB precursor selected from the group consisting of 1,3 -butanediol (BDO), ethyl acetoacetate, ethyl beta-hydroxybutyrate, and mixture thereof.

40. The method of claim 32, wherein the media comprises a ketone diester.

41. The method of claim 40, wherein the ketone diester is R, S 1,3 -butanediol di acetoacetate or R, S 1,3 butanediol acetoacetate diester.

42. The method of any one of claims 32-41, wherein the media further comprises a medium chain fatty acid, ester thereof, or derivative thereof.

43. The method of claim 42, wherein the medium chain fatty acid, ester thereof, or derivative thereof is selected from the group consisting of coconut oil, coconut milk powder, fractionated coconut oil, palm oil, palm kernel oil, caprylic acid, isolated hexanoic acid, isolated octanoic acid, isolated decanoic acid, ethoxylated triglyceride or derivative thereof, enone triglyceride orAttorney Docket No: 046483-7485W01(04066) derivative thereof, aldehyde triglyceride or derivative thereof, monoglyceride or derivative thereof, diglyceride or derivative thereof, triglyceride or derivative thereof, medium chain triglyceride salts, salt derivatives thereof, and mixtures thereof.

44. A population of T-cells, wherein the population of T-cells comprises an oxygen consumption rate (OCR) between about 1-200 pmol / min and / or an extracellular acidification rate (ECAR) of about 1-150 pmol / min.

45. The population of T-cells of claim 44, wherein the population of T-cells comprises CAR-T cells.

46. A method for improving chimeric antigen receptor (CAR)-T therapy, comprising administering to a subject in need thereof a therapeutically effective amount of a CAR-T therapy, wherein the subject is in a state of ketosis before and / or after administration of the therapeutically effective amount of the CAR-T therapy, wherein the subject is in the state of ketosis when the subject’s blood ketone levels are within a range of about 0.5 mmol / L and about 16 mmol / L.

47. The method of claim 46, wherein the subject is in the state of ketosis before and after administration of the therapeutically effective amount of the CAR-T therapy or a ketogenic composition a ketogenic composition comprising beta-hydroxybutyrate (BHB), a BHB salt, a BHB precursor, a ketone diester, or a combination thereof.

48. The method of claim 47, wherein the subject is in the state of ketosis for at least about 1-2 weeks before administration of the CAR-T therapy or the ketogenic composition.

49. The method of any one of claims 46-48, wherein the subject is in the state of ketosis for at least about 1 week following administration of the CAR-T therapy or the ketogenic composition.

50. The method of any one of claims 46-48, wherein the subject is in the state of ketosis for at least about 4 weeks following administration of the CAR-T therapy or the ketogenic composition.Attorney Docket No: 046483-7485W01(04066)51 . The method of any one of claims 46-50, wherein the subject is in the state of ketosis following administration of the ketogenic composition.

52. The method of any one of claims 46-50, wherein the subject is in the state of ketosis by being on a ketogenic diet.

53. The method of any one of claims 46-52, wherein the subject in the state of ketosis has increased serum level(s) of BHB, acetoacetate, and / or acetone compared to a subject that is not in a state of ketosis.

54. The method of any one of claims 46-52, wherein the subject in the state of ketosis has increased serum level(s) of one or more metabolites compared to a subject that is not in a state of ketosis, wherein the one or more metabolites are selected from the group consisting of BHB, docosanoic acid, a-linolenic acid, linoleic acid, erucic acid, tetradecanedioic acid, 10- hydroxy decanoic acid, stearic acid, 1-norleucine, arachidic acid, 1-norleucine, nicotinamide 1- oxide, d-saccharic acid, n-acetyl-d-alloisoleucine, uric acid, methylcysteine, oleamide, palmitoylcamitine, dl-P-leucine, oleic acid, and combinations thereof.

55. The method of claim 53 or 54, wherein one or more of the serum level(s) are increased by at least 50% compared to a subject that is not in a state of ketosis.

56. The method of claim 53 or 54, wherein one or more of the serum level(s) are increased by at least 2-fold compared to a subject that is not in a state of ketosis.

57. The method of claim 53 or 54, wherein one or more of the serum level(s) are increased by at least about 5-fold to 20-fold compared to a subject that is not in a state of ketosis.

58. The method of any one claims 53-57, wherein the subject that is not in a state of ketosis is the subject being administered the CAR-T therapy.

59. The method of any one of claims 46-58, wherein the CAR-T therapy is a therapeutically effective amount of CAR-T cells.Attorney Docket No: 046483-7485W01(04066)60. The method of any one of claims 46-58, wherein the CAR-T therapy is a vector comprising a nucleic acid molecule encoding a chimeric antigen receptor.

61. The method of claim 60, wherein the vector is a viral vector.

62. The method of claim 61, wherein the viral vector is a lentivirus, such as a pseudotyped lenti virus.

63. The method of claim 60, wherein the vector is a lipid nanoparticle (LNP) comprising the nucleic acid molecule encoding the chimeric antigen receptor.

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