WO2025101276A1

WO2025101276A1 - Compositions and methods for making and using immune cells

Info

Publication number: WO2025101276A1
Application number: PCT/US2024/048256
Authority: WO
Inventors: Craig Alan BYERSDORFER; Erica BRAVERMAN
Original assignee: University of Pittsburgh
Current assignee: University of Pittsburgh
Priority date: 2023-11-06
Filing date: 2024-09-25
Publication date: 2025-05-15
Anticipated expiration: 2026-05-06

Abstract

The present disclosure relates to methods and compositions for making a T cell with increased immune function comprising administering to said T cell a composition comprising a compound 991 and the uses thereof in increasing an immune response in a subject and/or treating a cancer in a subject.

Description

COMPOSITIONS AND METHODS FOR MAKING AND USING IMMUNE CELLS

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to, and the benefit of, U.S. Provisional Patent Application No. 63/596,392, filed November 6, 2023, which is incorporated by reference herein in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant number HL144556 awarded by the National Institute of Heath and W81XWH- 19- 1-0291 awarded by the Department of Defense. The government has certain rights in this invention.

REFERENCE TO SEQUENCE LISTING

The sequence listing submitted on September 25, 2024 as an .XML file entitled “10504- 095WOl_ST26” created on September 25, 2024 and having a file size of 4,896 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5).

BACKGROUND

Chimeric Antigen Receptor T cell (CART) therapy has had a significant impact on the treatment of relapsed/refractory acute B-cell lymphoblastic leukemia, with more than 90% of treated pediatric patients initially achieving remission. However, despite the success of this adoptive cellular therapy, up to 50% of patients relapse after CART treatment, limiting its utility as a long-term cure. Further, CARTs have seen limited success in other cancers, particularly solid tumors. While the reasons for this limited efficacy are many, one of the most prominent concerns relates to the functional status of the injected CART cells. The ex vivo expansion process drives significant activation and differentiation of CARTs, limiting their ability to form memory populations and negatively impacts their in vivo persistence. This combination results in leukemia relapse and restricted tumor clearance in other cancers. As such, identifying methods to augment the in vivo function and persistence of CARTs has become critical to improving their therapeutic efficacy.

What are needed are compositions and methods to improve T cell persistence and/or immune function in vivo. The compositions and methods disclosed herein address these and other needs.

SUMMARY

As discussed above, ex vivo expansion processes are necessary to generate sufficient cell numbers, but often prior processes promoted sustained activation and differentiation, negatively impacting in vivo persistence and function. Here, it is demonstrated that promoting AMPK activity during CART expansion metabolically reprograms cells without significantly limiting T cell yield, enhances in vivo anti-cancer efficacy, and improves CD4+ in vivo persistence. Importantly, AMPK agonism achieves these results without further modifying the expansion media, changing the CART construct, or genetically altering the cells. Altogether, these data highlight AMPK agonism as a potent and readily translatable approach to improve the metabolic profile and overall efficacy of cancer- targeting T cells.

Accordingly, disclosed herein are methods of making a T cell having an increased immune function comprising administering to the T cell a composition comprising a compound 991 and the method of use thereof in increasing an immune response in a subject or in treating a cancer in a subject.

Accordingly, in one aspect, disclosed herein is a method of making a T cell having an increased immune function comprising administering to the T cell a composition comprising a compound 991. In some embodiments, the increased immune function comprises increased in vivo persistence of the T cell. In some embodiments, the method further comprising administering IL- 2 to the T cell.

In some embodiments, the compound 991 is administered to the T cell at least two times over a period of between about 12 hours and about 84 hours. In some embodiments, the compound 991 is administered to the T cell at least two times over a period of between about 46 hours and about 50 hours. In some embodiments, the compound 991 is administered to the T cell at least two times over a period of about 48 hours. In some embodiments, the T cell is within a culture medium and all or substantially all of the compound 991 in the culture medium is removed prior to the second administration of the compound 991.

In some embodiments, the compound 991 is administered at a molar concentration of between about 0.5 pM and about 100 pM. In some embodiments, the Compound 991 is administered at a molar concentration of about 50 pM.

In some embodiments, the compound 991 is first administered about four to about six days after stimulation of the T cell. In some embodiments, the compound 991 is first administered about five days after stimulation of the T cell.

In some embodiments, the T cell is a CD4⁺ T cell. In some embodiments, the T cell is a CD8⁺ T cell. In some embodiments, the T cell is a CAR T cell, an effector T cell, an effector memory T cell, a TEMRA, a central memory T cell, an effector T regulatory cell (Treg), an effector memory Treg, a tumor-infiltrating lymphocyte (TIL) T cell, a cytotoxic T cell (CTL), a natural killer T-cell (NK T cell), a virus-specific T cell (VST) or a T memory stem cell (TSCM). In one aspect disclosed herein is a method of increasing an immune response in a subject comprising administering to the subject a therapeutically effective amount of a compound 991 T cell.

In one aspect disclosed herein is a method of treating a cancer in a subject comprising administering to the subject a therapeutically effective amount of a compound 991 T cell.

In some embodiments, the subject is a human. In some embodiments, the T cell is obtained from the human.

In some embodiments, the cancer is a hematological cancer. In some embodiments, the cancer is a leukemia.

DESCRIPTION OF DRAWINGS

Fig. 1(A, B) shows the structure and use of Compound 991. Figure 1A shows a chemical formula for Compound 991. Figure IB is a schematic showing creation of CAR T cells and treatment of those cells with Compound 991.

Fig. 2(A-D) shows that 991 treatment as shown in Figure 2(B, D), but not A7 treatment as shown in Figure 2(A, C), enhances multiple aspects of CART cell oxidative metabolism, including basal and maximal oxygen consumption rates. Human CART cells were generated from healthy human donor T cells through lentiviral transduction, and then expanded in either DMSO (untreated), A769662 (A7), or Compound 991 (991). CARTs then underwent restimulation overnight before assessment utilizing the mitostress kit for the Seahorse Metabolic Analyzer. ** p<0.01, *** p<0.001, **** pcO.OOOl.

Fig. 3(A, B) shows AMPK-agonist treated CARTs show decreased cytotoxicity vs untreated CARTs in vitro, which is mimicked by the cytotoxicity of 41BB vs CD28 CARTs. Figure 3A shows that CD28-costimulated CARTs were generated and expanded with or without 991. After expansion, cells were placed in the Incucyte against zzGreen+NALM6 targets, with green fluorescence followed to measure cell killing over 48 hours. Figure 3B shows that the experiment was repeated using CD28-costimulated and 4 IBB costimulated CARTs. In both experiments, mock transduced human T cells were plated against NALM6 targets as a control.

Fig. 4(A-C) shows improved leukemia clearance with 991 treated T cells. Numbers in ( ) represent # of leukemia- free mice at week 10/total mice. Squares represent CART treated with 25 pM Compound 991; triangles represent CART treated with 10 M Compound 991; Upside down triangles represent CART without 991 treatment; circles represent no CART.

Fig. 5(A, B) shows 991-treated CART cells outperform standard CARTs in vivo. Figure 5A shows an example of results following tumor burden with IVIS imaging. Figure 5B shows overall survival. N= 5-8 mice per group. ** p < 0.01, *** p<0.001. Fig 6 shows 991-treated CART cells outperform standard CAR T cells for in vitro cytotoxicity in tumor conditioned media. CD28-costimulated CARTs were generated and expanded with or without 991. After expansion, cells were placed in the Incucyte against zzGreen+NALM6 targets, with green fluorescence followed to measure cell killing. In this experiment, the co-culture media had first been ‘conditioned’ by growing NALM6 cells in it for 72 hours, prior to sterile filtering and addition to the CART and target NALM6 cells.

Fig. 7(A-E) shows that 991 treatment drives AMPK activity in Human T cells without restricting expansion. Figure 7 A shows a schematic of Compound 991 treatment protocol. Figure 7B shows that proteins from human T cells treated with 991 or DMSO control were precipitated on Days 7-9 and phosphorylation of AMPKa on Thrl72 (to detect AMPK activation) was measured by immunoblot. Accompanying densitometry was quantitated on cells obtained from multiple donors using ImageJ software, followed by normalization of 991-treated levels within each sample to DMSO controls. Figure 7C shows cell count of human T cells that were manually counted on Days 5, 7, 9, and 11 and counts plotted to demonstrate expansion over time. Figure 7(D, E) shows DMSO and 991-treated cells incubated with BrdU for 2 hours on Day 9 of culture as shown in Figure 7D or Day 11 of culture as shown in Figure 7E, followed by staining for BrdU incorporation. All data were obtained on 3 or more independent human donor samples. Numbers above the graphs represent statistical significance as determined by paired Student T-test.

Fig. 8(A-G) shows that 991-treated human T cells gain mitochondrial capacity. Figure 8 A shows resting Day 11 DMSO- and 991-treated T cells assessed for oxidative capacity utilizing the Seahorse Metabolic Analyzer. Bar graphs represent data from 2 individual human donors. Figure 8B shows DMSO- and 991-treated T cells incubated with MitoTracker Green to measure mitochondrial density. Bar graphs represent median fluorescence intensity (MFI) data for 4 human donors. Figure 8C shows that DMSO- and 991-treated cells were lysed as in Figure 7B and total PGCla protein measured via immunoblot. Densitometry is shown for 4 human donors. Figure 8D shows the re-stimulation schematic. Figure 8E shows that human T cells were assessed for oxidative capacity following re-activation with DYNABEADS for 24 hours utilizing the Seahorse Metabolic Analyzer. Figure 8F shows that re-activated human T cells were incubated with CellROX dye to measure reactive oxygen species. Figure 8G shows that cells were counted at the time of re-stimulation, and again 72 hours later, to determine percentage cell yield. Unless otherwise stated, bar graphs represent composite data from three or more independent human donors. In panels B-G, bar graph data from 991-treated cells was normalized back to DMSO- treated controls. Fig. 9(A-G) shows that AMPK agonist pre-treatment improves CART anti-leukemic activity and recipient survival. Figure 9(A, B) shows a schematic of CAR plasmid, as in Figure 9A, and CART transduction and agonist treatment protocol in Figure 9B. Figure 9C shows that resting Day 11 DMSO- and 991-treated CART cells were assessed for oxidative capacity utilizing the Seahorse Metabolic Analyzer. Bar graphs represent data from 3 individual human donors. Figure 9D shows that human CART cells were re-activated with NALM6 leukemia targets for 24 hours, followed by further assessment of oxidative capacity. Bar graphs represent data from 4 individual human donors and in Figure 9(C, D) are normalized back to DMSO-treated controls. Figure 9E shows a schematic of Nalm6 xenograft leukemia model. Figure 9(F, G) shows radiance measurements as in Figure 9F and survival as in Figure 9G in Leukemia only, DMSO-CART cell, and 991-CART cell recipients, n = 8 Leukemia only recipients, 6 DMSO CART recipients, and 11 991-treated CART recipients. Data are representative of two individual experiments.

Fig. 10(A-F) shows that transcriptomes of 991-treated T cells are enriched for cell cycle and metabolic gene sets. RNA was harvested from Day 11 T cells from three individual human donors and analyzed for gene expression differences by RNA sequencing. Figure 10(A, B) shows that transcript differences were plotted via log fold-change versus negative log P value, with data points meeting statistical significance highlighted in blue for CD4s, as in Figure 10A and CD8s as in Figure 10B. Figure 10(C-F) shows that gene sets were then ranked and GSEA performed using comparison to Hallmark, KEGG, and transcription factor databases through the GSEA software (see methods for further details). The highest ranked gene sets were in cell cycling as shown in Figure 10(C, D) and metabolism as shown in Figure 10(E, F), shown for CD4s and CD8s, respectively. Accompanying tables list additionally enriched cell cycle and metabolic gene sets.

Fig. ll(A-L) shows that AMPK agonism drives fatty acid oxidation and promotes generation of mitochondrially-protective metabolites. Figure 11 A shows that cells were incubated with FAOBlue dye for 2 hours, followed by flow cytometry analysis. Figure 1 IB shows that cells were pre-incubated +/- etomoxir, then incubated with puromycin for 30 minutes, followed by staining for puromycin incorporation. Bar graphs represent the MFI of etomoxir-treated group divided by the MFI of the control group for both DMSO and 991-treated cultures. Figure 11C shows that cells were incubated with Nile Red dye for 10 minutes followed by flow cytometry analysis. Figure 11D shows total CPT1A protein measured by immunoblot and densitometry normalized in each sample to DMSO controls. Figure 11(E, F) shows Vitamin B5 as in Figure HE and free carnitine levels, as shown in Figure HF, measured by mass spectrometry. Figure l l(G-L) shows mass spectrometry measured levels of intracellular proline as in Figure 11G, glycine as in Figure 11H, leucine as in Figure 111, glutamate as in Figure 11 J, aspartate as in Figure 1 IK, and threonine as in Figure 1 IL. All bar graphs represent data from 3 or more human donors.

Fig. 12(A-K) shows that AMPK agonism mimics cellular starvation. Figure 12(A, B) shows media recovered from 48-hour cultures (+/- 991) assessed for total glucose levels as in Figure 12A and lactate levels as in Figure 12B. Figure 12(C, D) shows Intracellular hexose as shown in Figure 12C and lactate as shown in Figure 12D content was measured by mass spectrometry in T cells on day 9 of culture. Figure I2(E, F) shows untargeted metabolite data analyzed using Metaboanalyst software. Pathways with an enrichment factor >1.5 are highlighted. Figure 12G shows proposed interactions between AMPK, mTOR, and ULK1. Figure 12H shows an immunoblot for phosphorylated and total Raptor levels on Days 7-9 of treatment. Bar graphs represent data from multiple donors, with 991-treated results normalized to DMSO controls. Figure 121 shows that cells were incubated with puromycin for 2 hours, followed by intracellular staining for puromycin incorporation. Figure 12J shows an immunoblot for phosphorylated ULK1 protein in day 9 cells, with values from multiple donors normalized to DMSO controls. Figure 12K shows that Day 7 cells +/- 991 were incubated with CYTO-ID dye for 30 minutes and incorporation assessed by flow cytometry. Incubation with rapamycin served as a positive control. Bar graphs represent values from three human donors, except for the CYTO-ID data shown in Figure 12K, which was two donors.

Fig. 13(A-F) shows that improved leukemia control correlates with increased numbers of 991-treated CD4+ CART cells. Figure 13A shows that timeline of evaluations in our xenograft leukemia model. Figure 13(B, C) shows mice injected intraperitoneally with BrdU, followed by spleen and bone marrow harvest 30-60 minutes later. BrdU incorporation was compared between DMSO- and 991-treated CAR T cells in the bone marrow as shown in Figure 13B and the spleen as shown in Figure 13C, both on Day 3 and up to one-week post-transfer. Figure 13(D, E) Total human CD4+ and CD8+ T cell counts were obtained from the spleen and bone marrow on Day 3 as shown in Figure 13D and after one week as shown in Figure 13E. Figure 13F shows CD4/CD8 ratios calculated in the spleen at one- and two- weeks post-injection. n=7 for both groups in the day 3 bone marrow samples (panels B and D), and n=8 for both day 3 spleen samples (panels C and D). n=10 mice in both groups at one-week post-injection (panels C, E, and F) and n=6 and 11 for the week 2 DMSO and 991-treated samples in Figure 13F, respectively.

Fig. 14(A-D) shows that 991 exposure does not change the memory or exhaustion phenotype of CART cells. Figure 14(A, B) shows Human CARTs taken on Dll of culture and stained for memory markers CD62L and CD45RO. Data analysis was divided into T cell subsets, CD4 as shown in Figure 14A and CD8 as shown in Figure 14B. Figure 14(C, D) shows human CARTs recovered from culture on Dll and stained for exhaustion markers PD1 and Tim3. Data are again divided into CD4 T cells as shown in Figure 14C and CD8 T cells as shown in Figure 14D.

Fig. 15 shows that 991 treatment does not increase ROS burden during treatment. Figure 15 shows T cells assessed on D7 of culture for ROS burden using the ROS-reactive dye, CellROX. Bar graphs represent data from 2 human donors.

Fig. 16 shows that Leukemia is absent in both DMSO- and 991-treated CART recipients by Day 3 post-CART injection. NSG mice were sacrificed 3 days after CART injection and cells of the bone marrow analyzed by flow for ongoing leukemia (using Zs-Green) and the presence of CART cells (using the CAR EGFR tag). Representative flow plots are shown for the leukemia only control, as well as the DMSO- and 991-treated CART groups.

Fig. 17 shows that 991 exposure does not change the CD4/CD8 ratio of CART cells prior to injection. Day 11 CART cells were stained for CD4 and CD8 and analyzed by flow. Bar graphs on the right represent composite data from 6 human donors.

DETAILED DESCRIPTION

Disclosed herein are methods and compositions for making a T cell with increased immune function comprising administering to said T cell a composition comprising to some degree compound 991. In some embodiments, the T cell is a is a CART cell. Also disclosed herein are methods of increasing an immune response in a subject comprising administering to the subject a therapeutically effective amount of a T cell contacted with compound 991. The compositions and methods have been shown to be surprisingly effective at creating T cells with maintained or increased growth in vitro, increased in vivo persistence and/or have increased levels of oxidative metabolism as compared to a control.

Terms used throughout this application are to be construed with ordinary and typical meaning to those of ordinary skill in the art. However, Applicant desires that the following terms be given the particular definition as provided below.

Terminology

As used in the specification and claims, the singular form "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a cell" includes a plurality of cells, including mixtures thereof.

The term “about” as used herein when referring to a measurable value such as an amount, a percentage, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, or ±1% from the measurable value.

"Activate", "activating", and "activation" mean to increase an activity, response, condition, or other biological parameter. This may also include, for example, a 10% increase in the activity, response, or condition, as compared to the native or control level. Thus, the increase can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.

“Administration” to a subject includes any route of introducing or delivering to a subject an agent (e.g., a T cell). Administration can be carried out by any suitable route, including oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra-joint, parenteral, intra-arteriole, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation, via an implanted reservoir, or via a transdermal patch, and the like. Administration includes self-administration and the administration by another.

As used herein, the term “comprising” is intended to mean that the compositions and methods include the recited elements, but not excluding others. “Consisting essentially of’ when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like. “Consisting of” shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions of this invention. Embodiments defined by each of these transition terms are within the scope of this invention.

A “control” is an alternative subject or sample used in an experiment for comparison purposes. A control can be "positive" or "negative." In some embodiments, the control described herein refers to a T cell to which compound 991 has not been administered.

“Differentiation” refers to the process by which immature and unspecialized cells mature and take on specialized forms and/or functions. Differentiation state can be determined based on a cell’ s expression profile. In some embodiments, a less differentiated T cell is CD62L⁺CD45RA⁺ or CD62L⁺CD27⁺.

The term “extracellular acidification rate” or “ECAR” is predominantly or wholly a measure of lactic acid secretion per unit time and is a proxy for a cell’s rate of glycolysis.

"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 of DNA and translation of mRNA results in the protein.

"Expression vector" or “vector” comprises 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., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.)

The “fragments,” whether attached to other sequences or not, can include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the activity of the fragment is not significantly altered or impaired compared to the nonmodified peptide or protein. These modifications can provide for some additional property, such as to remove or add amino acids capable of disulfide bonding, to increase its bio-longevity, to alter its secretory characteristics, etc. In any case, the fragment must possess a bioactive property of the sequence from which it is derived such as increasing in vivo persistence, decreasing differentiation, and/or increasing levels of oxidative metabolism in a T cell in which the fragment is expressed.

The term "gene" or "gene sequence" refers to the coding sequence or control sequence, or fragments thereof. A gene may include any combination of coding sequence and control sequence, or fragments thereof. Thus, a "gene" as referred to herein may be all or part of a native gene. A polynucleotide sequence as referred to herein may be used interchangeably with the term "gene”, or may include any coding sequence, non-coding sequence or control sequence, fragments thereof, and combinations thereof. The term "gene" or "gene sequence" includes, for example, control sequences upstream of the coding sequence (for example, the ribosome binding site).

As used herein, the term “expression” refers to either or both “gene expression” and “protein expression.” “Gene expression” refers to the process by which polynucleotides are transcribed into mRNA and “protein expression” refers to the process by which mRNA is translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell. “Gene overexpression” refers to the overproduction of the mRNA transcribed from the gene, at a level that is at least about 2.5 times higher, at least about 5 times higher, or at least about 10 times higher than the expression level detected in a control sample. “Protein overexpression” includes the overproduction of the protein product encoded by a gene at a level that is at least about 2.5 times higher, at least about 5 times higher, or at least about 10 times higher than the expression level detected in a control sample.

“In vitro T cell expansion period” or “ex vivo T cell expansion period” refer herein to a period of time in which one or more T cells reproduce and increase in number ex vivo. For example, when CAR T cells are administered to a subject, such as for a therapy or treatment, increasing the number of CAR T cells prior to the administration is important. Such expansion or reproduction is commonly achieved through CD3 and/or CD28 activation of a T cell followed by cultivation of the T cell in a media. In some embodiments, the media includes one or more cytokines. In some embodiments, the expansion period is for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days. In some embodiments, the expansion period is about or between 1 to 2 days. In some embodiments, the expansion period is about or between 1 to 3 days. In some embodiments, the expansion period is about or between 1 to 4 days. In some embodiments, the expansion period is about or between 1 to 5 days. In some embodiments, the expansion period is about or between 1 to 6 days. In some embodiments, the expansion period is about or between 1 to 7 days. In some embodiments, the expansion period is about or between 1 to 8 days. In some embodiments, the expansion period is about or between 1 to 9 days. In some embodiments, the expansion period is about or between 1 to 10 days. In some embodiments, the expansion period is about or between 1 to 11 days. In some embodiments, the expansion period is from 1 to 12 days. In some embodiments, the expansion period is about or between 1 to 13 days. In some embodiments, the expansion period is about or between 1 to 14 days. In some embodiments, the expansion period is about or between 1 to 15 days. In some embodiments, the expansion period is from 1 to 16 days. In some embodiments, the expansion period is about or between 1 to 17 days. In some embodiments, the expansion period is about or between 1 to 18 days. In some embodiments, the expansion period is about or between 1 to 19 days. In some embodiments, the expansion period is about or between 1 to 20 days. In some embodiments, the expansion period is about or between 1 to 21 days.

The term “in vivo persistence” refers to a cell’s survival time and/or effector function in vivo. Using the methods or the compositions herein, the in vivo persistence of a T cell is at least about 1.5 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 60 times, at least about 80 times, at least about 100 times, at least about 500 times, at least about 1000 times higher than a control T cell. In some embodiments, a T cell’s survival time is at least about 1.5 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 60 times, at least about 80 times, at least about 100 times, at least about 500 times, at least about 1000 times higher than a control T cell. In some embodiments, a T cell’s effector function is at least about 1.5 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 60 times, at least about 80 times, at least about 100 times, at least about 500 times, at least about 1000 times higher than a control T cell.

As used herein, “immune function” refers to one or more of 1) secretion of cytotoxins such as perforin and granzymes, 2) target cell killing, 3) secretion of activating cytokines such as IFN- y, GM-CSF and TNF-a, and 4) secretion of activating cytokines such as IL-4, IL-5, and IL-10. In some embodiments, an increase in immune function in a CD8⁺ T cell includes in an increase in either, both, or all three including secretion of cytotoxins, production and secretion of cytokines, and target cell killing. In some embodiments, an increase in immune function in a CD4+ T cell includes an increase in secretion of macrophage activating cytokines. In some embodiments, an increase in immune function in a CD4+ T cell includes an increase in secretion of B cell activating cytokines.

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).

As used herein, "operatively linked" can indicate that the regulatory sequences useful for expression of the coding sequences of a nucleic acid are placed in the nucleic acid molecule in the appropriate positions relative to the coding sequence so as to effect expression of the coding sequence. This same definition is sometimes applied to the arrangement of coding sequences and/or transcription control elements (e.g., promoters, enhancers, and termination elements), and/or selectable markers in an expression vector. The term "operatively linked" can also refer to the arrangement of polypeptide segments within a single polypeptide chain, where the individual polypeptide segments can be, without limitation, a protein, fragments thereof, linking peptides, and/or signal peptides. The term operatively linked can refer to direct fusion of different individual polypeptides within the single polypeptides or fragments thereof where there are no intervening amino acids between the different segments as well as when the individual polypeptides are connected to one another via one or more intervening amino acids. The term “oxidative metabolism” refers to the chemical process in which oxygen is used to make energy from carbohydrates. A cell’s oxidative metabolism level can be determined by any method known to those of skill in the art. In some embodiments, the level of oxidative metabolism is equivalent to the oxidative consumption rate (OCR), which can be determined using, for example, a Seahorse Extracellular Flux Analyzer. This can include, but is not limited to, measurement of basal OCR, maximal OCR, and spare respiratory capacity (SRC).

"Pharmaceutically acceptable" component can refer to a component that is not biologically or otherwise undesirable, i.e., the component may be incorporated into a pharmaceutical formulation of the invention and administered to a subject as described herein without causing significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the formulation in which it is contained. When used in reference to administration to a human, the term generally implies the component has met the required standards of toxicological and manufacturing testing or that it is included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration.

"Pharmaceutically acceptable carrier" (sometimes referred to as a “carrier”) means a carrier or excipient that is useful in preparing a pharmaceutical or therapeutic composition that is generally safe and non-toxic and includes a carrier that is acceptable for veterinary and/or human pharmaceutical or therapeutic use. The terms "carrier" or "pharmaceutically acceptable carrier" can include, but are not limited to, phosphate buffered saline solution, water, emulsions (such as an oil/water or water/oil emulsion) and/or various types of wetting agents.

As used herein, the term “carrier” encompasses any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations. The choice of a carrier for use in a composition will depend upon the intended route of administration for the composition. The preparation of pharmaceutically acceptable carriers and formulations containing these materials is described in, e.g., Remington's Pharmaceutical Sciences, 21st Edition, ed. University of the Sciences in Philadelphia, Lippincott, Williams & Wilkins, Philadelphia, PA, 2005. Examples of physiologically acceptable carriers include saline, glycerol, DMSO, buffers such as phosphate buffers, citrate buffer, and buffers with other organic acids; antioxidants including ascorbic acid; 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, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEENTM (ICI, Inc.; Bridgewater, New Jersey), polyethylene glycol (PEG), and PLURONICSTM (BASF; Florham Park, NJ). To provide for the administration of such dosages for the desired therapeutic treatment, compositions disclosed herein can advantageously comprise between about 0.1% and 99% by weight of the total of one or more of the subject compounds based on the weight of the total composition including carrier or diluent.

The term “pharmaceutically acceptable salts” refers to any acid or base addition salt whose counter-ions are non-toxic to the subject to which they are administered in pharmaceutical doses of the salts.

The term "polynucleotide" refers to a single or double stranded polymer composed of nucleotide monomers.

The term "polypeptide" refers to a compound made up of a single chain of D- or L-amino acids or a mixture of D- and L-amino acids joined by peptide bonds.

The terms “peptide,” “protein,” and “polypeptide” are used interchangeably to refer to a natural or synthetic molecule comprising two or more amino acids linked by the carboxyl group of one amino acid to the alpha amino group of another.

The term "promoter" as used herein is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence.

The terms "identity" and “identical to” shall be construed to mean the percentage of nucleotide bases or amino acid residues in the candidate sequence that are identical with the bases or residues of a corresponding sequence to which it is compared, after aligning the sequences and introducing gaps, if necessary to achieve the maximum percent identity for the entire sequence, and not considering any conservative substitutions as part of the sequence identity. Neither N- nor C-terminal extensions nor insertions shall be construed as reducing identity. A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) that has a certain percentage (for example, 80%, 85%, 90%, or 95%) of "sequence identity" to another sequence means that, when aligned over their full lengths, that percentage of bases (or amino acids) are the same in comparing the two sequences. This alignment and the percent sequence identity can be determined using software programs known in the art. In one embodiment, default parameters are used for alignment. In one embodiment a BLAST program is used with default parameters. In one embodiment, BLAST programs BLASTN and BLASTP are used with the following default parameters: Genetic code=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non- redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR.

The term “increased” or “increase” as used herein generally means an increase by a statically significant amount; for the avoidance of any doubt, “increased” means an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.

The term “reduced”, “reduce”, “suppress”, or “decrease” as used herein generally means a decrease by a statistically significant amount. However, for avoidance of doubt, “reduced” means a decrease by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (i.e. absent level as compared to a reference sample), or any decrease between 10-100% as compared to a reference level.

The term “subject” is defined herein to include animals such as mammals, including, but not limited to, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice and the like. In some embodiments, the subject is a human.

“Suppressor function” refers herein to a T cell’s suppression of the activation, proliferation or cytokine production of other immune cells (including but not limited to T cells, B cells and/or dendritic cells). A regulatory T cell, or a Treg, has suppressor function, and the present invention can be used to increase this suppressor function or to increase the number of Treg cells in a T cell population. A Treg can be any T cell that conveys a regulatory or suppressive function on another cell type and is not limited to specific definitions based on the expression of particular cell surface markers (for example CD4) or presence of particular transcription factors (for example FoxP3).

The terms “treat,” “treating,” “treatment,” and grammatical variations thereof as used herein, include partially or completely delaying, alleviating, mitigating or reducing the intensity of one or more attendant symptoms of a disorder or condition and/or alleviating, mitigating or impeding one or more causes of a disorder or condition. Treatments according to the invention may be applied preventively, prophylactically, pallatively or remedially. Prophylactic treatments are administered to a subject prior to onset (e.g., before obvious signs of cancer), during early onset (e.g., upon initial signs and symptoms of cancer), or after an established development of cancer. Prophylactic administration can occur for several days to years prior to the manifestation of symptoms of a disease (e.g., a cancer).

“Therapeutically effective amount” or “therapeutically effective dose” of a composition (e.g., a composition comprising an agent) refers to an amount that is effective to achieve a desired therapeutic result. In some embodiments, a desired therapeutic result is a mitigation of a cancer. In some embodiments, a desired therapeutic result is an increase in a T cell driven immune response. In some embodiments, a desired therapeutic result is an increase in a regulatory T cell (Treg) immune response. Therapeutically effective amounts of a given therapeutic agent will typically vary with respect to factors such as the type and severity of the disorder or disease being treated and the age, gender, and weight of the subject. The term can also refer to an amount of a therapeutic agent, or a rate of delivery of a therapeutic agent (e.g., amount over time), effective to facilitate a desired therapeutic effect, such as mitigation of a cancer. The precise desired therapeutic effect will vary according to the condition to be treated, the tolerance of the subject, the agent and/or agent formulation to be administered (e.g., the potency of the therapeutic agent, the concentration of agent in the formulation, and the like), and a variety of other factors that are appreciated by those of ordinary skill in the art. In some instances, a desired biological or medical response is achieved following administration of multiple dosages of the composition to the subject over a period of days, weeks, or years.

The word “vector” refers to any vehicle that carries a polynucleotide into a cell for the expression of the polynucleotide in the cell. The vector may be, for example, a plasmid, a phage particle, or a nanoparticle. Once transformed into a suitable host cell, the vector may replicate and function independently of the host genome, or may in some instances, integrate into the genome itself. In some embodiments, the vector is a DNA construct containing a DNA sequence which is operably linked to a suitable control sequence capable of affecting the expression of the DNA in a suitable host cell. Such control sequences can include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites, and sequences which control the termination of transcription and translation. In other embodiments, the vector is a lipid nanoparticle. Lipid nanoparticles can be used to deliver mRNA to a host cell for expression of the mRNA in the host cell.

Compositions and Methods of Making

Disclosed herein are compositions and methods of making a T cell having an increased immune function comprising contacting the T cell with a composition comprising a compound 991. This method of making has been shown to be surprisingly effective at increasing T cell oxidative metabolism levels, increasing T cell in vivo persistence as compared to a control, increasing suppressive functionality in regulatory T cells, and/or increasing the amount or percentage of CD4+ T cells in a T cell population in vitro and/or in vivo. Accordingly, included herein are methods of making a T cell population having an increased percentage of CD4+ T cells as compared to a control T cell population comprising contacting the T cell with a composition comprising a compound 991. In some embodiments, the T cell population having an increased percentage of CD4+ T cells is an in vitro population. In other embodiments, T cell population having an increased percentage of CD4+ T cells is an in vivo population, or a T cell population arising in vivo following administration of the T cell of the present invention to the subject. In some embodiments, the T cell is a chimeric antigen receptor (CAR) T cell (also referred to herein as “CART”) and/or the T cell population is a CAR T cell population.

As detailed below, the present disclosure highlights the novel use of a direct AMPK agonist, compound 991, to metabolically re-program human T cells. Exposing T cells to an AMPK agonist which binds directly to the AMPK heterotrimer created metabolically augmented cells with improved in vivo anti-leukemia activity. Interestingly, compound 991 exposure did not drive memory reprogramming to any significant extent but instead orchestrated a network of metabolic changes including increased autophagic flux, enhanced fatty acid oxidation, and generation of mitochondrially-protective metabolites. Together, these changes created CARTs with improved in vivo persistence, particularly within the CD4+ compartment.

Accordingly, as used herein, “immune function” refers to one of more of increased T cell oxidative metabolism levels, increased T cell in vivo persistence, increased suppressive functionality in regulatory T cells, and increased amount or percentage of CD4+ T cells in a T cell population, all as compared to a relevant control. The present disclosure includes a method of making a T cell having an increased in vivo persistence as compared to a control comprising contacting the T cell with a composition comprising a compound 991. In some embodiments, the T cell is a CD4+ T cell. The disclosure also includes a method of making a T cell population having an increased amount or ratio of CD4+ T cells, such as an increased amount of CD4+ T cells as compared to CD8+ T cells in the population, wherein the method comprises contacting the T cell with a composition comprising a compound 991. In some embodiments, the T cell population having an increased amount or ratio of CD4+ T cells is an in vitro population. In other embodiments, the T cell population having an increased amount or ratio of CD4+ T cells is an in vivo population. In some embodiments, the compound 991 can be any as described in PCT Publication WO 2010/036613, and/or U.S. Patent No. 8,394,969 which are incorporated by reference in their entireties. In some embodiments, the compound 991 is selected from:

or a pharmaceutically acceptable salt thereof. In some embodiments, the compound 991 has the structural formula of Formula I which is shown below:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound 991 is administered to the T cell during an in vitro T cell expansion period. “In vitro T cell expansion period” or “ex vivo T cell expansion period” refer herein to a period of time in which one or more T cells reproduce and increase in number ex vivo. For example, when CAR T cells are administered to a subject, such as for a therapy or treatment, increasing the number of CAR T cells prior to the administration can be important. Such expansion or reproduction is commonly achieved through CD3 and/or CD28 activation of a T cell followed by cultivation of the T cell in a media. In some embodiments, the media includes one or more cytokines. In some aspects, the cytokine is an IL-2.

The in vitro T cell expansion period can be for any amount of time appropriate for the particular T cell, such as a CAR T cell, and its subsequent in vivo use. In some embodiments, the in vitro T cell expansion period begins upon contacting the T cell with a CD3 and/or CD28 activator. As used herein “CD3 and/or CD28 activator” refers to a composition comprising a CD3 ligand and/or a CD28 ligand for binding to the T cell CD3 and/or T cell CD28, respectively. In some embodiments, the CD3 ligand is an anti-human CD3 antibody. In some embodiments, the CD28 ligand is an anti-human CD28 antibody. One example of a CD3 and/or CD28 activator is Human T-Activator CD3/CD28 DYNABEADS (Fisher Scientific (Thermo) 11132D). The in vitro T cell expansion period ends no later than the T cell administration to the subject.

The compound 991 is administered to the T cell one or more times during the in vitro T cell expansion period. In some embodiments, the compound 991 is administered to the T cell once during the in vitro T cell expansion period. In other embodiments, the compound 991 is administered to the T cell twice during the in vitro T cell expansion period. In other embodiments, the compound 991 is administered to the T cell three times during the in vitro T cell expansion period. In other embodiments, the compound 991 is administered to the T cell four times during the in vitro T cell expansion period. In other embodiments, the compound 991 is administered to the T cell five times during the in vitro T cell expansion period.

In some embodiments, the compound 991 is administered to the T cell about less than one day from the onset of the in vitro T cell expansion period. In some embodiments, the compound 991 is administered to the T cell between about 1 to 2 days from the onset of the in vitro T cell expansion period. In some embodiments, the compound 991 is administered to the T cell between about 1 to 3 days from the onset of the in vitro T cell expansion period. In some embodiments, the compound 991 is administered to the T cell between about 1 to 4 days from the onset of the in vitro T cell expansion period. In some embodiments, the compound 991 is administered to the T cell between about 1 to 5 days from the onset of the in vitro T cell expansion period. In some embodiments, the compound 991 is administered to the T cell between about 1 to 6 days from the onset of the in vitro T cell expansion period. In some embodiments, the compound 991 is administered to the T cell between about 1 to 7 days from the onset of the in vitro T cell expansion period. In some embodiments, the compound 991 is administered to the T cell between about 1 to 8 days from the onset of the in vitro T cell expansion period. In some embodiments, the compound 991 is administered to the T cell between about 1 to 9 days from the onset of the in vitro T cell expansion period. In some embodiments, the compound 991 is administered to the T cell between about 1 to 10 days from the onset or beginning of the in vitro T cell expansion period. In some embodiments, the compound 991 is administered to the T cell between about 1 to 11 days from the onset of the in vitro T cell expansion period. In some embodiments, the compound 991 is administered to the T cell between about 1 to 12 days from the onset or beginning of the in vitro T cell expansion period. In some embodiments, the compound 991 is administered to the T cell between about 1 to 13 days from the onset or beginning of the in vitro T cell expansion period. In some embodiments, the compound 991 is administered to the T cell between about 1 to 14 days from the onset of the in vitro T cell expansion period. In some embodiments, the compound 991 is administered to the T cell between about 1 to 15 days from the onset of the in vitro T cell expansion period. In some embodiments, the compound 991 is administered to the T cell between about 1 to 16 days from the onset of the in vitro T cell expansion period. In some embodiments, the compound 991 is administered to the T cell between about 1 to 17 days from the onset of the in vitro T cell expansion period. In some embodiments, the compound 991 is administered to the T cell between about 1 to 18 days from the onset or beginning of the in vitro T cell expansion period. In some embodiments, the compound 991 is administered to the T cell between about 1 to 19 days from the onset of the in vitro T cell expansion period. In some embodiments, the compound 991 is administered to the T cell between about 1 to 20 days from the onset of the in vitro T cell expansion period. In some embodiments, the compound 991 is administered to the T cell between about 1 to 21 days from the onset of the in vitro T cell expansion period.

In some embodiments, the compound 991 is administered to the T cell about 3 to 9 days from the onset of the in vitro T cell expansion period. In some embodiments, the compound 991 is administered to the T cell about 4 to 8 days from the onset of the in vitro T cell expansion period. In some embodiments, the compound 991 is administered to the T cell about 5 to 7 days from the onset of the in vitro T cell expansion period. In some embodiments, the compound 991 is administered to the T cell about 3 to 7 days from the onset of the in vitro T cell expansion period and again about 5 to 9 days from the onset of the in vitro T cell expansion period. In some embodiments, the compound 991 is administered to the T cell about 4 to 6 days from the onset of the in vitro T cell expansion period and again about 6 to 8 days from the onset of the in vitro T cell expansion period. In some embodiments, the compound 991 is administered to the T cell about 5 days from the onset of the in vitro T cell expansion period and again about 7 days from the onset of the in vitro T cell expansion period.

In some embodiments, the compound 991 is administered to the T cell about 2 to 19 days from the onset of the in vitro T cell expansion period. In some embodiments, the compound 991 is administered to the T cell about 4 to 17 days from the onset of the in vitro T cell expansion period. In some embodiments, the compound 991 is administered to the T cell about 6 to 15 days from the onset of the in vitro T cell expansion period. In some embodiments, the compound 991 is administered to the T cell about 8 to 13 days from the onset of the in vitro T cell expansion period and again about 9 to 11 days from the onset of the in vitro T cell expansion period.

In some embodiments of the methods disclosed herein, the compound 991 is contacted with the T cell at least two times over a period of between about 12 hours and about 84 hours. In some embodiments, the compound 991 is contacted with the T cell at least two times over a period of between about 24 hours and about 72 hours. In some embodiments, the compound 991 is contacted with the T cell at least two times over a period of between about 36 hours and about 60 hours. In some embodiments, the compound 991 is contacted with the T cell at least two times over a period of between about 44 hours and about 52 hours. In some embodiments, the compound 991 is contacted with the T cell at least two times over a period of between about 46 hours and about 50 hours. In some embodiments, the compound 991 is contacted with the T cell at least two times over a period of about 48 hours.

In some aspects of the present disclosure, the T cell is within a culture medium and all or substantially all of the compound 991 in the culture medium is removed prior to each subsequent administration of the compound 991 to the T cell. As used herein, “substantially all” refers to removal of at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the compound 991.

In some embodiments, the compound 991 is at a molar concentration of between about 25 pM and about 100 pM. In some embodiments, the compound 991 is at a molar concentration of between about 0.5 pM to 50 pM. In some embodiments, the compound 991 is at a molar concentration of between about 0.5 M to 45 M. In some embodiments, the compound 991 is at a molar concentration of between about 0.5 p .VI to 40 pM. In some embodiments, the compound 991 is at a molar concentration of between about 0.5 pM to 35 pM. In some embodiments, the compound 991 is at a molar concentration of between about 0.5 pM to 30 pM. In some embodiments, the compound 991 is at a molar concentration of between about 0.5 pM to 25 pM. In some embodiments, the compound 991 is at a molar concentration of between about 0.5 pM to 20 pM. In some embodiments, the compound 991 is at a molar concentration of between about 0.5 pM to 15 pM. In some embodiments, the compound 991 is at a molar concentration of between about 0.5 pM to 10 pM. In some embodiments, the compound 991 is at a molar concentration of between about 1 pM to 50 pM. In some embodiments, the compound 991 is at a molar concentration of between about 1 pM to 45 pM. In some embodiments, the compound 991 is at a molar concentration of between about 1 pM to 40 pM. In some embodiments, the compound 991 is at a molar concentration of between about 1 pM to 35 pM. In some embodiments, the compound 991 is at a molar concentration of between about 1 pM to 30 pM. In some embodiments, the compound 991 is at a molar concentration of between about 1 pM to 25 pM. In some embodiments, the compound 991 is at a molar concentration of between about 1 pM to 20 p M. In some embodiments, the compound 991 is at a molar concentration of between about 1 pM to 15 pM. In some embodiments, the compound 991 is at a molar concentration of between about 1 pM to 10 pM. In some embodiments, the compound 991 is at a molar concentration between 5 pM to 50 pM. In some embodiments, the compound 991 is at a molar concentration of between about 5 pM to 45 pM. In some embodiments, the compound 991 is at a molar concentration between 5 pM to 40 pM. In some embodiments, the compound 991 is at a molar concentration of between about 5 pM to 35 pM. In some embodiments, the compound 991 is at a molar concentration between 5 pM to 30 pM. In some embodiments, the compound 991 is at a molar concentration of between about 5 pM to 25 pM. In some embodiments, the compound 991 is at a molar concentration between 5 pM to 20 pM. In some embodiments, the compound 991 is at a molar concentration of between about 5 pM to 15 pM. In some embodiments, the compound 991 is at a molar concentration between 5 pM to 10 pM. In some embodiments, the compound 991 is at a molar concentration between 7.5 pM to 50 pM. In some embodiments, the compound 991 is at a molar concentration between 7.5 pM to 45 pM. In some embodiments, the compound 991 is at a molar concentration between 7.5 pM to 40 pM. In some embodiments, the compound 991 is at a molar concentration of between about 7.5 pM to 35 pM. In some embodiments, the compound

991 is at a molar concentration between 7.5 pM to 30 pM. In some embodiments, the compound 991 is at a molar concentration of between about 7.5 uM to 25 pM. In some embodiments, the compound 991 is at a molar concentration between 7.5 pM to 20 p M.

In some embodiments, the compound 991 is at a molar concentration of between about 7.5 pM to 15 pM. In some embodiments, the compound 991 is at a molar concentration between 7.5 pM to 10 pM. In some embodiments, the compound 991 is at a molar concentration between 10 pM to 50 pM. In some embodiments, the compound 991 is at a molar concentration between 10 pM to 45 pM. In some embodiments, the compound 991 is at a molar concentration between 10 pM to 40 pM. In some embodiments, the compound 991 is at a molar concentration of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 pM. In some embodiments, the compound 991 is at a molar concentration of about 10 pM.

In some embodiments, the compound 991 is at a molar concentration of between about 10 pM to 35 pM. In some embodiments, the compound 991 is at a molar concentration between 10 pM to 30 pM. In some embodiments, the compound 991 is at a molar concentration of between about 10 pM to 25 pM. In some embodiments, the compound 991 is at a molar concentration between 10 pM to 20 pM. In some embodiments, the compound 991 is at a molar concentration of between about 10 pM to 15 pM.

In some embodiments, the compound 991 is at a molar concentration between 12.5 pM to 50 pM. In some embodiments, the compound 991 is at a molar concentration between 12.5 pM to 45 pM. In some embodiments, the compound 991 is at a molar concentration between 12.5 pM to 40 pM. In some embodiments, the compound 991 is at a molar concentration of between about 12.5 pM to 35 pM. In some embodiments, the compound 991 is at a molar concentration between 12.5 pM to 30 pM. In some embodiments, the compound 991 is at a molar concentration of between about 12.5 pM to 25 pM. In some embodiments, the compound 991 is at a molar concentration between 12.5 pM to 20 pM. In some embodiments, the compound 991 is at a molar concentration of between about 12.5 pM to 15 pM. In some embodiments, the compound 991 is at a molar concentration of about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 pM. In some embodiments, the compound 991 is at a molar concentration of about 25 pM.

In some embodiments, the compound 991 is administered to the T cell twice during the in vitro T cell expansion period at a molar concentration of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 pM. In some embodiments, the compound 991 is administered to the T cell twice during the in vitro T cell expansion period at a molar concentration of about 10 pM. In some embodiments, the compound 991 is administered to the T cell twice during the in vitro T cell expansion period at a molar concentration of about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 pM. In some embodiments, the compound 991 is administered to the T cell twice during the in vitro T cell expansion period at a molar concentration of about 25 ,u M.

Included herein are methods further including contacting the T cell with IL-2 during the in vitro T cell expansion period. As used herein, “IL-2” refers to a polypeptide that, in humans, is encoded by the IL-2 gene. IL-2 stands for “interleukin 2,” and it is a cytokine. In some embodiments, the IL-2 polypeptide is that identified in one or more publicly available databases as follows: HGNC: 6001, NCBI Entrez Gene: 3558, Ensembl: ENS G00000109471, OMIM: 147680, UniProtKB/Swiss-Prot: P60568. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 1, or a polypeptide sequence having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% identity with SEQ ID NO: 1, or a polypeptide comprising a portion of SEQ ID NO: 1. The IL-2 polypeptide of SEQ ID NO: 1 may represent an immature or pre-processed form of mature IL-2, and accordingly, included herein are mature or processed portions of the IL_2 polypeptide in SEQ ID NO: 1. Included herein are methods wherein the compound 991 and the 11-2 are contacted with the T cell simultaneously. In other embodiments, the T cell is contacted with the compound 991 before it is contacted with the IL-2. In still other embodiments, the T cell is contacted with the compound 991 after it is contacted with the IL-2.

In some aspects, the first contact of the compound 991 with the T cell is about four to about six days the T cell’s first contact with a CD3 and/or CD28 activator. In some embodiments, the first contact of the compound 991 with the T cell is less than one day after the T cell’s first contact with a CD3 and/or CD28 activator. In some embodiments, the first contact of the compound 991 with the T cell is one day after the T cell’s first contact with a CD3 and/or CD28 activator. In some embodiments, the first contact of the compound 991 with the T cell is two days after the T cell’s first contact with a CD3 and/or CD28 activator. In some embodiments, the first contact of the compound 991 with the T cell is three days after the T cell’s first contact with a CD3 and/or CD28 activator. In some embodiments, the first contact of the compound 991 with the T cell is four days after the T cell’s first contact with a CD3 and/or CD28 activator. In some embodiments, the first contact of the compound 991 with the T cell is about five days after the T cell’s first contact with a CD3 and/or CD28 activator. In some embodiments, the first contact of the compound 991 with the T cell is six days after the T cell’s first contact with a CD3 and/or CD28 activator. In some embodiments, the first contact of the compound 991 with the T cell is seven days after the T cell’s first contact with a CD3 and/or CD28 activator. In some embodiments, the first contact of the compound 991 with the T cell is eight days after the T cell’s first contact with a CD3 and/or CD28 activator. In some embodiments, the first contact of the compound 991 with the T cell is nine days after the T cell’s first contact with a CD3 and/or CD28 activator. In some embodiments, the first contact of the compound 991 with the T cell is ten days after the T cell’s first contact with a CD3 and/or CD28 activator. In some embodiments, the first contact of the compound 991 with the T cell is eleven days after the T cell’s first contact with a CD3 and/or CD28 activator. In some embodiments, the first contact of the compound 991 with the T cell is twelve days after the T cell’s first contact with a CD3 and/or CD28 activator. In some embodiments, the first contact of the compound 991 with the T cell is thirteen days after the T cell’s first contact with a CD3 and/or CD28 activator. In some embodiments, the first contact of the compound 991 with the T cell is fourteen days after the T cell’s first contact with a CD3 and/or CD28 activator. In some embodiments, the first contact of the compound 991 with the T cell is fifteen days after the T cell’s first contact with a CD3 and/or CD28 activator. In some embodiments, the first contact of the compound 991 with the T cell is sixteen days after the T cell’s first contact with a CD3 and/or CD28 activator. In some embodiments, the first contact of the compound 991 with the T cell is seventeen days after the T cell’s first contact with a CD3 and/or CD28 activator. In some embodiments, the first contact of the compound 991 with the T cell is eighteen days after the T cell’s first contact with a CD3 and/or CD28 activator. In some embodiments, the first contact of the compound 991 with the T cell is nineteen days after the T cell’s first contact with a CD3 and/or CD28 activator. In some embodiments, the first contact of the compound 991 with the T cell is twenty days after the T cell’s first contact with a CD3 and/or CD28 activator. In some embodiments, the first contact of the compound 991 with the T cell is twenty-one days after the T cell’s first contact with a CD3 and/or CD28 activator.

The T cell can be a CD4⁺ T cell, a CD8⁺ T cell, a T helper cell (Th), a regulatory T cell (Treg), a conventional T cell (Tcon), an effector T cell (Teff), or a follicular T helper cell (Tfh). The CD4⁺ T helper T cell can be any of a variety of helper cells including a Thl, a Th2, a Th9, a Thl7, or a Th22 cell. For example, Thl releases IFN-y and TNF; Th2 releases IL-4 (an important survival factor for B-type lymphocytes and a differentiating factor for T lymphocytes), IL-5 and IL- 13; Th9 produces IL-9; Treg secretes IL- 10 (a cytokine with an immunosuppressive function, which in some cases maintains expression of the FOXP3 transcription factor.) and TGF-P; and Thl7 produces IL-17 (a cytokine playing an important role in host defense against bacteria, and fungi). In some aspects, the T cell is an effector T cell (CD25+, CD45RA+/-, CD45RO+/-, CD127- ), an effector memory T cell (CD25-, CD45RA-, CD45RO+, CD127+), a TEMRA (CD25-, CD45RA+, CD45RO+, CD127+), a central memory T cell (CD25+, CD45RA-, CD45RO +, CD127+), an effector T regulatory cell (Treg) (CD25+/-, CD45RA-, CD45RO+, CD127-, CTLA- 4+), an effector memory Treg (CD25+, CD45RA-, CD45RO+, CD127+, CTLA-4+), a T memory stem cell (TSCM) (CD45RA+, CCR7+, CD27+, CD95-t CXCR3-t), or a naive T cell (CD25-, CD45R+-, CD45RO-, CD127+). In some embodiments, the T cell is a tumor infiltrating lymphocyte (or TIL), or a T cell that is obtained from a tumor. In some embodiments, the T cell is a cytotoxic T cell (CTL). In some embodiments, the T cell is a natural killer T-cell (NK T cell). In some embodiments, the T cell is obtained from a subject to be treated.

Accordingly, in some embodiments, the T cell is a CD4⁺ T cell. In some embodiments, the T cell is a CD8⁺ T cell. In some embodiments, the T cell is an effector T cell such as an effector memory T cell, a TEMRA, a central memory T cell, a regulatory T cell (Treg), an effector memory Treg, or a T memory stem cell (TSCM).

In some embodiments, the T cell is a Chimeric Antigen Receptor (CAR) T cell. The term "chimeric antigen receptor (CAR)," as used herein, refers to artificial T-cell receptors, chimeric T-cell receptors, or chimeric immunoreceptors, for example, and encompass engineered receptors that graft an artificial specificity onto a particular immune effector cell (e.g., a T cell). In some embodiments, CARs comprise an intracellular domain, a transmembrane domain, and an extracellular domain comprising a tumor associated antigen binding region. Methods for making CAR are known in the art. See, e.g., U.S. Patent No: 9,540,445 and International Patent Application Publication No. W02014011987, both incorporated by reference herein in their entireties. As used herein, a T cell comprising a CAR is referred to as a “CART cell.” In some embodiments, the T cell is a cell bearing a cloned T cell receptor which would embody a known reactivity.

In some embodiments, the methods of making further comprise increasing a level of the AMPKy2 polypeptide in the T cell via transducing a vector into the T cell, wherein the vector comprises a polynucleotide sequence encoding the AMPKy2 polypeptide. AMP-activated protein kinase (AMPK) is a heterotrimeric kinase complex composed of a catalytic a subunit with serine/threonine kinase activity, as well as P and y subunits that regulate its activation and substrate specificity. AMPK activation is regulated by the binding of adenylate nucleotides (i.e., ATP, ADP and AMP) to the nucleotide-binding sites of the y subunit, which precedes activating phosphorylation events on the a and subunits. There are three isoforms of the y-subunits, isoforms 1, 2, and 3. “AMPKy2” refers herein to a polypeptide that synthesizes and hydrolyzes cyclic adenosine 5’ -diphosphate-ribose, and in humans, is encoded by the PRKAG2 gene. In some embodiments, the AMPKy2 polypeptide is that identified in one or more publicly available databases as follows: HGNC: 9386, Entrez Gene: 51422, Ensembl: ENSG00000106617, OMIM: 602743, and UniProtKB: Q9UGJ0. In some embodiments, the AMPKy2 polypeptide comprises SEQ ID NO:2. In some embodiments, the AMPKy2 polypeptide comprises SEQ ID NO: 3. In some embodiments, the AMPKy2 polypeptide comprises a polypeptide sequence having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% identity with SEQ ID NO:2 or SEQ ID NO: 3, or a polypeptide comprising a portion of SEQ ID NO:2 or SEQ ID NO: 3. The AMPKy2 polypeptide of SEQ ID NO:1 or SEQ ID NO: 3 may represent an immature or pre-processed form of mature AMPKy2, and accordingly, included herein are mature or processed portions of the AMPKy2 polypeptide in SEQ ID NO:2 and SEQ ID NO: 3. In some embodiments, the AMPKy2 described herein is a full-length polypeptide of AMPKy2 that comprises a polypeptide sequence at least about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, or about 99.5% identical to SEQ ID NO: 3. In some embodiments, the AMPKy2 described herein is a truncated version of AMPKy2 polypeptide that consists of a polypeptide sequence at least about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, or about 99.5% identical to SEQ ID NO: 2.

In some embodiments, the AMPKy2 polypeptide is operably linked to a degradation motif. “Degradation motif’ is used herein to refer to a polypeptide sequence that targets an operably linked amino acid sequence, e.g., an AMPKy2 polypeptide, for degradation. In some embodiments, the degradation motif is an E3 ubiquitination motif. In some embodiments, the degradation motif is activated for targeting upon a change in the motif’s conformation.

In some embodiments, the vector is a viral vector. "Viral vector" as disclosed herein means, in respect to a vehicle, any virus, virus-like particle, virion, viral particle, or pseudotyped virus that comprises a nucleic acid sequence that directs packaging of a nucleic acid sequence in the virus, virus-like particle, virion, viral particle, or pseudotyped virus. In some embodiments, the virus, virus-like particle, virion, viral particle, or pseudotyped virus is capable of transferring a vector (such as a nucleic acid vector) into and/or between host cells. In some embodiments, the virus, virus-like particle, virion, viral particle, or pseudotyped virus is capable of transferring a vector (such as a nucleic acid vector) into and/or between target cells, such as a hepatocyte in the liver of a subject. Importantly, in some embodiments, the virus, virus-like particle, virion, viral particle, or pseudotyped virus is capable of transporting into a nucleus of a target cell (e.g., a hepatocyte). The term “viral vector” is also meant to refer to those forms described more fully in U.S. Patent Application Publication U.S. 2018/0057839, which is incorporated herein by reference for all purposes. Suitable viral vectors include, e.g., adenoviruses (Mitani et al., Hum. Gene Ther. 5:941-948, 1994), adeno-associated virus (AAV) (Goodman et al., Blood 84: 1492-1500, 1994), vaccinia viruses, herpesviruses, baculoviruses and retroviruses (Agrawal et al., Exper. Hematol. 24:738-747, 1996), parvoviruses, and lentiviruses (Naidini et al., Science 272:263-267, 1996). In some embodiments, the viral vector is a lentiviral vector. In some embodiments, the AMPKy2 coding polynucleotide sequence is operatively linked to a second polynucleotide sequence that encodes a ribosomal skipping sequence (or self-cleaving peptide). In some embodiments, the ribosomal skipping sequence is introduced between the AMPKy2 polypeptide and a protein, wherein the protein can be located upstream of the N- terminus of the AMPKy2 polypeptide or downstream of the C-terminus of the AMPKy2 polypeptide. The ribosomal skipping sequence helps generate two proteins by having the ribosome fall off in between the two sequences. Accordingly, in some aspects, the vector comprises a ribosomal skipping sequence that is operatively linked to the AMPK/2 coding polynucleotide sequence. In some embodiments, the ribosomal skipping sequence is T2A. In some embodiments, the T2A comprises a sequence of at least about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, about 99.5% identical to SEQ ID NO: 5 or a fragment thereof.

In some embodiments, the vector further comprises additional promoter elements, e.g., enhancers that regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site of the nucleic acid sequence mentioned above (e.g., the nucleic acid sequence encoding AMPKy2 polypeptide), although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription.

In some embodiments, the T cells produced according to the methods described herein have one or more increased immune function as compared to a control T cell, those characteristics being selected from the group consisting of: 1) an increased oxidative metabolism, 2) less differentiated, 3) an increased extracellular acidification rate (ECAR), 4) an increased level of proliferation, 5) an increased in vivo survival time or persistence, and 6) an increased effector function either in vitro or in vivo. A cell’s oxidative metabolism level and ECAR can be determined by any method known to those of skill in the art. In some embodiments, the level of oxidative metabolism is equivalent to the oxidative consumption rate (OCR), which can be determined using, for example, a Seahorse Extracellular Flux Analyzer. ECAR can also be determined using a Seahorse Extracellular Flux Analyzer. In some embodiments, the T cell has an increased oxidative metabolism as compared to a control T cell. In some embodiments, the T cell is less differentiated than a control T cell. In some embodiments, the T cell has an increased ECAR as compared to a control T cell. In some embodiments, the T cell has an increased level of proliferation as compared to a control T cell. In some embodiments, the T cell has an increase in vivo survival time as compared to a control T cell. In some embodiments, the T cell has an increased effector function as compared to a control T cell, either in vitro or in vivo. In some embodiments, the T cell has an increased in vivo persistence as compared to a control.

Methods of Increasing an Immune Response and Treatment

As noted above, disclosed herein is a method of increasing an immune response in a subject comprising administering to the subject a therapeutically effective amount of a compound 991 T cell. The present disclosure shows that human T cells expanded in the presence of compound 991 activated AMPK without significantly limiting cellular expansion and gained both mitochondrial density and improved handling of reactive oxygen species (ROS). Importantly, receipt of 991-exposed CARTs improved in vivo cancer clearance, prolonged recipient survival, and increased CD4+ T cell yields at early times post-injection. Ex vivo, 991 agonist treatment mimicked nutrient starvation, increased autophagic flux, and promoted generation of mitochondrially-protective metabolites.

Accordingly, as used herein, “immune response” refers to a reaction by the subject’s immune cells to a foreign constituent such as a virus or bacteria or an abnormality such as a cancer. In some embodiments, the increased immune response comprises T cells having increased immune function as described above. The term “compound 991 T cell” refers to any of the herein described T cells contacted with a 991 compound according to any of the methods described herein.

Accordingly, included herein are methods of increasing an anti-cancer immune response in a subject. Also included are methods of treating a cancer in a subject. Each of these methods comprises administering to the subject a therapeutically effective amount of a compound 991 T cell. The present disclosure includes methods wherein the T cell is obtained from a subject, contacted with a 991 compound, and then returned to the subject. In some embodiments, the subject is a human.

Examples of increased immune responses can be increased killing effects on a target, such as a tumor, a pathogen or a vaccine, or increased suppressive effects on inflammation or autoimmunity. Accordingly, in some embodiments, the method of increasing an immune response can be used to treat cancers or infections. In these embodiments, treatment can be a reduction in the size of a tumor, of the number of tumors, and/or in the metastasis of a tumor in the subject. In some embodiments, the method of increasing an immune response can be used to treat diseases relating to increased inflammation, such as an inflammatory disease or autoimmune diseases. In some embodiments, treatment can be a decrease in bacterial load, an increased number of virustargeting T cells and/or short recovery time in the subject.

As used herein, “cancer” refers to any of a hematological cancer such as lymphoma, myeloma or leukemia, a melanoma, lung cancer (including lung adenocarcinoma, basal cell carcinoma, squamous cell carcinoma, large cell carcinoma, bronchioloalveolar carcinoma, bronchogenic carcinoma, non-small-cell carcinoma, small cell carcinoma, mesothelioma); breast cancer (including ductal carcinoma, lobular carcinoma, inflammatory breast cancer, clear cell carcinoma, mucinous carcinoma, serosal cavities breast carcinoma); colorectal cancer (colon cancer, rectal cancer, colorectal adenocarcinoma); anal cancer; pancreatic cancer (including pancreatic adenocarcinoma, islet cell carcinoma, neuroendocrine tumors); prostate cancer; prostate adenocarcinoma; ovarian carcinoma (ovarian epithelial carcinoma or surface epithelial- stromal tumor including serous tumor, endometrioid tumor and mucinous cystadenocarcinoma, sex-cord-stromal tumor); liver and bile duct carcinoma (including hepatocellular carcinoma, cholangiocarcinoma, hemangioma); esophageal carcinoma (including esophageal adenocarcinoma and squamous cell carcinoma); oral and oropharyngeal squamous cell carcinoma; salivary gland adenoid cystic carcinoma; bladder cancer; bladder carcinoma; carcinoma of the uterus (including endometrial adenocarcinoma, ocular, uterine papillary serous carcinoma, uterine clear-cell carcinoma, uterine sarcomas, leiomyosarcomas, mixed mullerian tumors); glioma, glioblastoma, medulloblastoma, and other tumors of the brain; kidney cancers (including renal cell carcinoma, clear cell carcinoma, Wilm's tumor); cancer of the head and neck (including squamous cell carcinomas); cancer of the stomach (gastric cancers, stomach adenocarcinoma, gastrointestinal stromal tumor); testicular cancer; germ cell tumor; neuroendocrine tumor; cervical cancer; carcinoids of the gastrointestinal tract, breast, and other organs; signet ring cell carcinoma; mesenchymal tumors including sarcomas, fibrosarcomas, haemangioma, angiomatosis, haemangiopericytoma, pseudoangiomatous stromal hyperplasia, myofibroblastoma, fibromatosis, inflammatory myofibroblastic tumor, lipoma, angiolipoma, granular cell tumor, neurofibroma, schwannoma, angiosarcoma, liposarcoma, rhabdomyosarcoma, osteosarcoma, leiomyoma, leiomysarcoma, skin, including melanoma, cervical, retinoblastoma, head and neck cancer, pancreatic, brain, thyroid, testicular, renal, bladder, soft tissue, adenal gland, urethra, cancers of the penis, myxosarcoma, chondrosarcoma, osteosarcoma, chordoma, malignant fibrous histiocytoma, lymphangiosarcoma, mesothelioma, squamous cell carcinoma; epidermoid carcinoma, malignant skin adnexal tumors, adenocarcinoma, hepatoma, hepatocellular carcinoma, renal cell carcinoma, hypernephroma, cholangiocarcinoma, transitional cell carcinoma, choriocarcinoma, seminoma, embryonal cell carcinoma, glioma anaplastic; glioblastoma multiforme,, neuroblastoma, medulloblastoma, malignant meningioma, malignant schwannoma, neurofibrosarcoma, parathyroid carcinoma, medullary carcinoma of thyroid, bronchial carcinoid, pheochromocytoma, Islet cell carcinoma, malignant carcinoid, malignant paraganglioma, melanoma, Merkel cell neoplasm, cystosarcoma phylloide, salivary cancers, thymic carcinomas, and cancers of the vagina among others. In some embodiments the cancer is a leukemia. In some aspects, the cancer is acute myelogenous leukemia or acute lymphoblastic leukemia.

In these methods, the dosage forms of the compositions disclosed herein can be adapted for administration by any appropriate route. Appropriate routes include, but are not limited to, oral (including buccal or sublingual), rectal, epidural, intracranial, intraocular, inhaled, intranasal, topical (including buccal, sublingual, or transdermal), vaginal, intraurethral, parenteral, intracranial, subcutaneous, intramuscular, intravenous, intraperitoneal, intradermal, intraosseous, intracardiac, intraarticular, intracavenous, intrathecal, intravitreal, intracerebral, gingival, subgingival, intracerebroventricular, and intradermal. Such formulations may be prepared by any method known in the art.

The disclosed methods can be performed any time prior to and/or after the onset of a disease (e.g., a cancer or an infection) or administration of a vaccine. In some aspects, the disclosed methods can be employed 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45,

44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19,

18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 years;12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or

1 months; 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7,

6, 5, 4, or 3 days; 60, 48, 36, 30, 24, 18, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3, or 2 hours prior to the onset of a disease (e.g., a cancer or an infection) or administration of a vaccine; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 75, 90, 105, 120 minutes; 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 18, 24, 30, 36, 48, 60 hours; 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 45, 60, 90 or more days; 4, 5, 6, 7, 8, 9, 10, 11, 12 or more months; 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 years after the onset of a disease (e.g., a cancer or an infection) or administration of a vaccine.

Dosing frequency for the T cell compositions disclosed herein, includes, but is not limited to, at least once every 12 months, once every 11 months, once every 10 months, once every 9 months, once every 8 months, once every 7 months, once every 6 months, once every 5 months, once every 4 months, once every 3 months, once every two months, once every month; or at least once every three weeks, once every two weeks, once a week, twice a week, three times a week, four times a week, five times a week, six times a week, or daily. In some embodiment, the interval between each administration is less than about 4 months, less than about 3 months, less than about 2 months, less than about a month, less than about 3 weeks, such as less than about any of 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day, less than about 2 weeks, such as less than about any of 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day, or less than less than about a week, such as less than about any of 6, 5, 4, 3, 2, or 1 day. In some embodiment, the dosing frequency for the T cells disclosed herein includes, but is not limited to, at least once a day, twice a day, or three times a day. In some embodiment, the interval between each administration is less than about 48 hours, 36 hours, 24 hours, 22 hours, 20 hours, 18 hours, 16 hours, 14 hours, 12 hours, 10 hours, 9 hours, 8 hours, or 7 hours. In some embodiment, the interval between each administration is less than about 24 hours, 22 hours, 20 hours, 18 hours, 16 hours, 14 hours, 12 hours, 10 hours, 9 hours, 8 hours, 7 hours, or 6 hours. In some embodiments, the interval between each administration is constant. For example, the administration can be conducted daily, every two days, every three days, every four days, every five days, or weekly. Administration can also be continuous and adjusted to maintaining a level of the compound within any desired and specified range.

All patents, patent applications, and publications referenced herein are incorporated by reference in their entirety for all purposes.

EXAMPLES

The following examples are set forth below to illustrate the compositions, methods, and results according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the present invention which are apparent to one skilled in the art.

Example 1: Methods

Virus Production: A CD19-targeting CAR, based on the YESCARTA protein sequence, was cloned into a pHR backbone (similar to Addgene #14858), followed by addition of a T2A linker and a truncated EGFR tag. Transformed bacterial cultures were grown overnight in Terrific Broth (Sigma Aldrich) and plasmids isolated using QIAGEN QIAmp Miniprep Plasmid Isolation Kit 250. HEK293Ts (ATCC) were cultured in DMEM media (Gibco #11966-025) containing 10% fetal bovine serum (FBS), Pen Strep, 2mM L-Glutamine, and MEM Non-Essential Amino Acids. Early passage cells were transfected using the Lipofectamine 3000 Transfection kit (Invitrogen) with 2500ng of RSV-REV, PMD-2G, and PRRE and 10,000ng of CAR-tEGFR plasmids. After 24 hours, supernatant was replaced with IMDM media (Gibco #12440-053) containing 10% FBS. Supernatant containing viral particles was harvested at 48 and 72 hours, combined with Lenti-Pac (GeneCopoeia), and incubated at 4 degrees C overnight. Viral supernatants were then centrifuged at 3500x (g) for 25 minutes at 4 degrees, resuspended in DMEM, and either frozen at -80C or used immediately.

T cell isolation, transduction, and culture:

In Figures 1-6, the T cells were stimulated with CD3/CD28 DYNABEADS (Fisher Scientific) and transduced with a CAR encoding polynucleotide as per a retronectin transduction protocol (Takara protocol). On Day 5, post-stimulation, all like wells are harvested together and a magnet is used to remove DYNABEADS. The cells are counted and resuspended in AIM-V medium supplemented with 5 % CTS Immune Cell SR, hereafter SR (Thermo Scientific) at a density of 1x106 cells per milliliter and then plated using 500 microliters per well on a 24 well plate. Frozen aliquots of compound 991 (10 mM) and sterile DMSO, used as control are obtained and diluted at a 1:200 dilution in AIM-V + 5 % SR + 200 IU Interleukin-2 (IL2) to create a 50 micromolar treatment mixture. For 4 milliliters of AIM-V media 20 micro liters of compound 991 or DMSO is added. The treatment mixture is then added to appropriate wells at 500 microliters per well to obtain a 1 : 1 dilution of cell suspension and treatment mixture and a final concentration of 25 micromolar of compound 991 and 100 IU of IL-2. The treated cells are placed at 37° Celsius for 48 hours. Thereafter, all like wells are harvested together, spun down, counted and resuspend at a density of 1x106 per milliliter in AIM-V media supplemented with 5 % SR, place at 1 milliliter per well on a 6 well plate. Frozen aliquots of compound 991 (10 mM) and sterile DMSO, used as control are obtained and diluted at a 1:200 dilution in AIM-V + 5 % SR + 200 IU Interleukin-2 (IL2) to create a 50 micromolar treatment mixture. For 4 milliliters of AIM-V media 20 microliters of compound 991 or DMSO is added. The treatment mixture is then added to appropriate wells at 500 microliters per well to obtain a 1:1 dilution of cell suspension and treatment mixture and a final concentration of 25 micromolar of compound 991 and 100 IU of IL2. Cells are replaced at 37° Celsius for 48 hours. Thereafter, all like wells are harvested together, spun down, counted and resuspend at a density of 1x106 per milliliter in AIM-V media supplemented with 5 % SR. Add equal volume of AIMV + 5%SR + 200IU IL2. The cells were then plated in flasks and placed at 37° Celsius for 48 hours and then harvested for use in downstream applications.

In other Figures, de-identified huffy coats were obtained from healthy human donors (Vitalant), diluted with PBS, layered over lymphocyte separation medium (MPbio), and centrifuged at 400 x g and 25 degrees for 20 minutes with no brake. The PBMC layer was removed and T cells isolated using the Miltenyi Biotec Human Pan T cell isolation kit. Purified T cells were resuspended in AIM-V +5% SR (Gibco #A25961-01) and plated with Human T- Activator CD3/CD28 DYNABEADS (Fisher Scientific (Thermo) 11132D) at a 2:1 ratio. For standard human T cells, cells were split on Day 3 with fresh media. For CART cell production, transduction was performed per manufacturer’s instructions utilizing retronectin coated plates (Takara) on Days 2 and 3. In both cases, cells were removed from DYNABEADS by magnetic separation on Day 5 post-stimulation and expanded in AIM-V media containing 5% SR and IL-2 at lOOIU/ml. Cells were re-plated with fresh media every 48 hours thereafter. Compound 991 (SelleckChem S8654) was reconstituted in DMSO and added to cultures at a final concentration of either 10 or 25pM on Days 5 and 7. Control cultures received an equal volume of DMSO. For the survival curve and radiance analysis in Fig. 9, lOuM and 25uM-treated samples were combined into one 991-treated supergroup to improve statistical power. For re-stimulation experiments, primary human T cells were re-plated with DYNABEADS at a 1:1 ratio for up to 72 hours; CART cells were re-stimulated with NALM6 targets at a 3: 1 ratio.

Mice, cell lines, and xenograft leukemia model: NSG (NOD.Cg- Prkdc^sadIl2rgt^mIwl^lISzi mice were purchased from Jackson Laboratories. Male and female mice were used interchangeably and housed in a specific pathogen-free facility. Recipient animals were 8-12 weeks old at the time of injection. The human NALM6 B-cell leukemia cell line was purchased from ATCC and transduced with a retroviral vector expressing Zs-Green and Luciferase. NSG mice were injected with le6 NALM6 cells and seven days later received 3e6 total CARTs, either pre-treated with DMSO or 991. The 'leukemia only’ control group received no CARTs. The experimental unit was a single animal. Leukemia burden was followed weekly by IVIS imaging, following intraperitoneal injection of 3 mg luciferin and imaging after 10 minutes. Any animals with a baseline radiance below le8 were considered leukemia-free, a decision made prior to the start of the experiment. One animal from the 991-treated group in Fig. 10 died while still being leukemia-free by IVIS imaging. Two bone marrow samples were lost to processing from the day 3 samples in Fig. 13(B-D). Twenty-five NSG mice were utilized for the survival curve and radiance data presented in Fig. 10(F, G), and 43 NSG mice were used for the in vivo experiments in Fig. 13. Sample size was determined based on previous experience using these models and the number of CART cells available at the time of injection. Mice were randomly assigned to treatment groups based on the order in which they were ear-punched (also randomly assigned) and wherever possible, recipients of all three treatment groups (leukemia- only, DMSO, and 991) were co-housed prior to and following Nalm6 and CART cell injection. Cells were administered in numeric order within the cage, assuring equivalent timing between all dosing groups. Technicians performing the IVIS imaging were not aware of the treatment allocation.

Protein Isolation and Immunoblot: T cells were counted, washed with PBS, and lysed with 10% trichloroacetic acid. Lysates were centrifuged at 16,000x(g) at 4*C for ten minutes, washed twice in ice cold acetone, resuspended in solubilization buffer (9M Urea containing 1% DTT and 2% Triton X and NuPAGE lithium dodecyl sulfate sample buffer 4X (Invitrogen) at a 3:1 ratio), and heated at 70*C for 10 minutes. Protein gel electrophoresis was performed on ice using NuPAGE 4-12% Bis-Tris Protein Gels (Invitrogen) at 135V. In some cases, protein samples were heated to 95C for 5 minutes prior to gel loading. Protein was transferred to INVITROLON 0.45pm PVDF membranes (Invitrogen) at 30V on ice for one hour. Membranes were blocked in Tris Buffered Saline-Triton containing 5% nonfat milk and immunoblotting performed according to the Cell Signaling Technologies Western Blot Protocol. Blots were stripped for 10 minutes (Restore PLUS Western Blot Stripping Buffer, Thermo) prior to re-probing. Blots were developed with Super Signal West Femto chemiluminescence reagents (Thermo, 34096), detected by CL-X Posure Film (Thermo), and scanned in grayscale with an Epson V600 scanner. Images were cropped using ImageJ Software (version 1.47T), inverted, and densitometry quantitated in an area encompassing the largest band, followed by quantitation of subsequent bands using the same 2- dimensional area.

Flow Cytometry: Cells were washed with PBS + 2% FBS before staining with antibodies at 1:100 dilution for 30 minutes. For intracellular stains, cells were fixed per manufacturer’s instructions using Fix/Perm kit (Invitrogen, Cat #: 88-8824-00) and then stained with antibodies at 1: 100 dilution. MitoTracker Green (Invitrogen) staining was performed at 50 nM in room temperature PBS for 15 minutes. CellROX (Invitrogen) staining (500 nM) was performed in culture medium for 30 minutes at 37 degrees. FAO blue (DiagnoCine Precision) staining was performed in serum-free AIMV at 15 M at 37deg for 2 hours. Nile red (Thermo) staining was performed in serum free AIMV at 0.5pg/ml at 37deg for 15 minutes. Cyto-ID (Enzo) staining was performed per manufacturer’s instructions (37 C x 30 minutes), with 500nM rapamycin added to control cultures at the time of staining. Puromycin (MedChemExpress) uptake was performed in AIMV +5%SR at lOpg/ml at 37deg for 30 minutes. In some cases, cells were pretreated with 8pM etomoxir (Cayman Chemical Company) in AIMV + 5%SR for 15 minutes at 37deg before puromycin addition. BrdU analysis was performed utilizing the Phase-Flow kit per manufacturer’s instructions (BioLegend). In vitro cells were cultured in BrdU at 0.5 l per ml of cell solution per manufacturer’s instructions for 2 hours at 37deg prior to staining. Flow data was captured on a BD Fortessa analyzer (BD Biosciences) and evaluated using FlowJo software (version 10.1, Tree Star). Cells were gated by forward and side scatter to identify the lymphocyte population followed by downstream analysis.

Seahorse Mito Stress Assay: The Seahorse XF Cell Mito Stress Test (Agilent, Santa Clara, CA; Catalog #103015-100) was run on a Seahorse XFe96 Bioanalyzer (Agilent) to determine basal and maximal oxygen consumption (OCR), spare respiratory capacity (SRC), and extracellular acidification rates (ECAR). T cells were plated in assay media (XF Base media (Agilent) with glucose (25mM), sodium pyruvate (2 mM) and L-glutamine (4 mM) (Gibco), pH 7.4 at 37 °C) on a Seahorse cell culture plate coated with Cell-Tak (Coming) at le5 (restim) or 1.5e5 (resting) cells/well. After adherence and equilibration, basal ECAR and OCR readings were taken for 30 min. Cells were then stimulated with oligomycin (2 pM), carbonyl cyanide 4- ( trifluoromethoxy) phenylhydrazone (FCCP, 1 pM), and rotenone/antimycin A (0.5 pM) to obtain maximal respiratory and control values. Assay parameters were: 3 min mix, no wait, 3 min measurement, performed at baseline and repeated after each injection (3 cycles total). SRC was calculated as the difference between basal and the maximal OCR value obtained after FCCP uncoupling. The XF Mito Stress Test report generator and Agilent Seahorse analytics were used to calculate parameters using Wave software (Agilent, Version 2.6.1.53).

RNA sequencing: Total RNA, isolated using the RNeasy Plus Mini Kit (Qiagen) in technical triplicates, was used to generate libraries using Illumina Stranded Total RNA Prep and sequenced on an Illumina Nextseq2000 at the Health Sciences Sequencing Core at the UPMC Children’s Hospital of Pittsburgh. Differentially expressed genes were generated using DEseq2, identifying genes >2-fold change in expression level and p value of 0.05 as determined by two- way ANOVA. Enrichment analysis was accomplished using GSEA software, a joint project of UC San Diego and Broad Institute [49], followed by comparison to datasets from publicly available databases.

Metabolomics: For metabolite analysis, cells were washed and flash frozen in liquid nitrogen in technical replicates of five. Through collaboration with the University of Pittsburgh Health Sciences Mass Spectrometry Core, cells underwent metabolite extraction via resuspension in ice-cold 80% methanol, followed by addition of standards and subsequent liquid chromatography-high resolution mass spectrometry analysis. Following untargeted metabolomic analysis, putative metabolite identifications with a p value <0.05 and fold-change >2, were validated with commercial standards based on retention time, accurate mass, and MS2 fragmentation. Pathway analysis was performed on the untargeted dataset using Metaboanalyst, with comparison to the Biocyc database (Biocyc.org). Statistics: Graphing and statistical analysis was performed using GraphPad Prism for Windows (version 9.3.0, San Diego, CA; www.graphpad.com). Unpaired two-tailed Student t test and two-way ANOVA analysis were used to determine statistical significance. Log-rank (Mantel- Cox) analysis defined survival curve differences. Unless noted otherwise, data are displayed as mean ± standard deviation.

Example 2

Healthy human T cells underwent lentiviral transduction with a CD19-targeting CAR containing the CD28 costimulatory domain as discussed above in relation to Figures 1-6. Transduced CARTs were expanded in complete media supplemented with IL-2 and either AMPK agonist Compound 991 (991) as discussed above, AMPK agonist A769662 (A7), or DMSO (control). After expansion, cells were assessed for metabolic function in vitro using the Seahorse Metabolic Analyzer. In vitro cytotoxicity against NALM6 targets expressing zzGreen was measured by monitoring mean fluorescence intensity using the Incucyte. For in vivo studies, NSG mice were injected on Day -7 with luciferase+ NALM6 leukemia cells followed by CARTs +/- agonist on Day 0, with luminescence followed weekly by IVIS imaging.

Compound 991 treatment enhanced CART oxidative metabolism over DMSO-treated controls, while A7 treatment significantly reduced initial oxygen consumption, leading to 991 treatment being chosen for further study (Fig. 2). Despite this metabolic advantage, 991-treated CARTs showed reduced in vitro cytotoxicity against NALM6 targets compared to DMSO-treated controls, which interestingly mimicked the cytotoxicity advantage of CD28-costimulated CARTs compared to 41BB-costimulated CARTs (Fig. 3). Given 41BB-CARTs show enhanced persistence over CD28-CARTs in vivo, further in vivo studies were pursued with agonist-treated CARTs. Indeed, despite the slower killing in vitro, 991-treated CARTs outperformed the DMSO- treated control CARTs in vivo, with significantly reduced luminescence by IVIS imaging and improved overall survival (Fig. 4).

Conclusions: AMPK agonism during in vitro expansion of CARTs with 991, but not A7, created metabolically desirable CARTs for immunotherapy. This was demonstrated by increased oxidative metabolism after expansion in vitro and improved leukemia clearance in vivo. However, anti-leukemia activity appeared to decrease with in vitro assessments. These studies identify AMPK as an attractive target in immunotherapy, with attention paid to how this pathway is activated, and also suggest the potentially limited utility of using in vitro cytotoxicity as a predictor of in vivo function against leukemia. Example 3

991 treatment facilitates AMPK activity without restricting expansion: It was hypothesized that expanding CART cells in the presence of a direct AMPK agonist would metabolically optimize them for in vivo function. To test this hypothesis, it was first interrogated whether Compound 991 treatment activated AMPK without restricting growth or viability. Human T cells were isolated and stimulated with anti-CD3/CD8 DYNABEADS for 5 days, removed from the beads, and split into control (DMSO) and 991 treated groups. The AMPK heterotrimer is active when the alpha subunit, containing the kinase domain, is phosphorylated on Thrl72. Dosing experiments indicated stable phosphorylation of AMPKa Thrl72 for 48 hours following 991 exposure, leading to a final treatment schedule where 991 was added to T cell cultures for two 48 hours cycles (96 hours of total exposure), followed by a 48-hour washout period (Fig. 7A). Measurement of AMPKa phosphorylation confirmed increased activation of AMPK following 991 treatment (Fig. 7B), with no significant impact on cell growth or expansion through day 11 (Fig. 7C). To assess whether equivalent cell numbers indicated similar proliferation or a combination of proliferative differences and a change in cell survival, T cell proliferation was assessed by measuring incorporation of the thymidine analogue Bromodeoxyuridine (BrdU). In line with the expansion data, there was no difference in BrdU uptake on day 9, following 96 hours of agonist treatment on Day 9 (Fig. 7D). Interestingly, when BrdU incorporation was measured at the end of the culture period on Day 11 , there was now a significant increase in the proliferation of 991-treated cells (Fig. 7E). Given AMPK’s well- documented roles in optimizing metabolic fitness, it was hypothesized this ongoing cell turnover was due to enhanced metabolic capacity, which then allowed for a sustained proliferative effort despite the increasing distance from their original stimulation. This study therefore sought to measure the impact of 991 exposure on subsequent metabolic reprogramming.

Example 4

991-treated human T cells gain mitochondrial capacity and efficiency: To gauge the impact of 991 treatment on T cell metabolism, the Seahorse Metabolic Analyzer Mitostress test was utilized to measure mitochondrial capacity. On Day 11 (48 hours post 991 removal), 991- treated cells increased their oxygen consumption rates (OCR) and spare respiratory capacity (SRC) (Fig. 8A). It was hypothesized these increases might be secondary to an increase in total mitochondria, particularly as AMPK is known to activate (PGCl ), a transcription factor responsible for promoting mitochondrial biogenesis. Staining with MitoTracker revealed increased mitochondrial density in 991-treated cells (Fig. 8B), which correlated with elevated PGCla expression during 991-treatment (Fig. 8C). To better understand if these metabolic changes would persist following subsequent stimulation, cells were restimulated on Day 11 and repeated the metabolic assessments (Fig. 8D). As shown in Fig. 8E, augmented mitochondrial activity continued following activation, with increases in both OCR and SRC. Of note, driving mitochondrial metabolism can also generate increased levels of reactive oxygen species (ROS), which can be damaging to cells at high levels. Reassuringly, enhanced AMPK signaling improved ROS handling, which was hypothesized to be contributing to the ability of 991 -treated cells to tolerate increased mitochondrial respiration. Consistent with this interpretation, 991 -pretreated cells had lower ROS burden following 24 hours of stimulation (Fig. 8F). Greater numbers of 991- treated cells were recovered following 72 hours of restimulation (Fig. 8G), consistent with improved ROS handling supporting an increase in cellular proliferation. Altogether, these data demonstrate that 991 -treatment facilitates mitochondrial biogenesis and enhances mitochondrial function, allowing for increased metabolic capacity and improved cellular expansion upon in vitro re-stimulation.

Example 5

AMPK agonist treatment improves CART anti-leukemia activity and prolongs survival in a xenograft model: With data supporting improved metabolic fitness in agonist- treated T cells, it was next tested whether 991 pre-treatment improved the function of CART cells targeting leukemia. Human CART cells were generated via lentiviral transduction utilizing a CD19- targeting CAR (Fig. 9A) and expanded in the presence of the 991 agonist on the same schedule as the polyclonal human T cells in Figs. 1 and 2 (Fig. 9B). It was first confirmed that 991- treatment similarly enhanced the metabolic capacity of CART using the Seahorse metabolic analyzer. 991-treated CARTs at rest (Fig. 9C), as well as those following overnight activation with CD 19+ NALM6 leukemia cells (Fig. 9D), enhanced their mitochondrial capacity. To measure in vivo CAR T cell efficacy, luciferase expressing NALM6 cells were transferred into immunodeficient NSG mice followed one week later by 3e6 CART cells (Fig. 9E). Standard CART cells transferred into NALMb-bearing NSG mice delayed leukemia growth compared to the leukemia-only control. However, all DMSO-treated CART cell recipients eventually succumbed to lethal leukemia. In sharp contrast, 991-treated CARTs dramatically improved leukemia control, with 54% of 991-treated CART recipients (6/11) remaining leukemia-free through the end of the experiment (Fig. 9F). This improved leukemia clearance led to a significant and remarkably reproducible improvement in recipient survival, with 73% of mice receiving 991- treated CART cells surviving until day 70 (Fig. 9G). Together, these data highlight that expanding human CARTs in the presence of AMPK agonist 991 creates a superior CART cell product, with a striking improvement in overall leukemia clearance and subsequent recipient survival in the preclinical model.

Example 6

991 treatment upregulates cell cycle and metabolic gene sets without inducing changes in memory or activation markers: Multiple groups have demonstrated that CARTs with memorylike phenotypes demonstrate improved anti-leukemia activity in vivo. However, no differences were found in memory phenotype or activation status in the 991-treated cells (Fig. 14(A-D)). Since AMPK signaling can also impact cellular transcriptomics, including through direct activation of transcription factors as well as downstream influence on histone deacetylases, bulk RNA sequencing of Day 11 DMSO- versus 991-treated human T cells was pursued. Only a handful of transcripts were significantly altered in either CD4+ and CD8+ T cells, using a p value of <0.05 and log2-fold change of 0.6 (Fig. 10(A-B)). However, gene set enrichment analysis (GSEA) uncovered multiple upregulated pathways, with the highest enrichment scores in both CD4 and CD8 T cells clustering within cell proliferation and cell cycle pathways (Fig. 10 (C-D)), consistent with the higher proliferative rates observed at the end of in vitro culture (Fig. 7E). The second most enriched gene sets highlighted metabolic pathways (Fig. 10 (E-F)), with pathways directly related to supporting increased proliferation, including pyrimidine and folate metabolism, as well as a notable enrichment of oxidative phosphorylation and fatty acid oxidation. These latter data are again consistent with the increased oxidative capacity of 991-treated cells (Fig. 8 (A-C)) and suggest that increases in cell cycle may result from the enhanced metabolic capacity of 991- treated cells. It was also hypothesized that metabolic rewiring downstream of AMPK was likely responsible for the functional advantage of 991-treated CARTs in vivo and we therefore sought to understand mechanistically how AMPK was directing metabolism to achieve such impressive results.

Example 7

AMPK agonism simultaneously drives fatty acid oxidation while promoting generation of mitochondrially-protective metabolites: AMPK is well-known for its role in supporting fatty acid oxidation (FAO) and long-chain fatty acids (LC-FAs) can bind directly to AMPK to facilitate its activity. Notably, these LC-FAs use the same binding site as Compound 991. It was therefore hypothesized that T cells treated with 991 would increase their utilization of FAO. Using the oxidation-sensitive dye FAO-blue, increased FAO activity was observed in agonist-treated T cells (Fig. 11 A). This upregulation correlated with a higher sensitivity to etomoxir, the carnitine palmitoyltransferase 1 A (CPT1A) and FAO inhibitor, which was read out by a greater reduction in protein translation following etomoxir treatment of agonist-treated cells (Fig. 1 IB). Next cells were stained for lipid droplets, which serve as storage depots for FA intermediates like triacyl glycerides, before being broken down into single-chain FAs for FAO. In line with an increase in FAO, agonist-treated cells also demonstrated reduced staining with the lipid sensitive dye Nile Red, indicating decreased lipid reserves in agonist-treated cells (Fig. 11C). Agonist-treated cells also increased expression of CPT1A (Fig. 1 ID), the enzyme which facilitates transport of LC- FAs into the mitochondria for subsequent beta oxidation. And despite such significantly upregulated FAO, there was no difference in ROS generation during agonist treatment as measured by CellROX staining (Fig. 15A).

Mass spectrometry analysis of intracellular metabolites in 991 -treated day 9 cells further identified increased abundance of Vitamin B5 (Fig. 1 IE) and carnitine (Fig. 1 IF), two additional intermediates necessary for generating fatty-acyl-coA moieties and transporting them across the mitochondrial membrane, respectively. Further inspection of the metabolite data also noted upregulation of multiple amino acids (AAs) known to play a role in mitochondrial health and fitness, including proline, glycine, and leucine (Fig. ll(G-I)). Precursors of these AAs, such as glutamate, aspartate, and threonine, were conversely decreased (Fig. l l(J-L)), suggesting that AMPK specifically directs production of mitochondrially protective AAs. Altogether, these metabolic data highlight AMPK’s roles, not only in promoting FAO, but also in augmenting production of metabolites with roles in maintaining mitochondrial health and function to support the desired metabolic programming.

Example 8

AMPK agonism mimics cellular starvation and upregulates autophagy to enhance metabolic fitness: Some of the earliest literature aimed at improving T cell fitness highlighted the utility of blocking glycolysis during cellular expansion. With GSEA also highlighting enriched glycolytic datasets in 991 -treated cells (Fig. 10(E-F)), it was next sought to understand the role of glycolysis downstream of AMPK agonism. Since AMPK is known to promote glucose uptake, first the amount of glucose remaining in the media after 48 hours of culture in the presence of 991, was quantified. In contrast to an expected AMPK-mediated increase in glucose uptake, more glucose was regularly found remaining in the media of 991 -treated cultures compared to DMSO- treated controls (Fig. 12A). Supporting this lack of glycolytic activity, media from 991-treated cultures also exhibited reduced lactate content (Fig. 12B), with a reduction in intracellular hexoses in 991-treated cells (Fig. 12C). However, the full intracellular metabolite analysis painted a different picture, with marked elevated levels of intracellular lactate (Fig. 12D). Combined with reduced lactate in the media, these data suggest 991-treated cells are continuing to undergo glycolysis, but are retaining the generated lactate intracellularly instead of secreting it. Interestingly, lactate build-up itself has been demonstrated to reduce cellular glucose uptake, which may explain the lack of an expected increase in glucose uptake following agonist treatment. With glycolysis ongoing, despite reduced glucose uptake, the study sought to understand where cells were sourcing their sugar carbons. To do this, pathway analysis was performed on untargeted metabolite data to delineate pathway changes in the cells. Interestingly, the top four most significantly upregulated metabolic pathways following 991 agonist treatment concerned the breakdown of alternative sugar sources, including glycogen (Fig. 12(E-F)). Pathways involving nucleotide metabolism and mitochondrial performance were also highlighted, again supporting the GSEA results and the observed metabolic activity of agonist-treated cells. Further, an increased reliance on intracellular sugar breakdown, alongside lactate retention, are both in line with cells exhibiting a nutrient starvation response. If enhanced AMPK signaling were indeed promoting a starvation response, it would be expected to find an increase in autophagic flux, as cells looked for a way to break down additional energy sources. Such a finding would be of particular interest since increased autophagy, in the setting of nutrient restriction, enforces metabolic efficiency in T cells during in vitro expansion.

Although retention of lactate itself promotes cellular autophagy, AMPK is also a well- known driver of autophagy, both by activating Unc-51 like kinase 1 (ULK1) and by restricting mTOR-mediated ULK inhibition through phosphorylation of the mTORcl complex protein, Raptor (Fig. 12G). mTOR inhibition was first tested by measuring total and phosphorylated Raptor levels, noting that a large role for AMPK is to target Raptor for phosphorylation-dependent degradation. In 991 -treated cells, phosphorylation of Raptor was significantly increased while total Raptor protein levels were significantly decreased (Fig. 12H). Without a fully functional mTORcl complex to signal the availability of amino acids, 991-treated cells regularly reduced their translational activity in line with their sense of lower amino acid levels (Fig. 121). Such a reduction in protein translation has independently been highlighted as a further mechanism to improve in vivo T cell function. Meanwhile, 991 -treatment increased phosphorylation of ULK1 (Fig. 12J), concomitant with an increase in cellular autophagy (Fig. 12K). Together, these data suggest that AMPK agonist treatment drives cellular programming reminiscent of the response to nutrient starvation, increasing availability of intracellular energy sources through autophagy while reducing high energy expenditure by decreasing protein translation.

Example 9

Improved leukemia control correlates with increased survival of 991 -treated CD4+ CART cells: The data herein shows that AMPK agonism metabolically reprograms cells towards pathways which facilitate cellular fitness. It was therefore hypothesized that the mechanism of improved leukemia clearance and subsequent improved survival in the pre-clinical model could be either enhanced initial CART expansion and/or prolonged in vivo persistence of the CARTs over time. Importantly, recent CART clinical data has highlighted the importance of CART cell persistence, particularly within the CD4+ compartment, to mediate effective long-term, leukemia- free survival. To investigate the etiology of improved leukemia clearance, leukemia dosing was repeated but a cohort of mice was sacrificed at either Day 3 or Day 5-7 (one week) post-CART injection (Fig. 13A). Recipients were injected with BrdU just prior to harvest to measure the active proliferation of previously transferred CART cells. T cells were enumerated from the bone marrow, where NALM6 leukemia cells first expand, as well as the spleen. There were no differences in proliferation of 991 -treated CART cells from the bone marrow at either timepoint (Fig. 13B), but a transient proliferative increase in DMSO CART cells in recipient spleens on Day 3 that was gone by one week (Fig. 13C) was noted. Importantly, recipients of either DMSO- or 991 -treated CART cells demonstrated no evidence of active leukemia in the bone marrow at either day 3 or one week (Fig. 13A). Despite increased BrdU positivity in splenic DMSO CARTs on Day 3, there was no difference in total DMSO-treated T cell numbers in the spleen or bone marrow at this time or at one week (Fig. 13D). In contrast, there was a significant increase in CD4+ 991 -treated CART cells in both the bone marrow and spleen by one week (Fig. 13E) and this CD4+ T cell advantage drove a notable elevation in the CD4/CD8 ratio, which increased further at the two-week time point (Fig. 13F). Importantly, there were no pre-injection differences in the CD4/CD8 ratios of DMSO- versus 991-treated CART products (Fig. 17A). Together, these data suggest that the increased leukemia control and subsequent effective survival in mice receiving 991 CART cells correlates with improved persistence of CART cells within the CD4+ compartment.

Example 10

Discussion: A lack of CART cell persistence limits their ability to function as an effective curative therapy. It is also well documented that driving ex vivo CART expansion in the presence of abundant nutrients reduces their functional ability upon in vivo transfer. However, engineering nutrient deficient media can be costly, limiting clinical translation. Meanwhile, metabolic rewiring via promotion of mitochondrial biogenesis and FAO, while reducing ROS production, are promising interventions to improve CART function, adding yet another layer of complexity to CART manufacturing. Ultimately, the ability to generate multiple metabolic changes with one treatment could create both a greater in vivo advantage and simultaneously reduce disruptions to current CART protocols. The data herein suggests that reinforcing AMPK signaling, via treatment with the direct agonist Compound 991, can achieve these goals. Compound 991 is a commercially available agonist which binds directly to the AMPK heterotrimer. A treatment protocol has been optimized which allows for maximal metabolic benefit without significantly restricting T cell expansion, a notable limitation of other methods. T cells expanded in 991 gain mitochondrial capacity, a trait which continues upon re-stimulation and without additional agonist treatment. This gain in mitochondrial capacity correlates with increased mitochondrial density, likely occurring through mechanisms downstream of the transcription factor PGCla. Impressively, despite increased mitochondrial activity in 991-treated T cells, ROS abundance was regularly reduced following restimulation, suggesting these cells also have heightened redox capacity.

Interestingly, despite the dramatic functional improvement, no consistent changes were found in the differentiation status of 991-treated T cells, including just prior to injection. This was surprising, in part because a different model of AMPK activation promoted formation of T cell central memory populations. These current studies, however, did uncover upregulation of both metabolic and cell cycle gene sets through GSEA. Coupled with the in vivo data that 991- pretreatment improves CART persistence, particularly of CD4+ cells, the results together suggest that the underlying metabolic phenotype and capacity, rather than T cell differentiation status, is the major determinant of long-term survival in the agonist-treated model presented herein. And while much debate has surrounded the role of memory T cells to prevent metabolic and functional exhaustion, the data herein, support a model where metabolic and functional optimization occurs independently from changes in memory phenotype.

Even given the variability of using T cells from random human donors, the metabolic changes occurring following 991 treatment were dramatic. AMPK normally signals in the setting of nutrient starvation and 991 treatment activated a metabolic program consistent with nutrient starvation despite adequate levels of nutrients in the media. This surprisingly led to reduced glucose uptake but intracellular lactate retention, with cells relying on intracellular sources of sugar carbons for the glycolysis they were pursuing. In line with intracellular nutrient utilization, agonist treatment encouraged autophagy while reducing energy expended through protein translation, adaptations implicated as beneficial for long-term cellular fitness. 991-treated cells also upregulated FAO, consistent with the binding pocket for Compound 991 on AMPK being the docking site for LC-FAs to increase AMPK-driven activity. Importantly, alongside increases in FAO, mitochondrial biogenesis, and autophagy, agonist treatment also simultaneously generated metabolites important for mitochondrial health and redox buffering. Indeed, glutamate, aspartate, and threonine all contribute to the generation of proline and glycine, the latter being products which have been highlighted in multiple models as supporting mitochondrial function and longevity. Many of these metabolites are also critical for other metabolic pathways, including the tricarboxylic acid cycle and nucleotide synthesis. In fact, redirecting energy expenditure towards nucleotide synthesis during amino acid restriction has been implicated in fueling the subsequent increase in proliferative burst observed following restoration of nutrient levels.

With an optimized metabolic profile, it was hypothesized that 991-treated CARTs would demonstrate both increased proliferative capacity and enhanced in vivo persistence. Quantification of proliferation at two early times post-injection identified no proliferative advantage to 991-treated CARTs in either the BM or spleen. There was a trend towards increased 991-treated CART cell numbers in the bone marrow on Day 3, but this difference did not reach statistical significance. Meanwhile, a surprising but transient increase in DMSO-treated CART proliferation occurred at 72 hours post-injection, but which was gone by one week. It is possible that leukemia clearance was slower in mice receiving DMSO-treated CART cells, allowing for the capture of the final proliferative expansion of a small population of short-lived effectors cells in the spleens of these animals. In support of this interpretation, the increased proliferation at 72 hours did not translate to numerically more DMSO-treated CART cells at one week, suggesting that any actively proliferating cells at 72 hours were short-lived. The far more interesting finding became evident between days 5 and 7, where significantly more 991-treated CD4+ CART cells were recovered from both the bone marrow and spleen. These increased numbers, in the absence of proliferative advantages, strongly suggest that 991 in vitro treatment increases the resiliency of T cells at early times post-transfer, allowing them to persist longer in vivo. Further, the widening CD4/CD8 ratio, which became more dramatic over time, further implies that human CD4 T cells were the subtype most positively impacted by agonist pretreatment. Recent clinical data from long-term survivors, highlighting persistence of the CD4+ compartment as a critical factor for effective cure, underscores the importance of these exciting results.

Altogether, it was demonstrated that expanding CARTs in the presence of the direct AMPK agonist Compound 991 metabolically reprograms them, both by encouraging cellular starvation pathways without actually starving the cells and promoting FAO alongside augmentations in mitochondrial health and capacity. CARTs resulting from this process demonstrate an impressive increase in their metabolic capabilities, translating to improved in vivo persistence, particularly of donor CD4+ cells. Thus, it was concluded that addition of Compound 991 to currently utilized culture methods represents an easily translatable intervention to metabolically optimize human T cells, creating products with the improved capacity to serve as an effective curative therapy.

Claims

CLAIMS What is claimed is:

1. A method of making a T cell having an increased immune function as compared to a control comprising administering to the T cell a composition comprising a compound 991.

2. The method of claim 1, wherein the increased immune function comprises increased in vivo persistence of the T cell.

3. The method of claim 1 or claim 2, wherein the compound 991 has the structural formula shown below or a pharmaceutically acceptable salt thereof

4. The method of any one of claims 1-3, wherein the compound 991 is administered to the T cell during an in vitro T cell expansion period.

5. The method of claim 4, wherein the compound 991 is administered to the T cell at least two times during the in vitro T cell expansion period.

6. The method of claim 5, wherein the compound 991 is administered to the T cell about 4 days to about 6 days after onset of the in vitro T cell expansion period and again about 6 days to about 8 days after onset of the in vitro T cell expansion period.

7. The method of claim 5, wherein the compound 991 is administered to the T cell about 5 days after onset of the in vitro T cell expansion period and again about 7 days after onset of the in vitro T cell expansion period.

8. The method of any one of claims 5-7, wherein the T cell is within a culture medium and all or substantially all of the compound 991 in the culture medium is removed prior to each subsequent administration of the compound 991.

9. The method of any one of claims 1-8, wherein the compound 991 is administered at a molar concentration of between about 0.5 pM and about 100 M.

10. The method of any one of claims 1-9, wherein the compound 991 is administered at a molar concentration of between about 5 pM and about 50 pM.

11. The method of any one of claims 1-10, wherein the compound 991 is administered at a molar concentration of between about 5 pM and about 30 pM.

12. The method of any one of claims 1-11, wherein the compound 991 is administered at a molar concentration of between about 10 pM and about 25 pM.

13. The method of any one of claims 1-12, further comprising administering IL-2 to the T cell.

14. The method of any one of claims 1-13, wherein the compound 991 is first administered about four to about six days after onset of the in vitro T cell expansion period.

15. The method of any one of claims 1-14, wherein the compound 991 is first administered about five days after onset of the in vitro T cell expansion period.

16. The method of any one of claims 1- 15, wherein the T cell is a CD4⁺ T cell.

17. The method of any one of claims 1- 16, wherein the T cell is a CAR T cell, an effector T cell, an effector memory T cell, a TEMRA, a central memory T cell, an regulatory T cell (Treg), an effector memory Treg, a tumor-infiltrating lymphocyte (TIL) T cell, a cytotoxic T cell (CTL), a natural killer T-cell (NK T cell), a virus-specific T cell (VST) or a T memory stem cell (TSCM).

18. The method of any one of claims 1-17, wherein the T cell is a CAR T cell.

19. A method of increasing an immune response in a subject comprising administering to the subject a therapeutically effective amount of a compound 991 T cell.

20. A method of treating a cancer in a subject comprising administering to the subject a therapeutically effective amount of a compound 991 T cell.

21. The method of claim 19 or claim 20, wherein the T cell is made according to the method of any one of claims 1-18.

22. The method of any one of claims 19-21, wherein the subject is a human.

23. The method of claim 22, wherein the T cell is obtained from the human.

24. The method of any one of claims 20-23, wherein the cancer is a hematological cancer.

25. The method of any one of claims 20-23, where the cancer is a leukemia.