FITNESS FOR IMMUNOTHERAPY

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No. 62/354,338 filed June

24, 2016, herein incorporated by reference.

FIELD

The present disclosure provides recombinant T cells that have increased expression of PGCla, TFAM, GABPA, and ERRa, and methods of their use, for example in the treatment of a tumor. Also provided are kits and compositions that can be used with such methods.

BACKGROUND

The immune system has evolved multiple cellular mechanisms for the detection and elimination of abnormal or stressed cells in a wide array of environments. Early detection of cancer, via immunosurveillance, can occur almost anywhere, facilitating destruction of early transformed cells expressing neoantigen. However, as cancers immunoedit and escape this initial immune detection, they also generate an extraordinarily immunosuppressive microenvironment which restricts T cell infiltration, activation, and effector function both through direct repression (via cytokines, adenosine, prostaglandins, glucose restriction, etc.) as well as the recruitment of immunosuppressive populations tasked with maintaining immune tolerance (Jiang et al., 2015). The result is an ineffectual antitumor immune response and consequent tumor progression.

Recent advances in cancer immunotherapy have revealed that the T cell response to cancer can be reinvigorated in a variety of ways, resulting in durable and effective benefit in a wide array of cancer types (La-Beck et al., 2015; Mahoney et al., 2015; Ribas, 2015). These include engineering chimeric antigen receptors to better target T cells to tumor antigens, personalized antigen vaccines to persistent neoepitopes, and, probably most prominently, antibody-mediated blockade of co-inhibitory 'checkpoint' molecules, like programmed death-1 (PD-1), cytotoxic T lymphocyte antigen 4 (CTLA-4), lymphocyte activation gene 3 (LAG-3), T cell immunoglobulin and mucin-containing gene 3 (Tim-3), among others (La-Beck et al., 2015). These molecules are highly upregulated on tumor-infiltrating T cells and are thought to act to negatively regulate T cell activation and effector function. This elevated and sustained expression of co-inhibitory molecules is indicative of a hyporesponsive phenotype, originally discovered in chronic viral infection, termed T cell 'exhaustion' (Wherry and Kurachi, 2015). Thought to occur from chronic activation and inflammation, antigen persistence results in continued TCR and cytokine signals, which promote upregulation of these receptors, resulting a hyporesponsiveness functionally similar to anergy but mechanistically distinct (Crespo et al., 2013; Schietinger and Greenberg, 2014). Importantly, T cells can still have an exhausted phenotype in the absence of co-inhibitory molecules (Legat et al., 2013; Odorizzi et al., 2015), shedding light on the fact that while these co-inhibitory molecules have been extensively studied at the molecular and biochemical levels, it is still unclear what the contribution of co-inhibitory molecule signaling is to the initiation and/or maintenance of the exhausted phenotype. Thus for improving the treatment of cancer, chronic viral infections, and other diseases, it is critical to understand the mechanisms behind the dysfunction in chronically activated T cells (Pauken and Wherry, 2015). This is especially important considering that, while checkpoint blockade has had remarkable success in the clinic, the majority of patients still do not respond to these therapies (La-Beck et al., 2015).

Carrying out effector function is a metabolically demanding process (Pearce et al., 2013). T cells must efficiently divide and replicate their genome very rapidly and with fidelity, synthesize high levels of cytokines, and deliver cytotoxic payload to target cells. Recent discoveries of T cells' dependence on nutrient sensing and availability and flux through various glycolytic pathways have shown that metabolism represents a key mechanism by which the immune system can be regulated (Delgoffe and Powell, 2015). They also suggest that the fate and function of T cells are intrinsically tied to their metabolism, and that a T cell (like any other cell) requires the machinery to generate bioenergetic intermediates to support proliferation and effector function (Delgoffe and Powell, 2015).

T cells utilize aerobic glycolysis, diverting glucose into cytosolic lactate fermentation rather than mitochondrial acetyl-CoA oxidation to support their expansion and proliferation during their effector phase (Pearce et al., 2013; Roos and Loos, 1970). The precise contributions of this pathway and teleologic reasoning for its function remain the subject of much study, but nevertheless the mitochondria remain an essential component of T cell metabolism. Effector T cells significantly upregulate oxidative phosphorylation activity and memory T cell precursors become increasingly dependent on mitochondria to mediate fatty acid oxidation over time (van der Windt et al., 2012; van der Windt et al., 2013). Furthermore, the mitochondria remain important organelles for biosynthesis, calcium buffering, and mediating programmed cell death (Rizzuto et al., 2012;

Wenner, 2012). Thus, while T cells may divert glucose metabolism away from mitochondrial pathways during activation, mitochondria are still critical for maintaining the health and integrity of the T cell in both effector and memory phases. While the effects of glucose deprivation in tumor microenvironments on glycolytic metabolism and T cell function have garnered much recent interest, the mitochondrial phenotype of T cells infiltrating tumors remains unclear (Chang et al., 2015; Ho et al., 2015; Siska and Rathmell, 2015; Zhao et al., 2015).

SUMMARY

It is shown herein that T cells infiltrating solid tumors display an overall phenotype of metabolic insufficiency, characterized most prominently by a crippling loss of mitochondrial function and mass. Loss of mitochondrial function in tumor-reactive T cells occurs specifically in the tumor microenvironment, largely independently of PD-1 blockade, and due to a defect in

PPAR-gamma coactivator la (PGC la) -programmed mitochondrial biogenesis. This defect is due in part to chronic Akt signaling, which represses Foxo transcription factor activity and consequent PGC la expression. Metabolic reprogramming of T cells through enforced PGC la expression rescues mitochondrial function, intratumoral T cell proliferation, and a superior antitumor response characterized by cytokine production and tumor control.

Provided herein are recombinant T cells that can be used to improve cellular

immunotherapy, such as chimeric antigen receptor (CAR) T cell therapy or T cell receptor (TCR) gene therapy. For example, the disclosure provides recombinant T cells containing a vector that encodes one or more of peroxisome proliferator- activated receptor (PPAR) gamma coactivator 1- alpha (PGC la), mitochondrial transcription factor A (Tfam), GA binding protein transcription factor alpha subunit GABPA), and estrogen-related receptor alpha (ERRa). Expression of the vector in the T cell results in increased expression of one or more of these genes, and thus increased activity of these proteins. In some examples, the PGC la, Tfam, GABPA, and/or ERRa expressed from the vector is a variant sequence that encodes a protein that is resistant to negative regulation. For example, the PGC la resistant to negative regulation may include a S572A mutation (based on the human sequence, S571A in mouse), the Tfam resistant to negative regulation may include a S55A, S56A, and/or S61A (see e.g., Lu et al. , Molecular Cell 49: 121-32, 2013) mutation, the GABPA resistant to negative regulation may include a T280E mutation (see e.g. , Jain and Jaiswal, /. Biol. Chem. 281 : 12132-42, 2006), and the ERRa resistant to negative regulation may include a S19A mutation(see e.g. , Tremblay et al. , Mol. Endocrinol. 22:570-84, 2008).

The recombinant T cell expressing PGC la, Tfam, GABPA, and/or ERRa may also include a chimeric antigen receptor (CAR), or a recombinant T cell receptor (TCR) (such as a TCR that targets the T cells to a tumor cells, such as WT1 for acute myeloid leukemia and chronic myeloid leukemia). The CAR or recombinant TCR can be expressed from the same or a different vector than the PGCla, Tfam, GABPA, and/or ERRa.

The vectors used to generate such recombinant T cells, such as a vector that includes a nucleic acid molecule encoding one or more of PGCla, Tfam, GABPA, and ERRa and a nucleic acid molecule encoding a CAR a recombinant TCR, are provided herein. Examples of vectors that can be used include viral vectors, such as a lentiviral vector or retrovirus.

Also provided are methods for producing such recombinant T cells, for example ex vivo. T cells used for such methods can be obtained from a subject having cancer that will later receive the generated recombinant T cells, or from a donor subject. Such methods can include introducing the vector encoding one or more of PGCla, Tfam, GABPA, and ERRa (which may also encode a CAR or TCR), into a T cell, thereby generating the recombinant T cell. In some examples, a separate vector encoding a CAR or TCR is introduced into the T cell, thereby generating the recombinant T cell. Such methods can further include culturing or growing the transformed recombinant T cells under conditions that permit expression of one or more of PGCla, Tfam, GABPA, and ERRa (and in some examples also a CAR or TCR). In some examples, the recombinant T cells are incubated or culture in the presence of interleukin 2 (IL-2), interleukin 15 (IL-15), and/or interleukin 7 (IL-7). In some examples, the recombinant T cells are cultured for a period of days or weeks (such as at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, or at least 6 weeks). In some examples, the method includes monitoring the recombinant T cells, for example determining the number of cells, determining or measuring the mitochondrial activity and/or mass of the recombinant T cells, and/or determining or measuring expression of one or more of PGCla, PD-1, LAG-3, Tim-3, PGCla, Tfam, GABPA, ERRa, CAR, and TCR.

The disclosed recombinant T cells can be used in cancer immunotherapy, for example to treat a tumor in vivo. Both solid and liquid tumors can be treated, such as a leukemia, lymphoma, colorectal cancer, melanoma, cervical cancer, lung cancer, ovarian cancer, bladder cancer, breast cancer, or head and neck cancer. For example, effective amount of the recombinant T cells are administered to the subject, thereby treating a tumor (such as a primary tumor and/or a metastasis) in the subject. In some examples, such subjects are also administered an effective amount of IL-2 to the subject before, after, or both before and after, administering the recombinant T cells. In some examples, the subject is administered an effective amount of nonmyeloablative chemotherapy or radiotherapy to deplete native lymphocytes prior to administering an effective amount of the disclosed recombinant T cells. In some examples, the subject is also treated with an effective amount of chemotherapy, radiation therapy, biologic therapy, or combinations thereof. Such methods can treat the tumor in the subject by reducing the volume or weight of the tumor, reducing the number of metastases, reducing the size or weight of a metastasis, or combinations thereof. In some examples the subject administered the disclosed recombinant T cells was previously treated unsuccessfully with a chemotherapy, radiation therapy, biologic therapy, or combinations thereof (e.g., the tumor in the subject did not significantly decrease in size or even increased in size, and/or metastasized). In some examples the subject has a tumor that was not responsive to a PD-1 antagonist or a PD-Ll antagonist (e.g., the tumor in the subject did not significantly decrease in size or even increased in size, and/or metastasized), such as an antibody that specifically binds PD-1 or PD-Ll, such as Atezolizumab, MPDL3280A, BNS-936558 (Nivolumab), Pembrolizumab, Pidilizumab, CT011, AMP-224, AMP-514, MEDI-0680, BMS-936559, BMS935559, MEDI-4736, MPDL-3280A, MSB-0010718C.

Also provided are compositions and kits that can be used with the disclosed methods. In some examples, the composition or kits include recombinant T cells and/or vectors disclosed herein, for example in combination with a pharmaceutically acceptable carrier. In one example, the kit, further includes one or more of a transfection reagent, culture medium, antibiotic, IL2, IL15, IL7, anti-CD3, and anti-CD28 (e.g., anti-CD3/anti-CD28 stimulatory beads). In some examples, in a kit, such reagents are present in a separate container.

The foregoing and other objects and features of the disclosure will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS FIGS. 1A-1E. (A) MitoTracker Deep Red FM, MitoTracker Green FM, and TMRE staining of CD8 ⁺ T cells from spleen and LNs. The indicated histogram represents T cells pre-incubated with 100-111 μΜ CCCP, which collapses membrane potential. Results from CCCP titration are tabulated below. (B) MitoTracker Green FM and intracellular VDAC staining of LN and TIL from dl4 B 16 tumors as in FIG. 1A. (C) MitoTracker Deep Red FM staining of naive (CD62L ^hi CD44 ^lD ), effector memory (CD62L ^lo CD44 ^hi ), and central memory (CD62L ^hi CD44 ^hi ) CD8 ⁺ T cells from LN and tumor-infiltrating compartments. (D) 2NBDG uptake and MitoTracker FM staining measurements from CD8 ⁺ and CD4 ⁺ T cells infiltrating day 18 MC38 tumors or (E) LLC tumors or from the nondraining or draining LN. Results represent 3 (A, B, D, E), or 6 (C) independent experiments. ** p < 0.01, **** p < 0.001 by unpaired (B) or paired (C) t test. Error bars indicate s.e.m.

FIGS. 2A-2E. Tumor-infiltrating CD8 ⁺ T cells display suppressed mitochondrial function and mass. (A), Representative flow cytogram of nondraining (ndLN), draining (dLN) lymph node, or tumor-infiltrating lymphocyte (TIL) preparations from C57/BL6 mice inoculated with B16 melanoma cells 12 d prior, gated on CD8 or CD4 as indicated. (B), Tabulated flow cytometric data from CD8 ⁺ T cells isolated from mice bearing the indicated tumor types. Each circle represents an individual animal. (C), Transmission electron microscopy of activated or tumor-infiltrating CD8 ⁺ T cells. (D) MitoTracker FM staining of CD8 ⁺ T cells from PBL or TIL of HNSCC patients. (E) OCR trace (left) and metabolic analysis panels (middle, right) from CD8 ⁺ T cells isolated from the indicated sites from B16-bearing animals. T cells activated 24 h with anti- CD3/anti-CD28 (Teff) are included as a control. Spare respiratory capacity is calculated as the difference between initial, basal OCR values and the maximal OCR values achieved after FCCP uncoupling. Data represent the mean or are representative of 3-5 independent experiments. *, p <0.05, ** p <0.01, *** p < 0.001 by unpaired t-test. Error bars indicate s.e.m.

FIGS. 3A-3D. Loss of mitochondrial function and mass is not simply a phenotype of robust activation in vivo. (A), Representative flow cytogram and tabulated results of 2NBDG uptake and MitoTracker FM measurements of OT-I T cells injected into B 16 ^OVA -bearing or VV ^OVA -infected, or uninfected congenically mismatched hosts for 6 d. Plots are gated on CD8 ⁺ T cells and the congenic allele marker (Thyl). (B) Seahorse measurements (OCR, SRC, and ECAR) from cells in A. (C) ATP measurements from purified donor cells from A. (D) Flow cytogram of glucose uptake and mitochondrial mass of OT-I (Thyl.l ^"1" ) T cells adoptively transferred into B16 ^OVA bearing mice for 6 days, isolated from either LN or tumor, then transferred into VV ^OVA - infected mice for 6 days. Flow cytogram depicts splenic CD8 ⁺ Thyl.l ⁺ cells. *, p <0.05, ** p <0.01, *** p < 0.001 by unpaired t-test. Results represent four (A-C) or three (D) experiments. Circles represent individual animals. UI=uninfected V ^OVA =VV ^OVA -infected (1 x 10 ⁶ PFU IP), spl = spleen. Error bars indicate s.e.m.

FIGS. 4A-4B. Tumor infiltrating T cells do not efficiently elaborate cytokines. (A) Cytokine production of OT-I T cells injected as in FIG. 3. Some were rechallenged with peptide overnight and others were stimulated with PMA/ionomycin, as tabulated below from TIL or spleen of VV ^OVA infected mice. (B) VDAC staining of OT-I T cells transferred as in A. Error bars indicate s.e.m.

FIGS. 5A-5E. T cell mitochondrial dysfunction is induced upon entry into the tumor microenvironment. (A) Representative flow cytogram and tabulated data of LN and TIL of naive, CTV-labeled, OT-I (Thyl.l ⁺ ) CD8 ⁺ T cells transferred into B 16 ^OVA -bearing mice (5-7mm tumors), or the spleens of the same progenitor cells transferred into B6 mice infected with 1 x 10 ⁶ PFU yyov ^A _{for 72} ft. Cells were stained with MitoTracker Deep Red FM. (B) As in A, but with the mitochondrial membrane potential dye TMRE. (C) As in A, but with the cellular ROS indicator DCFDA. (D) As in A, but cells were permeabilized and stained intracellularly for LC3B. (E)

Representative data from experiments as in A, but some mice received mitophagy inhibitor m-divi- 1. Results represent the mean of three or four independent experiments, with n=7-9 mice per group. ** p <0.01, *** p < 0.001, *** p < 0.0001 by two-way ANOVA. Error bars indicate s.e.m.

FIGS. 6A-6B. OT-I T cells do not lose mitochondrial activity when

activated by tumor cells in vitro. (A) Flow cytogram and tabulated data of CTV-labeled OT-I splenocytes cocultured with either 25 ng/niL SIINFEKL peptide or in a 1:4 ratio with B 16 ^OVA cells, seeded 8 h prior to coculture, in the presence of 10 U/mL IL-2. (B) As in A, but using previously activated, purified effector OT-I T cells. Results represent the mean of 3 (of 8) independent experiments. Error bars indicate s.e.m.

FIGS. 7A-7G. Mitochondrial dysfunction in intratumoral T cells is progressive and correlates with coinhibitory molecule expression in mouse and human tumors. (A),

Representative flow cytogram of CD8 ⁺ T cells isolated from dl4 B 16-bearing C57/BL6 mice. (B) Flow cytogram showing mitochondrial mass and glucose competency of CD8 ⁺ T cell subsets. (C,D) Tabulated data from B. (E) ATP measurements from CD8 ⁺ T cells sorted directly ex vivo from tumors based on the indicated expression. Results are compared to LN CD8 ⁺ CD44 ^hl cells (Teff). (F) Cytogram of coinhibitory molecules and (G) mitochondrial/glucose status of CD8 ⁺ cells from PBMC or TIL from HNSCC patients. Data represent the mean or are representative of 3-5 independent experiments. *, p <0.05, ** p <0.01, *** p < 0.001 by unpaired t-test. Error bars indicate s.e.m.

FIGS. 8A-8D. Mitochondrial mass loss is a characteristic of exhausted T cells. (A)

VDAC staining of LN or TIL CD8 ⁺ T cells from B 16-bearing mice expressing co-inhibitory molecules PD-1 and LAG-3. (B) Co-inhibitory molecule expression on CD8 ⁺ and CD4 ⁺ T cells that are LN-resident or infiltrating MC38 or (C) LLC tumors on day 18 post inoculation (7-10 mm diameter tumors). (D) CD8 ⁺ T cells were sorted flow cytometrically from B 16-bearing animals based on MitoFM staining and washed extensively. Cells were then stimulated with anti-CD3/anti- CD28 for 6 h in the presence of a protein transport inhibitor, and then stained intracellularly for cytokines. Results for IFNy staining are tabulated to the right. Results represent the mean of four independent experiments. Error bars indicate s.e.m.

FIGS. 9A-9D. PD-1 blockade does not rescue metabolic dysfunction in intratumoral T cells. (A) Representative flow cytogram of CD8 ⁺ T cells from LN and TIL preparations in B 16- bearing mice receiving thrice- weekly injections of anti-PDl or its isotype control. (B) Tabulated results from A as well as MC38-bearing mice. Each dot represents a mouse in this experiment. (C) Percentage of mice experiencing tumor regression in several experiments conducted as in B. (D) Flow cytogram and tabulated values of MitoTracker FM staining during cell division of OT-I T cells transferred into established B16 ^OVA tumors under the cover of anti-PDl or its isotype control. Data are representative of five (A,B) or three (C,D) of independent experiments. Error bars indicate s.e.m.

FIGS. lOA-lOC. Regulatory T cells do not mediate mitochondrial dysfunction in tumor -infiltrating CD8 ⁺ T cells (A), Flow cytogram of CD4 ⁺ T cells from B16-bearing

Foxp3 ^{GFP Cre ERT2} or Foxp3 ^{DTR GFP} mice treated for 3 days with diphtheria toxin. (B), Representative flow cytogram and tabulated data of MitoTracker FM staining in CD8 ⁺ T cells from mice in A. (C), Flow cytogram and tabulated data of CellTrace Violet labeled CD8 ⁺ T cells stimulated with anti- CD3 and APCs in the presence or absence of flow-cytometrically purified T _reg cells (CD4 ⁺ GFP ⁺ cells from a Foxp3 ^GRP mouse). Results are representative of (A), or represent the mean of (B, C) two of three independent experiments. Error bars indicate s.e.m.

FIGS. 11A-11H. Intratumoral T cell mitochondrial biogenesis is repressed by chronic Akt-mediated repression of PGCla (A) Flow cytogram (left) and tabulated data (right) of PGCla intracellular staining in CD8 ⁺ T cells isolated from nondraining or draining LNs or TIL

preparations from B 16 or MC38 bearing mice. Shaded histogram indicates isotype control. (B) Flow cytogram of PGCla expression in CTV-labeled, naive OT-I T cells adoptively transferred into B16 ^OVA bearing mice for 72h. Tabulation for multiple experiments is to the right. (C) Flow cytogram of cytokine production of congenically mismitached WT OT-I T cells transferred into B 16 ^OVA -bearing or VV ^OVA -infected mice for 96 h, then restimulated with SIINFEKL peptide. (D) Representative and tabulated phospho-Akt (S473) and phospho-Foxol(T24)/3a(T32) staining of the indicated cell populations in mice bearing 14-day B16 tumors. MFI is reported. (E) Representative flow cytogram and tabulated data indicating PGCla staining in pAkt low or high cells. (F) MFI of pAkt staining in naive OT-I T cells, or OT-I T cells transferred for 3 or 6 days into a B16 ^OVA - bearing or VV ^OVA infected mouse. (G) PGCla levels and (H) mitochondrial mass of CD8 ⁺ T cells from LN and TIL of 14-day B16-bearing mice treated for 60 h with Akt inhibitor VIII or its vehicle. Results are representative of 5 (A,B, D) 3 (C, E,G, H) or 2 (F) independent experiments. *, p < 0.05, ** p <0.01, *** p < 0.001 by unpaired t-test (A, F, G, H) or paired t-test (D, E, G, H). *** p <0.001 by two-way ANOVA. Error bars indicate s.e.m.

FIGS. 12A-12G. PGCla is repressed progressively upon entry into the tumor microenvironment. (A) qPCR analysis of Ppargcla (encoding PGCla) from CD8 ⁺ T cells sorted from the indicated compartments based on co-inhibitory molecule expression from day 14 B16 tumors. Results are normalized to cyclophilin B expression and scaled to LN-resident CD4 ⁺ T cells. (B) PGCla MFI (per division) of congenically mismatched, CTV-labeled OT-I T cells transferred into B16 ^OVA bearing mice for 72 h under the cover of anti-PDl treatment or its isotype control. (C) of MitoFM staining of CD8 ⁺ T cells isolated from Ppargcl a ^m Cd4 ^Cre mice or Cd4 ^Cre controls after in vitro expansion. (D) Metabolic flux measurements (Seahorse) from the cells in C. (E)

Representative flow cytogram and tabulated results of PGCla versus T-bet and (F) Ki67 staining. (G) Representative flow cytogram depicting phospho-Akt (S473) activation as a function of PD-1 status. Results are representative of three (A, C, E, F), five (B, G), or two (D) independent experiments. * p <0.05, **** p <0.0001 by unpaired (C) or paired (E,F) t test. Error bars indicate s.e.m.

FIGS. 13A-13E. Bolstering mitochondrial biogenesis improves intratumoral T cell function. (A) Metabolic analysis of OT-I T cells retrovirally transduced with an empty mCherry vector or one encoding PGCla. MitoTracker FM staining at various timepoints post transduction is indicated. OCR, SRC, and ECAR values are from day 5-7 post transduction. (B) Representative flow cytogram of LN- and TIL-resident OT-I T cells transduced as in A and transferred into B16 ^OVA bearing C57/BL6 mice. Proportion of the transferred cells in LN and TIL and tabulated MitoTracker FM staining is reported. (C) Flow cytogram depicting cytokine synthesis in OT-I T cells transferred as in b and restimulated directly ex vivo with cognate peptide. Results are tabulated to the right. (D) Tumor growth plot of B16 ^OVA bearing mice treated therapeutically upon detection of palpable tumors on day 7 with 250,000 (<4mm ² ) or 500,000 (>4mm ² ) of PGCla or control- expressing cells (E) Survival plot from e. n= 15-17 mice per group. Results represent 6 (A-B), 4 (C), or 3 (D,E) independent experiments. ** p < 0.01, *** p < 0.001 by unpaired t-test (A-C), two- way ANOVA with repeated measures (D), or log-rank test (E). Error bars indicate s.e.m.

FIGS. 14A-14C. Enforced PGCla expression results in general increases in mitochondrial function. (A) Fuel usage test of PGCla- or EV-expressing OT-I T cells. Cells were uncoupled with FCCP and then subjected to sequential inhibition of pyruvate oxidation (UK5099), fatty acid oxidation (etomoxir), and glutaminolysis (BPTES). Results are displayed as % of FCCP- uncoupled OCR sensitive to the appropriate inhibitor. (B) IFNy and TNFa production of EV or PGCla expressing T cells prior to adoptive transfer. (C) Expression of PD-1 and LAG-3 on LN or TIL-resident reprogrammed T cells. Results represent the mean of three (A, B) or are representative of five (C) independent experiments. Error bars indicate s.e.m.

SEQUENCE LISTING SEQ ID NOS: 1 and 2: Exemplary human ERRa nucleic acid and protein sequence, respectively (see GenBank® Accession Nos. NM_004451.4 and NP_004442.3, respectively). SEQ ID NOS: 3 and 4: Exemplary human GABPA nucleic acid and protein sequence, respectively (see GenBank® Accession Nos. NM_001197297.1 and NP_001184226.1, respectively).

SEQ ID NOS: 5 and 6: Exemplary human PGCla nucleic acid and protein sequence, respectively (see GenBank® Accession Nos. NM_001330751.1 and NP_001317680.1, respectively).

SEQ ID NOS: 7 and 8: Exemplary human Tfam nucleic acid and protein sequence, respectively (see GenBank® Accession Nos. NM_001270782.1 and NP_001257711.1, respectively).

DETAILED DESCRIPTION

Unless otherwise noted, technical terms are used according to conventional usage.

Definitions of common terms in molecular biology can be found in Benjamin Lewin, Genes VII, published by Oxford University Press, 1999; Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994; and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995; and other similar references.

As used herein, the singular forms "a," "an," and "the," refer to both the singular as well as plural, unless the context clearly indicates otherwise. As used herein, the term "comprises" means "includes." Thus, "comprising a nucleic acid molecule" means "including a nucleic acid molecule" without excluding other elements. It is further to be understood that any and all base sizes given for nucleic acids are approximate, and are provided for descriptive purposes, unless otherwise indicated. Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described below. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. All references, including patent applications and patents, and sequences associated with the GenBank® Accession Numbers listed (as of June 24, 2016) are herein incorporated by reference.

In order to facilitate review of the various embodiments of the disclosure, the following explanations of specific terms are provided:

Administration: To provide or give a subject an agent, such as a disclosed recombinant T cell or other therapeutic agent, by any effective route. Exemplary routes of administration include, but are not limited to, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, intratumoral, and intravenous), transdermal, intranasal, and inhalation routes. Cancer: A malignant tumor characterized by abnormal or uncontrolled cell growth. Other features often associated with cancer include metastasis, interference with the normal functioning of neighboring cells, release of cytokines or other secretory products at abnormal levels and suppression or aggravation of inflammatory or immunological response, invasion of surrounding or distant tissues or organs, such as lymph nodes, etc. "Metastatic disease" refers to cancer cells that have left the original tumor site and migrate to other parts of the body for example via the bloodstream or lymph system.

Chimeric antigen receptor (CAR): Artificial, engineered T cell receptors, which graft an arbitrary specificity onto an immune effector cell. Typically, these receptors are used to graft the specificity of a monoclonal antibody onto a T cell; with transfer of their coding sequence facilitated by vectors. Using adoptive cell transfer, CARs can be useful to treating cancer. For example, T cells (obtained from the patient or from a donor) are modified so that they express receptors specific to the patient's particular cancer. The modified T cells, which can then recognize and kill the cancer cells, are introduced into the patient. First generation CARs typically included the intracellular domain from the CD3 ζ- chain, which is the primary transmitter of signals from endogenous TCRs. Second generation CARs added intracellular signaling domains from various costimulatory protein receptors (e.g., CD28, 41BB, ICOS) to the cytoplasmic tail of the CAR to provide additional signals to the T cell. Third generation CARs combine multiple signaling domains, such as CD3z-CD28-41BB or CD3z-CD28-OX40, to augment potency. Any of these CARs can be used herein.

Contact: Placement in direct physical association, including a solid or a liquid form. Contacting can occur in vitro or ex vivo, for example, by adding a reagent to a sample (such as one containing recombinant T cells), or in vivo by administering to a subject.

Effective amount: The amount of an agent (such as recombinant T cells disclosed herein) that is sufficient to effect beneficial or desired results.

A effective amount (also referred to as a therapeutically effective amount) may vary depending upon one or more of: the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art. The beneficial therapeutic effect can include enablement of diagnostic determinations; amelioration of a disease, symptom, disorder, or pathological condition; reducing or preventing the onset of a disease, symptom, disorder or condition; and generally counteracting a disease, symptom, disorder or pathological condition. In one embodiment, an "effective amount" (e.g., of recombinant T cells disclosed herein) is an amount sufficient to reduce the volume/size of a tumor, the weight of a tumor, the number of metastases, reduce the volume/size of a metastasis, the weight of a metastasis, or combinations thereof, for example by at least 10%, at least 20%, at least 25%, at least 50%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or at least 99% (as compared to no administration of the therapeutic agent). In one embodiment, an "effective amount" (e.g., of a vector encoding a target gene(s), such as TCR, CAR, PGCla, Tfam, GAB PA, and/or ERRa) is an amount sufficient to increase the activity and/or expression of the target gene(s) in an exhausted tumor-infiltrating T cell, for example by at least 10%, at least 20%, at least 25%, at least 50%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 99%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, or at least 600% (as compared to no administration of the therapeutic agent).

Estrogen-related receptor alpha (ERRa): (e.g., OMIM 601998): Also known as NR3B 1, is an orphan nuclear receptor involved in the activation of mitochondrial genes and mitochondrial biogenesis. ERRa sequences are publically available, for example from the GenBank® sequence database (e.g., Accession Nos. NP_004442.3, P11474.3, AAH63795.2, JAA39845.1 , AAB51250.1 , AAQ17212.1 , and AAS20260.1 provide exemplary ERRa protein sequences, while Accession Nos. M 004451.4, NM 001282450.1, NM_001282451.1, NM_007953.2 and NM 001008511.2 provide exemplary ERRa nucleic acid sequences). One of ordinary skill in the art can identify additional ERRa nucleic acid and protein sequences, including ERRa variants, such as those having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, or at least 99% sequence identity to these GenBank® sequences. In some examples, a variant ERRa is one that is resistant to negative regulation. Examples of a mutation that can be made to ERRa to increase its resistance to negative regulation includes but is not limited to S 19A.

Increase or Decrease: A statistically significant positive or negative change, respectively, in quantity from a control value (such as a value representing no therapeutic agent). An increase is a positive change, such as an increase at least 50%, at least 100%, at least 200%, at least 300%, at least 400% or at least 500% as compared to the control value. A decrease is a negative change, such as a decrease of at least 20%, at least 25%, at least 50%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% decrease as compared to a control value. In some examples the decrease is less than 100%, such as a decrease of no more than 90%, no more than 95%, or no more than 99%.

Isolated: An "isolated" biological component (such as T cells, a nucleic acid molecule, or a protein) has been substantially separated, produced apart from, or purified away from other biological components in the cell or tissue of an organism in which the component occurs, such as other cells (e.g., RBCs), chromosomal and extrachromosomal DNA and RNA, and proteins. Nucleic acids and proteins that have been "isolated" include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids and proteins. Isolated T cells, or isolated recombinant T cells, in some examples are at least 50% pure, such as at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 100% pure.

Mitochondrial transcription factor A (Tfam or mtTFA): (e.g., OMIM 600438): A mitochondrial transcription factor involved in the activation of mitochondrial transcription and participates in mitochondrial genome replication. Tfam binds mitochondrial promoter DNA to aid transcription of the mitochondrial genome. Tfam sequences are publically available, for example from the GenBank® sequence database (e.g., Accession Nos. NP_003192.1, NP_001257711.1, P 033386.1, AAH62022.1, P 001123683.1, and NP_001029188.2 provide exemplary Tfam protein sequences, while Accession Nos. CR407653.1, NM 003201.2, NM 001270782.1, NM_009360.4 and NM_031326.1 provide exemplary Tfam nucleic acid sequences). One of ordinary skill in the art can identify additional Tfam nucleic acid and protein sequences, including Tfam variants, such as those having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, or at least 99% sequence identity to these GenBank® sequences. In some examples, a variant Tfam is one that is resistant to negative regulation. Examples of mutations that can be made to Tfam to increase its resistance to negative regulation include but are not limited to 55A, S56A, and S61A

GA binding protein transcription factor, alpha subunit (GABPA) (aka E4TF1-10,

Nft2, Nrf2, Rch04A07): (e.g., OMIM 600609): One of three GA-binding protein transcription factor subunits which functions as a DNA-binding subunit, likely involved in activation of cytochrome oxidase expression and nuclear control of mitochondrial function. GABPA sequences are publically available, for example from the GenBank® sequence database (e.g., Accession Nos. P 001184226.1, NM_008065.2, NP_001102311.1, and NP_001253514.1 provide exemplary GABPA protein sequences, while Accession Nos. NM_001197297.1, NP_032091.2,

NM_001108841.1, and NM_001266585.1 provide exemplary GABPA nucleic acid sequences). One of ordinary skill in the art can identify additional GABPA nucleic acid and protein sequences, including GABPA variants, such as those having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, or at least 99% sequence identity to these GenBank® sequences. In some examples, a variant GABPA is one that is resistant to negative regulation. An examples of a mutation that can be made to GABPA to increase its resistance to negative regulation includes but is not limited to T280E. Operably linked: A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence (such as a TCR, CAR, PGCla, Tfam, GABPA, or ERRa coding sequence). Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.

Programmed cell death protein 1 (PD-1 or CD279) (e.g., OMIM 600244): A cell surface receptor that belongs to the immunoglobulin superfamily and is expressed on T cells and pro-B cells. PD-1 binds two ligands, PD-L1 and PD-L2. The human form is a 268 aa type 1

transmembrane protein. PD-1 is an inhibitory receptor that mediates T-cell exhaustion. PD-1 sequences are publically available, for example from the GenBank® sequence database (e.g., Accession Nos. NP_005009.2 (mature peptide is aa 21-288), CAA48113.1, NP_001301026.1 (mature peptide is aa 25-288), and CAA48113.1 (mature peptide is aa 21-288) provide exemplary PD-1 protein sequences, while Accession Nos. L27440.1, NM_005018.2, X67914.1, AB898677.1 and EU295528.2 provide exemplary PD-1 nucleic acid sequences). One of ordinary skill in the art can identify additional PD-1 nucleic acid and protein sequences, including PD-1 variants, such as those having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, or at least 99% sequence identity to these GenBank® sequences.

Peroxisome proliferator-activated receptor (PPAR) gamma coactivator 1 -alpha (PGCla or PPARGCla): (e.g., OMIM 604517): A transcriptional coactivator that regulates the genes involved in energy metabolism. PGC-la is a regulator of mitochondrial biogenesis and function. PGC-la sequences are publically available, for example from the GenBank® sequence database (e.g., Accession Nos. NP_001317680.1, NP_037393.1, NP_032930.1, and NP_112637.1 provide exemplary PGC-la protein sequences, while Accession Nos. NM_001330751.1,

NM_013261.3, NM_008904.2, and NM_031347.1 provide exemplary PGC-la nucleic acid sequences). One of ordinary skill in the art can identify additional PGC-la nucleic acid and protein sequences, including PGC-la variants, such as those having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, or at least 99% sequence identity to these GenBank® sequences. In some examples, a variant PGC-la is one that is resistant to negative regulation. An examples of a mutation that can be made to PGC-la to increase its resistance to negative regulation includes but is not limited to S572A.

Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers useful in this invention are conventional. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975), describes compositions and formulations suitable for pharmaceutical delivery of a therapeutic agent, such as a vector or recombinant T cell disclosed herein.

In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

Promoter: An array of nucleic acid control sequences which direct transcription of a nucleic acid. A promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements which can be located as much as several thousand base pairs from the start site of transcription.

Examples of promoters include, but are not limited to the SV40 promoter, the CMV enhancer-promoter, and the CMV enhancer/ -actin promoter. Both constitutive and inducible promoters are included (see e.g. , Bitter et al. , Methods in Enzymology 153:516-544, 1987). Also included are those promoter elements which are sufficient to render promoter-dependent gene expression controllable for cell-type specific, tissue- specific, or inducible by external signals or agents; such elements may be located in the 5' or 3' regions of the gene. Promoters produced by recombinant DNA or synthetic techniques can also be used to provide for transcription of the nucleic acid sequences.

Recombinant: A recombinant nucleic acid molecule is one that has a sequence that is not naturally occurring (e.g., a mutated PGCla, Tfam, GABPA, or ERRa) or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination can be accomplished by routine methods, such as chemical synthesis or by the artificial manipulation of isolated segments of nucleic acids, such as by genetic engineering techniques. Similarly, a recombinant protein is one encoded for by a recombinant nucleic acid molecule. Similarly, a recombinant or transgenic cell is one that contains a recombinant nucleic acid molecule and expresses a recombinant protein.

Sequence identity of amino acid sequences: The similarity between amino acid (or nucleotide) sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologs of a polypeptide will possess a relatively high degree of sequence identity when aligned using standard methods.

Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, /. Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Set U.S.A. 85:2444, 1988; Higgins and Sharp, Gene 73:237, 1988; Higgins and Sharp, CABIOS 5: 151, 1989; Corpet et al., Nucleic Acids Research 16: 10881, 1988; and Pearson and Lipman, Proc. Natl. Acad. Set U.S.A. 85:2444, 1988. Altschul et al., Nature Genet. 6: 119, 1994, presents a detailed consideration of sequence alignment methods and homology calculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol.

215:403, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. A description of how to determine sequence identity using this program is available on the NCBI website on the internet.

Variants of a native PGCla, Tfam, GABPA, or ERRa protein or coding sequences (such as a protein (or coding sequence thereof) with increased resistance to negative regulation) are typically characterized by possession of at least about 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity counted over the full length alignment with the amino acid sequence using the NCBI Blast 2.0, gapped blastp set to default parameters. For comparisons of amino acid sequences of greater than about 30 amino acids, the Blast 2 sequences function is employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of 1). When aligning short peptides (fewer than around 30 amino acids), the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties). Proteins with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 95%, at least 98%, or at least 99% sequence identity. When less than the entire sequence is being compared for sequence identity, homologs and variants will typically possess at least 80% sequence identity over short windows of 10-20 amino acids, and may possess sequence identities of at least 85% or at least 90% or at least 95% depending on their similarity to the reference sequence. Methods for determining sequence identity over such short windows are available at the NCBI website on the internet. One of skill in the art will appreciate that these sequence identity ranges are provided for guidance only; it is entirely possible that strongly significant homologs could be obtained that fall outside of the ranges provided. Thus, a variant PGCla, Tfam, GAB PA, or ERRa protein or nucleic acid sequence can have at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to any of the sequences shown in the GenBank® Accession Nos. provided herein.

Subject: A vertebrate, such as a mammal, for example a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. In one

embodiment, the subject is a non-human mammalian subject, such as a monkey or other non- human primate, mouse, rat, rabbit, pig, goat, sheep, dog, cat, horse, or cow. In some examples, the subject has a tumor, such as a cancer, that can be treated using the recombinant T cells disclosed herein. In some examples, the subject is a laboratory animal/organism, such as a mouse, rabbit, or rat.

T cells: White blood cells containing a T cell receptor on their cell surface, which play a role in cell-mediated immunity. In some examples, a T cell is obtained from the peripheral blood of a mammalian subject (such as one having cancer), for example via leukapheresis.

T cell receptor (TCR): A receptor found on the surface of T lymphocytes (or T cells) responsible for recognizing fragments of antigen as peptides bound to major histocompatibility complex (MHC) molecules. The TCR is composed of two different protein chains/ In humans, in 95% of T cells the TCR consists of an alpha (a) and beta (β) chain, whereas in 5% of T cells the TCR consists of gamma and delta (γ/δ) chains. This ratio changes during ontogeny and in diseased states as well as in different species. When the TCR engages with antigenic peptide and MHC (peptide/MHC), the T lymphocyte is activated through signal transduction, that is, a series of biochemical events mediated by associated enzymes, co-receptors, specialized adaptor molecules, and activated or released transcription factors. In one example, a TCR is a recombinant TCR, such as one used in TCR-engineered T cells.

Therapeutic agent: Refers to one or more molecules or compounds that confer some beneficial effect upon administration to a subject. The beneficial therapeutic effect can include enablement of diagnostic determinations; amelioration of a disease, symptom, disorder, or pathological condition; reducing or preventing the onset of a disease, symptom, disorder or condition; and generally counteracting a disease, symptom, disorder or pathological condition.

Transduced and Transformed: A virus or vector "transduces" a cell when it transfers nucleic acid into the cell. A cell is "transformed" or "transfected" by a nucleic acid transduced into the cell when the nucleic acid molecule becomes stably replicated by the cell, either by

incorporation of the nucleic acid into the cellular genome, or by episomal replication. Numerous methods of transfection are known to those skilled in the art, such as: chemical methods (e.g., calcium-phosphate transfection), physical methods (e.g., electroporation, microinjection, particle bombardment), fusion (e.g., liposomes), receptor-mediated endocytosis (e.g., DNA-protein complexes, viral envelope/capsid-DNA complexes) and by biological infection by viruses such as recombinant viruses {Wolff, J. A., ed, Gene Therapeutics, Birkhauser, Boston, USA (1994)} . In the case of infection by retroviruses, the infecting retrovirus particles are absorbed by the target cells, resulting in reverse transcription of the retroviral RNA genome and integration of the resulting provirus into the cellular DNA.

Transgene: An exogenous gene supplied by a vector. In one example, a transgene includes one or more PGCla, Tfam, GABPA, or ERRa coding sequences, for example in combination with a CAR or TCR coding sequence.

Treating, Treatment, and Therapy: Any success or indicia of success in the attenuation or amelioration of an injury, pathology or condition, including any objective or subjective parameter such as abatement, remission, diminishing of symptoms or making the condition more tolerable to the patient, slowing in the rate of degeneration or decline, making the final point of degeneration less debilitating, improving a subject's physical or mental well-being, or prolonging the length of survival. The treatment may be assessed by objective or subjective parameters; including the results of a physical examination, blood and other clinical tests, and the like. In some examples, treatment with the disclosed methods results in a decrease in the number, volume, and/or weight of a tumor and/or metastases.

Tumor, neoplasia, malignancy or cancer: A neoplasm is an abnormal growth of tissue or cells which results from excessive cell division. Neoplastic growth can produce a tumor. The amount of a tumor in an individual is the "tumor burden" which can be measured as the number, volume, or weight of the tumor. A "non-cancerous tissue" is a tissue from the same organ wherein the malignant neoplasm formed, but does not have the characteristic pathology of the neoplasm. Generally, noncancerous tissue appears histologically normal. A "normal tissue" is tissue from an organ, wherein the organ is not affected by cancer or another disease or disorder of that organ. A "cancer-free" subject has not been diagnosed with a cancer of that organ and does not have detectable cancer.

Exemplary tumors, such as cancers, that can be treated using the disclosed recombinant T cells include solid tumors, such as breast carcinomas (e.g. lobular and duct carcinomas, such as a triple negative breast cancer), sarcomas, carcinomas of the lung (e.g., non-small cell carcinoma, large cell carcinoma, squamous carcinoma, and adenocarcinoma), mesothelioma of the lung, colorectal adenocarcinoma, stomach carcinoma, prostatic adenocarcinoma, ovarian carcinoma (such as serous cystadenocarcinoma and mucinous cystadenocarcinoma), ovarian germ cell tumors, testicular carcinomas and germ cell tumors, pancreatic adenocarcinoma, biliary adenocarcinoma, hepatocellular carcinoma, bladder carcinoma (including, for instance, transitional cell carcinoma, adenocarcinoma, and squamous carcinoma), renal cell adenocarcinoma, endometrial carcinomas (including, e.g., adenocarcinomas and mixed Mullerian tumors (carcinosarcomas)), carcinomas of the endocervix, ectocervix, and vagina (such as adenocarcinoma and squamous carcinoma of each of same), tumors of the skin (e.g., squamous cell carcinoma, basal cell carcinoma, malignant melanoma, skin appendage tumors, Kaposi sarcoma, cutaneous lymphoma, skin adnexal tumors and various types of sarcomas and Merkel cell carcinoma), esophageal carcinoma, carcinomas of the nasopharynx and oropharynx (including squamous carcinoma and adenocarcinomas of same), salivary gland carcinomas, brain and central nervous system tumors (including, for example, tumors of glial, neuronal, and meningeal origin), tumors of peripheral nerve, soft tissue sarcomas and sarcomas of bone and cartilage, head and neck squamous cell carcinoma, and lymphatic tumors (including B-cell and T- cell malignant lymphoma).. In one example, the tumor is an

adenocarcinoma.

The disclosed recombinant T cells can also be used to treat liquid tumors, such as a lymphatic, white blood cell, or other type of leukemia. In a specific example, the tumor treated is a tumor of the blood, such as a leukemia (for example acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), hairy cell leukemia (HCL), T-cell prolymphocytic leukemia (T-PLL), large granular lymphocytic leukemia, and adult T-cell leukemia), a lymphoma (such as Hodgkin's lymphoma or non-Hodgkin' s lymphoma), or a myeloma.

Under conditions sufficient for: A phrase that is used to describe any environment that permits a desired activity. In one example the desired activity is increased expression or activity of one or more PGCla, Tfam, GABPA, and ERRa, for example in a T cell. In one example the desired activity is treatment of a tumor in vivo, for example using the disclosed recombinant T cells.

Vector: A nucleic acid molecule as introduced into a host cell (such as a T cell), thereby producing a transformed host cell. A vector may include nucleic acid sequences that permit it to replicate in the host cell, such as an origin of replication. A vector may also include one or more PGCla, Tfam, GABPA, or ERRa coding sequences, for example in combination with a CAR or TCR coding sequence, and/or selectable marker genes and other genetic elements known in the art. A vector can transduce, transform or infect a cell, thereby causing the cell to express nucleic acids and/or proteins other than those native to the cell. A vector optionally includes materials to aid in achieving entry of the nucleic acid into the cell, such as a viral particle, liposome, protein coating or the like.

Overview

While tumor-specific T cells recognize cancer cells, they are often rendered dysfunctional due to an immunosuppressive microenvironment. As T cell activation is bioenergetically demanding, we hypothesized that T cell dysfunction in cancer is due in part to metabolic insufficiency caused by the dearth nutrient conditions of the tumor microenvironment. It is shown herein that T cells demonstrate a crippling loss of mitochondrial function and mass when infiltrating murine and human tumors . Persistent loss of mitochondrial function occurs specifically in the tumor microenvironment, and is not merely a product of robust activation. Tumor infiltrating T cells (TILs) show a progressive loss of PGCla, a transcriptional coactivator programming mitochondrial biogenesis, induced in part by chronic Akt signaling in tumor- specific CD8+ T cells. Reprogramming tumor-specific T cells through enforced expression of PGCla results in superior intratumoral metabolic and effector function. The data herein support a model in which signals present in the tumor microenvironment repress T cell oxidative metabolism, resulting in effector cells with metabolic needs that cannot be met. As such, the metabolic status of the tumor microenvironment may predict the response to immunotherapy. The results indicate that modulation or reprogramming of the altered metabolism of tumor-infiltrating T cells represents a potential strategy to reinvigorate dysfunctional T cells for the treatment of cancer.

The data herein place the persistence and function of mitochondria as central to sustained effector function of T cells, especially those under continual stimulation like in cancer or chronic viral infections. Observed most prominently in CD8 ⁺ T cells specifically in the tumor

microenvironment, we demonstrate a progressive and persistent loss of mitochondrial function and mass. Importantly, at least in the timecourse of rapidly growing tumor, this effect could be observed concomitantly with PD- 1 upregulation but largely independently of treatment with anti- PD-1, with only mild and temporary increases observed in response to of this particular checkpoint. Rather, Akt signaling associated with chronic activation results in repression of oxidative metabolism, thus driving an unsustainable metabolic program. Thus, the data herein bring to light the metabolic nature of T cell dysfunction, in cancer and in general.

While glucose metabolism, especially aerobic glycolysis, has been heavily studied as a key mediator of T cell effector fate and function, the results herein indicate that mitochondrial function and mass are dynamically regulated and required to maintain optimal effector function. This is consistent with data suggesting mitochondrial membrane potential may predict sternness in tumor- infiltrating T cells, and that cytokine production may be increased in T cells that display high mitochondrial activity (Sukumar et al., 2016).

There is some debate as to whether MitoTracker Deep Red FM is a potentiometric dye or one that stains for mitochondrial mass irrespective of membrane potential (Xiao et al., 2016). The data herein employing uncoupling agents such as CCCP indicate while Deep Red FM may show some sensitivity for membrane potential at high doses, this pales in comparison to results achieved with TMRE, a true potentiometric dye (FIG. 1A). The corollary data herein employing

MitoTracker Green and VDAC antibodies indeed confirm that T cells infiltrating tumors exhibit losses of mitochondrial function and total mass, consistent with repressed PGCl-mediated mitochondrial biogenesis. While effector T cells have been shown to possess fewer mitochondria than their memory counterparts (van der Windt et al., 2012; van der Windt et al., 2013), it is unclear how chronic stimulation might alter this fate. The data herein suggest that the continued, inflammatory activation of T cells in cancer promotes a persistent defect in mitochondrial biogenesis, mediated in part by Akt-controlled inhibition of Foxo-programmed PGCla

transcription. While Akt does repress oxidative metabolism, more traditional roles for Akt suggest its potential as an in vivo therapeutic target may be limited, as T cells may require Akt and downstream signaling in situ to mediate effector functions (Macintyre et al., 2011). However, recent studies have revealed that this may not be the case. Akt inhibition has been employed in preclinical and translational settings as a means to reinvigorate TIL expansion, in part through modulating oxidative metabolism (Crompton et al., 2015). In addition, T cells lacking the mammalian target of rapamycin complex 2, the kinase for the hydrophobic motif (S473) of Akt, show superior effector function and increased oxidative metabolism, suggesting that full Akt activation may not be acutely required for effector function (Pollizzi et al., 2015). Thus, the data herein identify PGCla as a crucial mechanistic link between Akt and repressed oxidative metabolism in tumor-infiltrating T cells. In addition, Akt inhibitors, which are currently being evaluated in clinical trials as anti-cancer agents, may have immunomodulatory effects that could be synergistic with immunotherapies.

PD-1 inhibits mTORCl signaling (which has metabolic consequences), and modulates metabolism at the genetic level (Wherry and Kurachi, 2015). However, we only observed association between PD-1 upregulation and mitochondrial insufficiency, with mild and short-lived effects on mitochondrial metabolism arising from anti-PD-1 treatment. In the tumor

microenvironment, there may be too many other signals to be offset by PD- 1 blockade alone, or that other co-inhibitory molecules like LAG-3 or TIGIT may play additional roles in inhibiting oxidative metabolism in the tumor microenvironment. Likewise, we fail to see any effect of regulatory T cell suppression on mitochondrial function and mass, both using a genetic model of regulatory T cell depletion, Foxp3 ^{DTR GFP} mice treated with diphtheria toxin in established tumors, as well as coculture of purified T _reg cells with activated CD8 ⁺ T cells.

The data herein point to PGCla as a key node of signal integration tying a diverse array of cellular signals (including Akt signaling) to mitochondrial biogenesis. Type I II interferons, TNF, IL-12, energy charge, and low NAD ⁺ /NADH or oleate/palmitate ratios have been shown to repress PGCla expression, localization or transcriptional activity through a variety of signaling pathways (Alvarez-Guardia et al., 2010; Haghikia et al., 2015; Kauppinen et al., 2013; Kim et al., 2007b; Palomer et al., 2009; Scarpulla, 2011). Indeed, PGCla is post-translationally modified by a number of signaling pathways important for T cell biology (Akt, p38-MAPK, AMPK, SIRT1, PRMT1) (Fernandez-Marcos and Auwerx, 2011). Thus, the balance of these signals in the inflammatory milieu may determine PGCla activity and its ability to program mitochondrial biogenesis. Furthermore, these results indicate that determining the metabolic status of tumor- infiltrating T cells as well as the presence of various PGCla repressors may predict responses to immunotherapeutic regimens like checkpoint blockade.

The results herein add to a growing number of reports that collectively suggest that the T cell dysfunction observed in chronic activation (T cell exhaustion) has underpinnings in basic cellular processes like metabolism. Unlike anergy (induced by minimal signaling, TCR ligation alone, in a non- inflammatory environment), persistent, inflammatory activation in cancer and chronic viral infections promote an effector state that T cells cannot sustain (Jiang et al., 2015; Schietinger and Greenberg, 2014; Wherry and Kurachi, 2015). This is especially perilous in the tumor microenvironment, as chronic inflammatory signals might drive a sustained reliance on glycolysis in a tissue site where glucose levels are extremely low (Siska and Rathmell, 2015). It was observed that T cells also show depressed glucose uptake compared to LN-resident cells (FIGS. 2A-2E) and glycolysis compared to matched, virus-activated cells (FIG. 3B), suggesting that, generally, T cell 'exhaustion' is characterized by metabolic insufficiency (Chang et al., 2015; Ho et al., 2015; Zhao et al., 2015). Teleologically, T cells in 'metabolic distress' might upregulate co-inhibitory molecules as a means to prevent terminal loss of metabolic sufficiency or survival, a model consistent with results obtained in chronic viral infection (Staron et al., 2014). These crippling metabolic defects can be persistent even when removed from that microenvironment (FIG. 3D) which may provide a potential explanation for situations in which concomitant tumor immunity is lost at distal sites.

Finally, the data herein support modulation and reprogramming of the metabolic state as a viable strategy for the improvement of immunotherapy for cancer. While advances in checkpoint blockade and other types of immunotherapy have revealed that the mechanisms blocking immune cell activation can be altered by therapeutic intervention, tumors present a harsh microenvironment that is immunosuppressive by its basic nature. Further, the data suggest that the metabolic status of individual tumor microenvironments, which can vary from model to model or patient to patient, may help predict the response to immunotherapies like checkpoint blockade. While these results demonstrate that the direct metabolic reprogramming of T cells can have efficacy, other strategies to remodel the metabolism of the microenvironment itself can be used to create a more permissive environment for T cell activity.

Metabolism is central to cellular function, so it is largely unsurprising that T cells fail in nutrient-poor conditions. These result have shed light on the fact that during chronic activation as in cancer, T cells are driven to proliferate and perform effector function at the expense of their continued persistence and longevity. Development of strategies to modify the bioenergetics of T cells or the tumor microenvironment itself has the promise to improve and synergize with other forms of immunotherapy to increase the efficacy of the treatment of cancer.

Recombinant T Cells

Provided herein are recombinant T cells that can be used to improve cellular

immunotherapy, such as chimeric antigen receptor (CAR) T cell therapy or T cell receptor (TCR)- engineered T cells. For example, the disclosure provides recombinant T cells containing a vector that encodes one or more of peroxisome proliferator-activated receptor (PPAR) gamma coactivator 1-alpha (PGCla), mitochondrial transcription factor A (Tfam), GA binding protein transcription factor alpha subunit (GABPA), and estrogen-related receptor alpha (ERRa). Expression of the vector in the T cell results in increased expression of one or more of these genes, and thus increased activity of these proteins. In some examples, the PGCla, Tfam, GABPA, and/or ERRa expressed from the vector is a variant sequence that encodes a protein that is resistant to negative regulation.

The recombinant T cell expressing PGCla, Tfam, GABPA, and/or ERRa may also include a chimeric antigen receptor (CAR), or a recombinant T cell receptor (TCR) (such as a TCR that targets the T cells to a tumor cells, such as WT1 for acute myeloid leukemia and chronic myeloid leukemia). The CAR or recombinant TCR can be expressed from the same or a different vector than the PGC 1 a, Tfam, GABPA, and/or ERRa.

Thus, in some examples, expression of PGCla in the T cell increases PGCla protein expression and/or activity in the recombinant T cells by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500% or at least 600%. In some examples, expression of Tfam in the T cell increases Tfam protein expression and/or activity in the recombinant T cells by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500% or at least 600%. In some examples, expression of GABPA in the T cell increases GABPA protein expression and/or activity in the recombinant T cells by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500% or at least 600%. In some examples, expression of ERRa in the T cell increases ERRa protein expression and/or activity in the recombinant T cells by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500% or at least 600%.

In some examples, expressing PGCla, Tfam, GABPA, and/or ERRa in the recombinant T cells increases mitochondrial activity (e.g., oxidative metabolism) in the recombinant T cells by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500% or at least 600%. In some examples, expressing PGCla, Tfam, GABPA, and/or ERRa in the recombinant T cells increases mitochondrial mass in the recombinant T cells by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500% or at least 600%. In some examples, combinations of these effects are achieved.

The vectors used to generate such recombinant T cells, such as a vector that includes a nucleic acid molecule encoding one or more of PGCla, Tfam, GABPA, and ERRa and a nucleic acid molecule encoding a CAR a recombinant TCR, are provided herein. Examples of vectors that can be used include viral vectors, such as a lentiviral vector or retrovirus.

1. PGCla, Tfam, GABPA, and ERRa

The PGCla, Tfam, GABPA, or ERRa coding sequence in the vector can be native or variant PGCla, Tfam, GABPA, or ERRa sequence. Native PGCla, Tfam, GABPA, and ERRa sequences are provided above via GenBank® Accession Nos. for several species. Thus, in some examples, the vector introduced into the T cell includes a native PGCla, Tfam, GABPA, and/or ERRa coding sequence. In some examples, the vector introduced into the T cell includes a non- native PGCla, Tfam, GABPA, and/or ERRa coding sequence, but encodes a native PGCla, Tfam, GABPA, and/or ERRa protein sequence (e.g., a coding sequence that is degenerate). Variant PGCla, Tfam, GABPA, or ERRa proteins, including variants of the protein sequences provided above via GenBank® Accession Nos., can contain one or more mutations, such as a single insertion, a single deletion, a single substitution. In some examples, the variant PGCla, Tfam, GABPA, or ERRa protein includes 1-20 insertions, 1-20 deletions, 1-20 substitutions, and/or any combination thereof (e.g., single insertion together with 1-19 substitutions). In some examples, the disclosure provides a variant of any native PGCla, Tfam, GABPA, or ERRa protein having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 additional amino acid changes. In some examples, a variant PGCla, Tfam, GABPA, or ERRa protein includes 1-8 insertions, 1-15 deletions, 1-10 substitutions, and/or any combination thereof (e.g., 1-15, 1-4, or 1-5 amino acid deletions together with 1-10, 1-5 or 1-7 amino acid substitutions). In some examples, the disclosure provides a variant PGCla, Tfam, GABPA, or ERRa protein having 1, 2, 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 or 30 amino acid changes. In one example, such variant peptides are produced by manipulating the nucleotide sequence encoding a peptide using standard procedures such as site-directed mutagenesis or PCR. Such variants can also be chemically synthesized.

One type of modification includes the substitution of amino acids for amino acid residues having a similar biochemical property, that is, a conservative substitution (such as 1-4, 1-8, 1-10, or 1-20 conservative substitutions). Typically, conservative substitutions have little to no impact on the activity of a resulting peptide. For example, a conservative substitution is an amino acid substitution in any native PGCla, Tfam, GABPA, or ERRa protein sequence, which does not substantially affect the native function of the protein. An alanine scan can be used to identify which amino acid residues in a PGCla, Tfam, GABPA, or ERRa protein can tolerate an amino acid substitution. In one example, the native function of PGCla, Tfam, GABPA, or ERRa is not altered by more than 25%, for example not more than 20%, for example not more than 10%, when an alanine, or other conservative amino acid, is substituted for 1-4, 1-8, 1-10, or 1-20 native amino acids. Examples of amino acids which may be substituted for an original amino acid in a protein and which are regarded as conservative substitutions include: Ser for Ala; Lys, Gin, or Asn for Arg; Gin or His for Asn; Glu for Asp; Ser for Cys; Asn for Gin; Asp for Glu; Pro for Gly; Asn or Gin for His; Leu or Val for He; He or Val for Leu; Arg or Gin for Lys; Leu or lie for Met; Met, Leu or Tyr for Phe; Thr for Ser; Ser for Thr; Tyr for Trp; Trp or Phe for Tyr; and lie or Leu for Val.

More substantial changes can be made by using substitutions that are less conservative, e.g., selecting residues that differ more significantly in their effect on maintaining: (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation; (b) the charge or hydrophobicity of the polypeptide at the target site; or (c) the bulk of the side chain. The substitutions that in general are expected to produce the greatest changes in polypeptide function are those in which: (a) a hydrophilic residue, e.g., serine or threonine, is substituted for (or by) a hydrophobic residue, e.g., leucine, isoleucine, phenylalanine, valine or alanine; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g., lysine, arginine, or histidine, is substituted for (or by) an electronegative residue, e.g., glutamic acid or aspartic acid; or (d) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having a side chain, e.g., glycine. The effects of these amino acid substitutions (or other deletions and/or additions) can be assessed by analyzing the function of the mutant variant PGCla, Tfam, GAB PA, or ERRa protein by analyzing the native function of the protein.

In one example, the PGCla, Tfam, GABPA, and/or ERRa expressed from the vector is a variant form that is resistant to negative regulation. In one example, the variant PGCla, Tfam, GABPA, or ERRa has an increased ability to be resistant to negative regulation, such as an increase of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 200%, at least 300%, at least 400%, or at least 500% relative to a native protein. In one example, negative regulation is measured by transcriptional activity and effects on mitochondrial biogenesis. Examples of mutations that can be made to a PGCla, Tfam, GABPA, and/or ERRa protein to increase its resistance to negative regulation are shown in Table 1 :

Table 1 : Exemplary Mutations

2. Exemplary CARs

In some examples, the disclosed recombinant T cells can include a chimeric antigen receptor (CAR). CARs can be used to engineer T cells that target a tumor cell surface molecule for cancer immunotherapy. The CAR can be expressed from the same vector as PGCla, Tfam, GABPA, and/or ERRa, or from a different vector. CARs are fusion proteins that include antigen recognition moieties and T-cell activation domains. Thus, the recombinant T cells provided herein that have increased PGCla, Tfam, GABPA, and/or ERRa expression, can also be genetically modified to express CARs. For example, most B-cell malignancies express CD 19, and thus adoptive transfer of anti-CD19 CAR T cells can be used to treat such tumors, such as lymphomas and leukemias.

CARs generally include an ectodomain (which includes a signal peptide, an antigen recognition region, and a spacer that provides flexibility and optimizes T cell and target cell engagement), a transmembrane domain (e.g., hydrophobic alpha helix, such as CD3-zeta or CD2, and optionally intracellular signaling domains), and an endodomain. A review of CARs and their design is provided in Jensen and Riddell (Curr Opin Immunol. 33:9-15, 2015, herein incorporated by reference) and Dotti et al., Immunol Rev. 257(l):doil0.1111/imr.l2131 2014, herein incorporated by reference).

In one example, the ectodomain of CAR includes an antibody or antibody fragment (e.g. , an scFv) specific for a tumor associated antigen (TAA). In one example, the ectodomain comprises a monoclonal antibody fragment. Examples of TAAs that can be targeted by CAR are shown in Table 2 and below (as well as Morello et al., Cancer Discov 6: 133-46, 2016, herein incorporated by reference).

Table 2: Exemplary CAR Targets

TAA Tumor

CD19 Liquid (e.g., leukemia, lymphoma)

HER1 Adenocarcinoma (e.g., colorectal cancer,

head and neck cancer)

HER2 breast cancer, ovarian cancer, stomach

cancer, uterine cancer

CD20 Non-Hodgkin lymphoma

CD25 T-cell lymphoma

CD33 Acute myelogenous leukemia

CD52 chronic lymphocytic leukemia

CEA colorectal cancer, some gastric cancers,

biliary cancer

Cancer antigen 125 (CA125) ovarian cancer, mesothelioma, breast

cancer

Alpha-fetoprotein (AFP) hepatocellular carcinoma

Lewis Y colorectal cancer, biliary cancer TAG72 adenocarcinomas including colorectal,

pancreatic, gastric, ovarian, endometrial,

mammary, and non-small cell lung cancer

Vascular endothelial growth Colorectal cancer

factor

Hypoglycosylated MUC1 Epithelial cancers

In one example, the TAA is a member of the EGF receptor family (e.g., HER1 , 2, 3, and 4) or cytokine receptor (e.g., CD20, CD25, IL-13R, CD5, CD52, etc.). TAAs are proteins that are unique to cancer cells or are much more abundant on them, as compared to other cells, such as normal cells. For example HER2 is primarily found in breast cancers, while HER1 is primarily found in adenocarcinomas, which can be found in many organs, such as the pancreas, breast, prostate and colon.

Exemplary TAAs that can be targeted by a CAR, include but are not limited to: any of the various MAGEs (Melanoma- Associated Antigen E), including MAGE 1 (e.g., GenBank Accession Nos. M77481 and AAA03229), MAGE 2 (e.g., GenBank Accession Nos. LI 8920 and AAA17729), MAGE 3 (e.g., GenBank Accession Nos. U03735 and AAA17446), MAGE 4 (e.g., GenBank Accession Nos. D32075 and A06841.1), etc.; any of the various tyrosinases (e.g., GenBank Accession Nos. U01873 and AAB60319); mutant ras; mutant p53 (e.g. , GenBank Accession Nos. X54156, CAA38095 and AA494311); p97 melanoma antigen (e.g., GenBank Accession Nos. M12154 and AAA59992); human milk fat globule (HMFG) associated with breast tumors (e.g., GenBank Accession Nos. S56151 and AAB 19771); any of the various BAGEs (Human B melanoma- Associated Antigen E), including BAGE1 (e.g., GenBank Accession No. Q13072) and BAGE2 (e.g., GenBank Accession Nos. NM_182482 and NP_872288), any of the various GAGEs (G antigen), including GAGE1 (e.g., GenBank Accession No. Q13065) or any of GAGE2-6;

various gangliosides, CD25 (e.g., GenBank Accession Nos. NP_000408.1 and NM_000417.2).

Other TAAs that can be targeted by a CAR include HPV 16/18 and E6/E7 antigens associated with cervical cancers (e.g. , GenBank Accession Nos. NC_001526, FJ952142.1 , ADB94605, ADB94606, and U89349), mucin (MUC 1)-KLH antigen associated with breast carcinoma (e.g., GenBank Accession Nos. J03651 and AAA35756), CEA (carcinoembryonic antigen) associated with colorectal cancer (e.g., GenBank Accession Nos. X98311 and

CAA66955), gplOO (e.g., GenBank Accession Nos. S73003 and AAC60634) associated with for example melanoma, MARTI antigens associated with melanoma (e.g., GenBank Accession No. NP_005502), cancer antigen 125 (CA125, also known as mucin 16 or MUC16) associated with ovarian and other cancers (e.g., GenBank Accession Nos. NM_024690 and NP_078966); alpha- fetoprotein (AFP) associated with liver cancer (e.g., GenBank Accession Nos. NM_001134 and NP_001125); Lewis Y antigen associated with colorectal, biliary, breast, small-cell lung, and other cancers; tumor-associated glycoprotein 72 (TAG72) associated with adenocarcinomas; and the PSA antigen associated with prostate cancer (e.g., GenBank Accession Nos. X14810 and CAA32915).

Other exemplary TAAs that can be targeted by a CAR include, but are not limited to, PMSA (prostate membrane specific antigen; e.g., GenBank Accession Nos. AAA60209 and AAB81971.1) associated with solid tumor neovasculature, as well prostate cancer; HER-2 (human epidermal growth factor receptor 2, e.g., GenBank Accession Nos. M16789.1 , M16790.1, M16791.1, M16792.1 and AAA58637) associated with breast cancer, ovarian cancer, stomach cancer and uterine cancer, HER-1 (e.g., GenBank Accession Nos. NM_005228 and NP_005219) associated with lung cancer, anal cancer, and gliobastoma as well as adenocarcinomas; NY-ESO-1 (e.g. GenBank Accession Nos. U87459 and AAB49693) associated with melanoma, sarcomas, testicular carcinomas, and other cancers, hTERT (aka telomerase) (e.g., GenBank Accession. Nos. NM_198253 and NP_937983 (variant 1), NM_198255 and NP_937986 (variant 2)); proteinase 3 (e.g., GenBank Accession Nos. M29142, M75154, M96839, X55668, NM 00277, M96628, X56606, CAA39943 and AAA36342), and Wilms tumor 1 (WT-1 , e.g. GenBank Accession Nos. NM_000378 and NP_000369 (variant A), NM_024424 and NP_077742 (variant B), NM_024425 and NP_077743 (variant C), and NM_024426 and NP_077744 (variant D)).

In one example the TAAs targeted by a CAR is CD52 (e.g., GenBank Accession. Nos.

AAH27495.1 and CAI15846.1) associated with chronic lymphocytic leukemia; CD33 (e.g., GenBank Accession. Nos. NM_023068 and CAD36509.1) associated with acute myelogenous leukemia; and CD20 (e.g., GenBank Accession. Nos. NP_068769 NP_031667) associated with Non-Hodgkin lymphoma.

In one example the endodomain includes one or more costimulatory domains fused to

CO3C,, such as CD28, CD137, or OX-40, such as a CD3zeta-CD28-41BB or CD3-zeta-CD28- OX40.

3. Exemplary TCR sequences

In some examples, the disclosed recombinant T cells can include a recombinant T cell receptor (TCR). The TCR can be expressed from the same vector as PGCla, Tfam, GABPA, and/or ERRa, or from a different vector. Introducing mutation-specific TCR genes into T cells for adoptive transfer allow, for tumor-specific therapy. A review of TCRs and their design is provided in Blankenstein et al. (Curr Opin Immunol. 33 : 112-9, 2015, herein incorporated by reference). Like CARs, TCRs that target a TAA (such as those listed above) can be used. In other examples, provided in Table 3, a normal cell can be targeted, where the normal cells are dispensable. Harris and Kranz (Trends Pharmacol. Sci. 37:220-30, 2016, herein incorporated by reference) provides an comparison of TCRs and CARs.

Table 3: Exemplary TCRs

Making Recombinant T cells

1. Obtaining T cells

In some examples, T cells are obtained from a subject to be treated, such as one having cancer. In some examples, T cells are obtained from a donor subject. T cells can be obtained from circulating peripheral blood. In some examples, aphersis or leukapheresis is used. In some examples, PBMCs are obtained, and T cells enriched for by using anti-CD3/anti-CD28 beads. In some examples, T cells are obtained from a tumor (e.g., tumor infiltrating lymphocytes).

2. Vectors

Nucleic acid molecules encoding a native or variant PGCla, Tfam, GAB PA, and/or ERRa protein can be incorporated into a vector. Similarly, nucleic acid molecules encoding a CAR or recombinant TCR can be incorporated into a vector, which may be the same or a different vector than the one containing the native or variant PGCla, Tfam, GAB PA, and/or ERRa coding sequence. Nucleic acid sequences coding for g a native or variant PGCla, Tfam, GAB PA, and/or ERRa such as those having at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to those shown in a GenBank® Accession No.

provided herein, can be routinely generated. Similarly, nucleic acid molecules coding for a CAR or recombinant TCR, such as those having at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a known a CAR or recombinant TCR, can be routinely generated. In addition, one of skill can readily construct a variety of clones containing functionally equivalent nucleic acids, such as nucleic acids which differ in sequence but which encode the same protein sequence. In some examples, such a sequence is optimized for expression in a host cell, such as a host T cell used to express the desired protein(s).

Nucleic acid molecules include DNA, cDNA and RNA sequences which encode a peptide. Silent mutations in the coding sequence result from the degeneracy (i.e., redundancy) of the genetic code, whereby more than one codon can encode the same amino acid residue. Thus, for example, leucine can be encoded by CTT, CTC, CTA, CTG, TTA, or TTG; serine can be encoded by TCT, TCC, TCA, TCG, AGT, or AGC; asparagine can be encoded by AAT or AAC; aspartic acid can be encoded by GAT or GAC; cysteine can be encoded by TGT or TGC; alanine can be encoded by GCT, GCC, GCA, or GCG; glutamine can be encoded by CAA or CAG; tyrosine can be encoded by TAT or TAC; and isoleucine can be encoded by ATT, ATC, or ATA.

Codon preferences and codon usage tables for a particular species can be used to engineer isolated nucleic acid molecules encoding a PGCla, Tfam, GAB PA, ERRa, CAR and/or recombinant TCR protein that take advantage of the codon usage preferences of that particular species. For example, the PGCla, Tfam, GABPA, ERRa, CAR and/or recombinant TCR protein expressed from the vector(s) can be designed to have codons that are preferentially used by a particular organism of interest (e.g., in one whom the recombinant T cells are introduced).

A nucleic acid encoding a PGCla, Tfam, GABPA, ERRa, CAR and/or recombinant TCR protein can be cloned or amplified by in vitro methods, such as the polymerase chain reaction (PCR), the ligase chain reaction (LCR), the transcription-based amplification system (TAS), the self- sustained sequence replication system (3SR) and the Q replicase amplification system (QB). A wide variety of cloning and in vitro amplification methodologies are well known to persons skilled in the art. In addition, nucleic acids encoding sequences encoding a PGCla, Tfam, GABPA, ERRa, CAR and/or recombinant TCR protein can be prepared by cloning techniques. Examples of appropriate cloning and sequencing techniques, and instructions sufficient to direct persons of skill through cloning are found in Sambrook et al. (ed.), Molecular Cloning: A

Laboratory Manual 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring, Harbor, N.Y., 1989, and Ausubel et al., (1987) in "Current Protocols in Molecular Biology," John Wiley and Sons, New York, N.Y..

Nucleic acid sequences encoding a PGCla, Tfam, GABPA, ERRa, CAR and/or recombinant TCR protein can be prepared by any suitable method including, for example, cloning of appropriate sequences or by direct chemical synthesis by methods such as the phosphotriester method of Narang et al., Meth. Enzymol. 68:90-99, 1979; the phosphodiester method of Brown et al., Meth. Enzymol. 68: 109-151, 1979; the diethylphosphoramidite method of Beaucage et al., Tetra. Lett. 22: 1859-1862, 1981; the solid phase phosphoramidite triester method described by Beaucage & Caruthers, Tetra. Letts. 22(20): 1859- 1862, 1981, for example, using an automated synthesizer as described in, for example, Needham-VanDevanter et al., Nucl. Acids Res. 12:6159- 6168, 1984; and, the solid support method of U.S. Patent No. 4,458,066. Chemical synthesis produces a single stranded oligonucleotide. This can be converted into double stranded DNA by hybridization with a complementary sequence, or by polymerization with a DNA polymerase using the single strand as a template. One of skill would recognize that while chemical synthesis of DNA is generally limited to sequences of about 100 bases, longer sequences may be obtained by the ligation of shorter sequences.

In one example, a PGCla, Tfam, GAB PA, ERRa, CAR and/or recombinant TCR protein is prepared by inserting the cDNA which encodes the PGCla, Tfam, GABPA, ERRa, CAR and/or recombinant TCR protein into a vector. The insertion can be made so that the protein(s) is read in frame so that the protein(s) is produced. Techniques for preparing recombinant vectors (e.g. , plasmid or virus) containing a heterologous nucleic acid sequence encoding the PGCla, Tfam, GABPA, ERRa, CAR and/or recombinant TCR protein are known.

The nucleic acid coding sequence for a PGCla, Tfam, GABPA, ERRa, CAR and/or recombinant TCR protein can be inserted into an expression vector including, but not limited to a plasmid, virus or other vehicle that can be manipulated to allow insertion or incorporation of sequences and can be expressed in a T cell. Methods of expressing coding sequences from a vector are known in the art. Biologically functional viral and plasmid DNA vectors capable of expression and replication in a T cell are known in the art. The expression vector can contain additional elements necessary for the transfer and subsequent replication of the expression vector containing the PGCla, Tfam, GABPA, ERRa, CAR and/or recombinant TCR protein coding sequence in the T cell. Examples of such elements include, but are not limited to, origins of replication and selectable markers, such as a thymidine kinase gene or an antibiotic resistance marker, a 'suicide gene' like an inducible caspase 9, or a truncated (nonfunctional) EGFR.

Nucleic acid sequences encoding a PGCla, Tfam, GABPA, ERRa, CAR and/or recombinant TCR protein can be operatively linked to expression control sequences. In one example, the PGCla, Tfam, GABPA, and/or ERRa protein is expressed from the same expression control sequence as the CAR and/or recombinant TCR protein. An expression control sequence operatively linked to a PGCla, Tfam, GABPA, ERRa, CAR and/or recombinant TCR protein coding sequence is ligated such that expression of the PGCla, Tfam, GABPA, ERRa, CAR and/or recombinant TCR protein coding sequence is achieved under conditions compatible with the expression control sequences. Exemplary expression control sequences include, but are not limited to appropriate promoters, enhancers, transcription terminators, a start codon (i.e. , ATG) in front of a PGCla, Tfam, GABPA, ERRa, CAR and/or recombinant TCR protein-encoding gene, splicing signal for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons. Examples of expression control elements that can be used include, but are not limited to, lac system, operator and promoter regions of phage lambda, and promoters derived from polyoma, adenovirus, retrovirus or SV40. Additional operational elements include, but are not limited to, leader sequence, termination codons, polyadenylation signals and any other sequences necessary for the appropriate transcription and subsequent translation of the nucleic acid sequence encoding the PGCla, Tfam, GABPA, ERRa, CAR and/or recombinant TCR protein in the T cell. In one example, the promoter is a lentiviral promoter. In one example, an IRES is used to drive expression. In some examples, two promoters are used.

Viral vectors can be prepared that encode a PGCla, Tfam, GABPA, ERRa, CAR and/or recombinant TCR protein. In one example, PGCla, Tfam, GABPA, and/or ERRa, are expressed from the same vector as a CAR and/or recombinant TCR protein. In one example, PGCla, Tfam, GABPA, and/or ERRa, are expressed from a separate vector as a CAR and/or recombinant TCR protein. Exemplary viral vectors that can be used include, but are not limited to, polyoma, SV40, adenovirus, vaccinia virus, adeno-associated virus, herpes viruses including HSV and EBV, Sindbis viruses, alphaviruses and retroviruses of avian, murine, and human origin. Baculovirus

(Autographa californica multinuclear polyhedrosis virus; AcMNPV) vectors can also be used. Other suitable vectors include orthopox vectors, avipox vectors, fowlpox vectors, capripox vectors, suipox vectors, lentiviral vectors, alpha virus vectors, and poliovirus vectors. Specific exemplary vectors are poxvirus vectors such as vaccinia virus, fowlpox virus and a highly attenuated vaccinia virus (MVA), adenovirus, baculovirus and the like. Pox viruses of use include orthopox, suipox, avipox, and capripox virus. Orthopox include vaccinia, ectromelia, and raccoon pox. One example of an orthopox of use is vaccinia. Avipox includes fowlpox, canary pox and pigeon pox. Capripox include goatpox and sheeppox. In one example, the suipox is swinepox. Other viral vectors that can be used include other DNA viruses such as herpes virus and adenoviruses, and RNA viruses such as retroviruses and polio.

3. Expression of Recombinant Nucleic Acid Molecules

Exemplary methods that can be used to introduce a vector that includes a PGCla, Tfam, GABPA, ERRa, CAR and/or recombinant TCR protein coding sequence into a eukaryotic cell, such as a T cell, include but are not limited to: calcium phosphate coprecipitates; mechanical procedures such as microinjection, electroporation, insertion of a plasmid encased in liposomes, and infection with a viral vector (see for example, Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982). After introducing the vector that includes a PGCla, Tfam, GABPA, ERRa, CAR and/or recombinant TCR protein coding sequence into the T cell, the T cell can be cultured under conditions that permit expression of PGCla, Tfam, GABPA, ERRa, CAR and/or recombinant TCR protein(s). Such methods can include incubating or culturing the recombinant T cells in the presence of a culture media under conditions that allow for expansion of the recombinant T cells. In some examples, the method of ex vivo expansion can include incubating the recombinant T cells in a culture medium, such as serum-free media (e.g., AIM V® medium) or human AB serum. The method of ex vivo expansion can further include contacting the recombinant T cells with a gamma- chain cytokine (such as interleukin 2 (IL-2) and/or IL-15), anti-CD3, anti-CD28, or combinations thereof. In some examples, the anti-CD3 and the anti-CD28 are present on a bead. The amount of anti-CD3, anti-CD28 and IL-2 can vary, such as from 3-10 μg/mL anti-CD3 (immobilized), 2-10 μg/mL anti-CD28, and 10 U/mL to 6000 U/mL IL-2. In some examples, additional agents are present in the ex vivo culture, such as gamma chain cytokines (as well as reagents provided in US 5126132). In typical examples, the recombinant T cells are expanded ex vivo at 37°C.

In some examples, the recombinant T cells are incubated ex vivo to allow for sufficient expansion (e.g. , reproduction of the recombinant T cells) as needed for transplantation, such as a period of days or weeks (such as at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, or at least 6 weeks). In some examples, the recombinant T cells are allowed to expand until millions or billions of recombinant T cells are obtained, such as tens of billions recombinant T cells.

In some examples, the method includes monitoring the expanding recombinant T cells, for example determining the number of cells, determining or measuring the mitochondrial activity (e.g., oxidative metabolism), determining or measuring the mitochondrial mass of the TILs, determining or measuring nucleic acid and/or protein expression of one or more of PGCla, Tfam, GABPA, ERRa, CAR and/or recombinant TCR.

Methods of Using Recombinant T Cells

The recombinant T cells provided herein, for example generated using the disclosed methods, can be used in cancer immunotherapy, for example to treat a tumor in vivo. Solid and liquid tumors can be treated with the disclosed methods. Specific examples of tumors that can be treated include, but are not limited to, a leukemia, lymphoma, colorectal cancer, melanoma, cervical cancer, lung cancer, ovarian cancer, bladder cancer, breast cancer, or head and neck cancer. Other examples are provided herein. For example, an effective amount of the disclosed recombinant T cells (such as at least lxlO ⁶ recombinant T cells, at least 2xl0 ⁶ recombinant T cells, at least 5xl0 ⁶ recombinant T cells, at least lxlO ⁷ recombinant T cells, at least lxlO ⁸ recombinant T cells, at least lxlO ⁹ recombinant T cells, at least lxlO ¹⁰ recombinant T cells, at least 5xl0 ¹⁰ , or at least 5xlO ⁿ recombinant T cells) are administered to the subject, thereby treating a tumor (such as a primary tumor and/or a metastasis) in the subject. In some examples, the recombinant T cells are administered intravenously. In one example, the method further includes administering an effective amount of nonmyeloablative chemotherapy or radiotherapy to deplete native lymphocytes prior to administering an effective amount of the recombinant T cell (but prior to harvesting T cells if the T cells used are from the same patient). In some examples, such subjects are also administered an effective amount of IL-2 (such as 10,000 to 100,000 units/kg body weight) to the subject before, after, or both before and after, administering the disclosed recombinant T cells.

In some examples the subject administered the disclosed recombinant T cells was previously treated unsuccessfully with a chemotherapy, radiation therapy, biologic therapy, or combinations thereof (e.g., the tumor in the subject did not significantly decrease in size or even increased in size, and/or metastasized). In some examples the subject has a tumor that was not responsive to a PD-1 antagonist or a PD-L1 antagonist (e.g., the tumor in the subject did not significantly decrease in size or even increased in size, and/or metastasized), such as an antibody that specifically binds PD-1 or PD-L1, such as Atezolizumab, MPDL3280A, BNS-936558

(Nivolumab), Pembrolizumab, Pidilizumab, CT011, AMP-224, AMP-514, MEDI-0680, BMS- 936559, BMS935559, MEDI-4736, MPDL-3280A, MSB-0010718C.

Additional Therapies

The subject treated with the disclosed recombinant T cells can receive one or more additional therapies, such as one or more of an effective amount of chemotherapy an effective amount of radiotherapy (for example administration of radioactive material or energy (such as external beam therapy) to the tumor site to help eradicate the tumor or shrink it), an effective amount of a biologic, and surgery (for example surgical resection of the cancer or a portion of it).

In one example, the subject is further treated with one or more chemotherapeutic agents. Chemotherapeutic agents include any chemical agent with therapeutic usefulness in the treatment of diseases characterized by abnormal cell growth, such as cancer. One of skill in the art can readily identify a chemotherapeutic agent of use (see for example, Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 in Harrison's Principles of Internal Medicine, 14th edition; Perry et al , Chemotherapy, Ch. 17 in Abeloff, Clinical Oncology 2 ^nd ed., © 2000 Churchill Livingstone, Inc; Baltzer, L., Berkery, R. (eds): Oncology Pocket Guide to Chemotherapy, 2nd ed. St. Louis, Mosby- Year Book, 1995; Fischer, D.S., Knobf, M.F., Durivage, H.J. (eds): The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 1993; Chabner and Longo, Cancer Chemotherapy and Biotherapy: Principles and Practice (4th ed.). Philadelphia: Lippincott Willians & Wilkins, 2005; Skeel, Handbook of Cancer Chemotherapy (6th ed.). Lippincott Williams & Wilkins, 2003). Combination chemotherapy is the administration of more than one agent to treat cancer.

Examples of chemotherapeutic agents that can be used include alkylating agents, antimetabolites, natural products, or hormones and their antagonists. Examples of alkylating agents include nitrogen mustards (such as mechlorethamine, cyclophosphamide, melphalan, uracil mustard or chlorambucil), alkyl sulfonates (such as busulfan), nitrosoureas (such as carmustine, lomustine, semustine, streptozocin, or dacarbazine). Specific non-limiting examples of alkylating agents are temozolomide and dacarbazine. Examples of antimetabolites include folic acid analogs (such as methotrexate), pyrimidine analogs (such as 5-FU or cytarabine), and purine analogs, such as mercaptopurine or thioguanine. Examples of natural products include vinca alkaloids (such as vinblastine, vincristine, or vindesine), epipodophyllotoxins (such as etoposide or teniposide), antibiotics (such as dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, or mitocycin C), and enzymes (such as L-asparaginase). Examples of miscellaneous agents include platinum coordination complexes (such as cis-diamine-dichloroplatinum II also known as cisplatin), substituted ureas (such as hydroxyurea), methyl hydrazine derivatives (such as procarbazine), and adrenocrotical suppressants (such as mitotane and aminoglutethimide). Examples of hormones and antagonists include adrenocorticosteroids (such as prednisone), progestins (such as

hydroxyprogesterone caproate, medroxyprogesterone acetate, and magestrol acetate), estrogens (such as diethylstilbestrol and ethinyl estradiol), antiestrogens (such as tamoxifen), and androgens (such as testerone proprionate and fluoxymesterone).

Examples of commonly used chemotherapy drugs include Adriamycin, Alkeran, Ara-C,

BiCNU, Busulfan, CCNU, Carboplatinum, Cisplatinum, Cytoxan, Daunorubicin, DTIC, 5-FU, Fludarabine, Hydrea, Idarubicin, Ifosfamide, Methotrexate, Mithramycin, Mitomycin,

Mitoxantrone, Nitrogen Mustard, Taxol (or other taxanes, such as docetaxel), Velban, Vincristine, VP- 16, while some more newer drugs include Gemcitabine (Gemzar), Herceptin, Irinotecan (Camptosar, CPT-11), Leustatin, Navelbine, Rituxan STI-571, Taxotere, Topotecan (Hycamtin), Xeloda (Capecitabine), Zevelin and calcitriol. Non-limiting examples of immunomodulators that can be used include AS-101 (Wyeth-Ayerst Labs.), bropirimine (Upjohn), gamma interferon (Genentech), GM-CSF (granulocyte macrophage colony stimulating factor; Genetics Institute), IL- 2 (Cetus or Hoffman-LaRoche), human immune globulin (Cutter Biological), IMREG (from Imreg of New Orleans, La.), SK&F 106528, and TNF (tumor necrosis factor; Genentech).

Additional therapeutic agents that can be used include microtubule binding agents, DNA intercalators or cross-linkers, DNA synthesis inhibitors, DNA and/or RNA transcription inhibitors, antibodies, enzymes, enzyme inhibitors, gene regulators, angiogenesis inhibitors. These agents (which are administered at a therapeutically effective amount) and treatments can be used alone or in combination. Methods and therapeutic dosages of such agents are known to those skilled in the art, and can be determined by a skilled clinician.

Microtubule binding agent refers to an agent that interacts with tubulin to stabilize or destabilize microtubule formation thereby inhibiting cell division. Examples of microtubule binding agents that can be used in conjunction with the disclosed therapy include, without limitation, paclitaxel, docetaxel, vinblastine, vindesine, vinorelbine (navelbine), the epothilones, colchicine, dolastatin 15, nocodazole, podophyllotoxin and rhizoxin. Analogs and derivatives of such compounds also can be used and are known to those of ordinary skill in the art. For example, suitable epothilones and epothilone analogs are described in International Publication No. WO

2004/018478. Taxoids, such as paclitaxel and docetaxel, as well as the analogs of paclitaxel taught by U.S. Pat. Nos. 6,610,860; 5,530,020; and 5,912,264 can be used.

Suitable DNA and/or RNA transcription regulators, including, without limitation, actinomycin D, daunorubicin, doxorubicin and derivatives and analogs thereof also are suitable for use in combination with the disclosed therapies. DNA intercalators and cross-linking agents that can be administered to a subject include, without limitation, cisplatin, carboplatin, oxaliplatin, mitomycins, such as mitomycin C, bleomycin, chlorambucil, cyclophosphamide and derivatives and analogs thereof. DNA synthesis inhibitors suitable for use as therapeutic agents include, without limitation, methotrexate, 5-fluoro-5'-deoxyuridine, 5-fluorouracil and analogs thereof. Examples of suitable enzyme inhibitors include, without limitation, camptothecin, etoposide, formestane, trichostatin and derivatives and analogs thereof. Suitable compounds that affect gene regulation include agents that result in increased or decreased expression of one or more genes, such as raloxifene, 5-azacytidine, 5-aza-2'-deoxycytidine, tamoxifen, 4-hydroxytamoxifen, mifepristone and derivatives and analogs thereof.

The disclosed methods can further include administering to the subject a therapeutically effective amount of an immunotherapy. Non- limiting examples of immunomodulators that can be used include AS-101 (Wyeth-Ayerst Labs.), bropirimine (Upjohn), gamma interferon (Genentech), GM-CSF (granulocyte macrophage colony stimulating factor; Genetics Institute), IL-2 (Cetus or Hoffman-LaRoche), human immune globulin (Cutter Biological), IMREG (from Imreg of New Orleans, La.), SK&F 106528, and TNF (tumor necrosis factor; Genentech). The immunotherpautic agent can be a PD-1 antagonist or a PD-L1 antagonist, such as an antibody that specifically binds PD-1 or PD-L1, such as Atezolizumab, MPDL3280A, BNS-936558 (Nivolumab), Pembrolizumab, Pidilizumab, CT011, AMP-224, AMP-514, MEDI-0680, BMS-936559, BMS935559, MEDI-4736, MPDL-3280A, MSB-0010718C. The immunotherpautic agent can also be a CTLA-4, LAG-3, or B7-H3 antagonist, such as Tremelimumab, BMS-986016, and MGA271.

Non-limiting examples of anti-angiogenic agents include molecules, such as proteins, enzymes, polysaccharides, oligonucleotides, DNA, RNA, and recombinant vectors, and small molecules that function to reduce or even inhibit blood vessel growth. Examples of suitable angiogenesis inhibitors include, without limitation, angiostatin Kl-3, staurosporine, genistein, fumagillin, medroxyprogesterone, suramin, interferon-alpha, metalloproteinase inhibitors, platelet factor 4, somatostatin, thromobospondin, endostatin, thalidomide, and derivatives and analogs thereof. For example, in some embodiments the anti-angiogenesis agent is an antibody that specifically binds to VEGF (e.g. , Avastin, Roche) or a VEGF receptor (e.g. , a VEGFR2 antibody). In one example the anti-angiogenic agent includes a VEGFR2 antibody, or DMXAA (also known as Vadimezan or ASA404; available commercially, e.g. , from Sigma Corp., St. Louis, MO) or both. The anti-angiogenic agent can be bevacizumab, sunitinib, an anti-angiogenic tyrosine kinase inhibitors (TKI), such as sunitinib, xitinib and dasatinib. These can be used individually or in any combination.

Exemplary kinase inhibitors include Gleevac, Iressa, and Tarceva, sunitinib, sorafenib, anitinib, and dasatinib that prevent phosphorylation and activation of growth factors. Antibodies that can be used include Herceptin and Avastin that block growth factors and the angiogenic pathway. These can be used individually or in combination.

In some examples, the additional therapeutic agent administered is a biologic, such as a monoclonal antibody, for example, 3F8, Abagovomab, Adecatumumab, Afutuzumab, Alacizumab , Alemtuzumab, Altumomab pentetate, Anatumomab mafenatox, Apolizumab, Arcitumomab, Bavituximab, Bectumomab, Belimumab, Besilesomab, Bevacizumab, Bivatuzumab mertansine, Blinatumomab, Brentuximab vedotin, Cantuzumab mertansine, Capromab pendetide,

Catumaxomab, CC49, Cetuximab, Citatuzumab bogatox, Cixutumumab, Clivatuzumab tetraxetan, Conatumumab, Dacetuzumab, Detumomab, Ecromeximab, Eculizumab, Edrecolomab,

Epratuzumab, Ertumaxomab, Etaracizumab, Farletuzumab, Figitumumab, Galiximab, Gemtuzumab ozogamicin, Girentuximab, Glembatumumab vedotin, Ibritumomab tiuxetan, Igovomab,

Imciromab, Intetumumab, Inotuzumab ozogamicin, Ipilimumab, Iratumumab, Labetuzumab, Lexatumumab, Lintuzumab, Lorvotuzumab mertansine, Lucatumumab, Lumiliximab, Mapatumumab, Matuzumab, Mepolizumab, Metelimumab, Milatuzumab, Mitumomab,

Morolimumab, Nacolomab tafenatox, Naptumomab estafenatox, Necitumumab, Nimotuzumab, Nofetumomab merpentan, Ofatumumab, Olaratumab, Oportuzumab monatox, Oregovomab, Panitumumab, Pemtumomab, Pertuzumab, Pintumomab, Pritumumab, Ramucirumab,

Rilotumumab, Rituximab, Robatumumab, Satumomab pendetide, Sibrotuzumab, Sonepcizumab, Tacatuzumab tetraxetan, Taplitumomab paptox, Tenatumomab, TGN1412, Ticilimumab

(tremelimumab), Tigatuzumab, TNX-650, Trastuzumab, Tremelimumab, Tucotuzumab

celmoleukin, Veltuzumab, Volociximab, Votumumab, Zalutumumab, or combinations thereof.

In some examples, the subject is administered an effective amount of nonmyeloablative chemotherapy or radiotherapy to deplete native lymphocytes prior to administering an effective amount of the recombinant T cells. For example, the subject may receive an effective amount of nonmyeloablative chemotherapy, such as administration of one or more of cisplatin, fludarabine, idarubicin, melphalan, ara-C, 2-chlorodeoxyadenosine, antithymocyte globulin, and

cyclophosphamide (such as 10 to 50 mg/kg body weight). In some examples, the subject receives an effective amount of solid tumor irradiation, thymic irradiation, or total body irradiation (e.g., 2 Gy), or combinations thereof. Other specific examples are provided in Phan and Rosenberg, Cancer Control 20:289-97, 2013.

In some examples, following administration of the recombinant T cells, the subject is administered one or more of an effective amount of tacrolimus, cyclosporine, and/or methotrexate.

Clinical Response

Such methods can treat the tumor in the subject by reducing the volume or weight of the tumor, reducing the number of metastases, reducing the size or weight of a metastasis, or combinations thereof. In some examples a metastasis is cutaneous or subcutaneous. Thus, in some examples, administration of disclosed recombinant T cells treats a tumor in a subject by reducing the size or volume of the tumor by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99%, for example as compared to no administration of disclosed recombinant T cells or administration of recombinant T cells not containing a vector expressing a native or variant PGCla, Tfam, GAB PA, and/or ERRa coding sequence. In some examples, administration of disclosed recombinant T cells treats a tumor in a subject by reducing the weight of the tumor by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99%, for example as compared to no administration of disclosed recombinant T cells or administration of recombinant T cells not containing a vector expressing a native or variant PGCla, Tfam, GABPA, and/or ERRa coding sequence. In some examples, administration of disclosed recombinant T cells treats a tumor in a subject by reducing the size or volume of a metastasis by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99%, for example as compared to no administration of disclosed recombinant T cells or administration of recombinant T cells not containing a vector expressing a native or variant PGCla, Tfam, GABPA, and/or ERRa coding sequence. In some examples, administration of disclosed recombinant T cells treats a tumor in a subject by reducing the number of metastases by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% for example as compared to no administration of disclosed recombinant T cells or administration of recombinant T cells not containing a vector expressing a native or variant PGCla, Tfam, GABPA, and/or ERRa coding sequence. In some examples, combinations of these effects are achieved.

Compositions and Kits

Also provided are compositions and kits that can be used with the disclosed methods. In some examples, the composition or kit includes one or more disclosed recombinant T cells and/or vectors, for example with a pharmaceutically acceptable carrier. The kits can include additional reagents, such as one or more of anti-CD3, anti-CD28, IL-2, and IL-15. In some examples, in a kit, such reagents are present in a separate container. In one example, anti-CD3 and anti-CD28 are in the same container, and may be present on a bead for example. In some examples, the kit further includes one or more of a transfection reagent, culture medium, antibiotic, IL-2, IL-15, and IL-7, optionally wherein such reagents are present in separate containers. In some examples the kit or composition includes media in which the recombinant T cells can be cultured or expanded ex vivo, such as AIM V® media.

Example 1

Materials and Methods

This example provides the materials and methods used to generate the data discussed in the Examples below.

Mice

All animal work was done in accordance with the Institutional Animal Care and Use Committee of the University of Pittsburgh. All mice were housed in specific pathogen free conditions prior to use. C57/BL6, SJ/L (Thy 1.1), Ppargclcf*. Cd4 ^Cre , Foxp3 ^{GFP Cre ERT2} , Foxp3 ^{DTR GFP} and OT-I mice were obtained from The Jackson Laboratory.

Cell lines, antibodies, and other reagents

B16-F10 and LLC were obtained from ATCC. MC38 was obtained from Dario Vignali. B 16 ^OVA (M05) was obtained from Per Basse and Lou Falo. OVA-expressing Vaccinia virus was originally generated by Yewdell and Bennink and obtained from Jonathan Powell. Most antibodies for flow cytometry were obtained from BioLegend. MitoTracker Green FM, MitoTracker Deep Red FM, tetramethylrhodamine ester (TMRE), and H2-DCFDA were obtained from ThermoFisher. VDAC antibody was obtained from Abeam. LC3B, pAkt, pFoxol/3a antibodies were obtained from Cell Signaling Technologies and detected after surface staining with simultaneous fixation and permabilization in 1.5% PFA made up in IX Permeabilization buffer (eBioscience). 2-NBD- glucose, m-divi-1, and Akt inhibitor VIII were purchased from Cayman Chemical. PGCla antibody (H-300) was obtained from Santa Cruz Biotechnology, and was detected using the Foxp3 Fix/Perm kit (eBioscience) and Alexa Fluor 647 or Alexa Fluor 488-conjugated anti-rabbit IgG (Jackson Immunoresearch). Anti-PD-1 blocking antibody (J43) and its hamster IgG control were obtained from Bio-X-Cell. CellTrace Violet was from eBioscience, and CFSE was from BioLegend.

T cell isolations from lymph node and tumor and adoptive transfer

Spleen and lymph node CD8 ⁺ T cells were isolated from 6-8 week-old OT-I (Thyl.2 or Thyl.l) mice. Tissue was harvested, mechanically disrupted, and incubated with a biotinylated antibody cocktail consisting of antibodies (BioLegend) to B220, CDl lb, CDl lc, CD16/32, CD19, CD25, CD105, NKl.l, TCRy5, and CD4. After a wash step, cells were incubated with streptavidin-coated magnetic nanoparticles (BioLegend). After washing, CD8 ⁺ cells were isolated by applying a magnetic field and removing untouched cells. In some experiments, these OT-I CD8 ⁺ T cells were also labeled with the proliferation dye CellTrace Violet according to the manufacturer' s protocol. Mice bearing B16 ^OVA tumors or immunized with Vaccinia ^OVA received cells intravenously. To obtain single-cell suspensions of tumor infiltrating lymphocytes, tumor bearing mice were sacrificed and tumors were harvested. Excised, whole tumors were injected repeatedly using 20G needles with 2mg/mL collagenase type VI, 2U/mL hyluronidase (Dispase), and lOU/mL DNAse I (Sigma) in buffered RPMI with 10% FBS and incubated for 30 min at 37°C. Tumors were then mechanically disrupted between frosted glass slides and filtered to remove particulates, then vortexed for 2 minutes. In many experiments (especially prior to sorting), tumor homogenates were debulked of tumor cells using CD105-biotin mediated magnetic depletion. Patients and specimens

Peripheral venous blood samples were obtained from HNC patients with stage III/IVA disease previously untreated. Tumor biopsies or surgical tumor specimens were preserved for a maximum of 12 hours in complete media until tumor infiltrating lymphocytes were isolated.

Human tumor infiltrating lymphocyte (TIL) isolation

Fresh tumors from patients with HNC were minced into small pieces manually or using a gentleMACS dissociator (Miltenyi Biotec), then transferred to 70 μιη cell strainers (BD) and mechanically separated using the plunger of a 5-mL syringe. The cells passing through the cell strainer were collected, washed and subjected to Ficoll-Hypaque gradient centrifugation. After centrifugation, mononuclear cells were recovered and immediately used for experiments.

Human PBMC isolation from peripheral blood

Blood from healthy donors (Western Pennsylvania blood bank) or patients with HNC treated with cetuximab during or within 1 month of treatment (UPCI clinical trial #08-013 NCT 01218048). Lymphocytes were purified by Ficoll-Paque PLUS centrifugation following standard protocol (Amersham Biosciences), pulsed with 2NBDG, and stained for flow cytometry. Metabolism assays

Single-cell metabolic capacity was assayed by flow cytometry. Specifically, 2-NBD-glucose (Cayman Chemical) and MitoTracker FM dyes (ThermoFisher) were utilized to assay the propensity of cells to take up glucose or generate intermediates via their mitochondria.

Nondraining and draining lymph node or tumor preparations were pulsed with 20 μΜ 2-NBDG in 5% FBS-containing media for 30 min at 37°C. Cells were surface stained and loaded with MitoTracker FM dyes or TMRE to measure mitochondrial mass and function.

A Seahorse XFe96 Bioanalyzer was utilized to measure metabolic flux in real-time. T cells were plated on Cell-Tak coated Seahorse culture plates (50,000-100,000 T cells/well) in assay media consisting of minimal, unbuffered DMEM supplemented with 1% BSA and 25 mM glucose, ImM pyruvate, and 2 mM glutamine. Basal extracellular acidification and oxygen consumption rates were taken for 30 minutes. Cells were stimulated with oligomycin (2 μΜ), FCCP (0.5 μΜ), 2-deoxy glucose (100 mM) and rotenone/antimycin A (100 μΜ) to obtain maximal respiratory and control values. Spare respiratory capacity is measured as the difference between basal OCR values and maximal OCR values obtained after FCCP uncoupling. Electron microscopy

CD8 ⁺ T cells were sorted from LNs and TIL and fixed in 4% glutaraldehyde, then sectioned and stained for electron microscopy as previously described. (Li et al., 2013)

Retroviral expression

PGCla was originally generated by Dr. B. Spiegelman, obtained from Addgene (plasmid 1026) (Monsalve et al., 2000), and cloned into an MSCV-driven retroviral expression vector which also encodes an IRES-mCherry cassette, from Dario Vignali. This vector was transiently transfected into Phoenix ecotropic cells. OT-I T cell were stimulated with SIINFEKL peptide at 250 ng/mL in the presence of lOOU/mL IL-2 for 24 h. Retroviral supernatants were harvested, and filtered, and supplemented with 6 μg/mL polybrene. OT-I T cell cultures were spinduced with the retroviral supernatant for 90 min at 2500 rpm. 24 h later spinduction this was repeated. Cells were then expanded and sorted for mCherry expression prior to analysis and adoptive transfer.

B16 ^0VA in vitro stimulation

Freshly isolated OT-I splenocytes or purified, previously activated OT-I CD8 ⁺ T cells were CellTrace Violet labeled and plated at various ratios on B 16 or B 16 ^OVA cells in 10% RPMI for 72 h. Proliferation and mitochondrial mass of the T cells was examined flow cytometrically.

Treg cell suppression assay

Lymph nodes and spleens from Foxp3 reporter mice (Foxp3 ^{DTR GFP} ) were sorted based on expression of CD4 and GFP, then cocultured with CellTrace Violet-labeled CD8 ⁺ T cells from WT mice at a 1 :4 ratio (T _r e _g :Te _ff cell) in the presence of antigen presenting cells (CD4 ^" CD8 ^" splenic cells at a 2: 1 APC:T cell ratio) and 1 μg/mL anti-CD3. After 72 h, proliferation and mitochondrial mass of the CD8 ⁺ T cells were analyzed by flow cytometry.

Fuel usage test

100,000 previously activated, transduced T cells (Generated in FIG. 13) were plated on CellTak- coated Seahorse plates in minimal, unbuffered Seahorse media containing glucose, and glutamine. Basal measurements were taken and then cells were uncoupled with FCCP and subjected to inhibition by UK5099, etomoxir, and BPTES. FIG. 13A shows the percentage of the total FCCP uncoupled OCR inhibited by these agents. Example 2

Tumor infiltrating T cells display decreased mitochondrial mass

To assay the metabolic capacity of tumor-infiltrating T cells, flow cytometric analyses were used. Mitochondrial function and mass were measured using MitoTracker Deep Red FM (a membrane permeable, carbocyanine -based dye for mitochondria used previously to stain mitochondrial mass) (Cottet-Rousselle et al., 2011) and competency for glucose uptake using fluorescently labeled 2-NBD-glucose (2NBDG) in T cells infiltrating implantable tumors. While MitoTracker Deep Red has been shown to be membrane potential sensitive in some systems, uncoupling using CCCP showed that, using our staining protocols, MitoTracker Deep Red was highly resistant to collapse of membrane potential, especially compared to TMRE, a well-known membrane-potential sensitive dye (FIG. 1A).

C57/BL6 mice were inoculated with B16 melanoma, and at day 12 (the height of the antitumor immune response), lymph node and tumor preparations were pulsed with 2-NBDG and stained for flow cytometric analysis. While T cells in the lymph nodes, both non-draining (ndLN) and tumor draining (dLN), effectively take up glucose and have relatively high MitoTracker FM staining, CD8 ⁺ T cells tumor infiltrating lymphocytes (TIL) show a dramatic reduction of mitochondrial mass as well as the ability to take up glucose (FIGS. 2A, 2B). To confirm the phenotype observed was due to loss of mitochondrial mass and not only mitochondrial depolarization, we observed similar losses employing both MitoTracker Green FM staining (another carbocyanine-based dye) and antibodies to the mitochondrial outer membrane protein Voltage Dependent Anion Channel (VDAC) (FIG. IB). T cells of various effector and memory phenotypes have been shown to have distinct mitochondrial masses (van der Windt et al., 2013), which was confirmed with dyes; however, these differences are substantially less compared to those observed within the tumor microenvironment (FIG. 1C). For clarity, throughout this study, we primarily gate solely on CD8 ⁺ T cells and without further subdivision unless explicitly stated. This response was largely specific to CD8 ⁺ T cells, as CD4 ⁺ T cells retain most of their mitochondrial mass in B16 tumors. This phenotype was common to three different implantable tumor models inoculated into B6 mice, including MC38 and LLC, although with some notable differences. Mitochondrial mass loss was observed in a proportion of CD4 ⁺ T cells in LLC, and no significant differences in glucose uptake was observed (FIG. 2B and FIGS. ID and IE). This loss of mitochondrial mass was confirmed by transmission electron microscopy, which revealed that tumor-infiltrating CD8 ⁺ T cells not only show lower mitochondrial mass, but also abnormal mitochondrial morphology (FIG. 2C). The metabolic status of T cells infiltrating human head and neck squamous cell carcinoma (HNSCC) was examined, and a similar loss of mitochondrial staining when compared to peripheral blood T cells was observed (FIG. 2D).

The metabolic output of tumor-infiltrating T cells was examined using a Seahorse extracellular flux analyzer. Metabolic flux analysis of effector, LN -resident, or tumor-infiltrating CD8 ⁺ T cells confirmed a persistent defect in oxidative phosphorylation, with significant loss of spare respiratory capacity (a measure of mitochondrial reserve, measured as the difference between basal and uncoupled maximal oxygen consumption) compared to naive, LN-resident cells or previously activated effector T cells (FIG. 2E). This results in an increased dependence on glycolytic metabolism, as evidenced by increased extracellular acidification rate (FIG. 2E). Thus, T cells infiltrating mouse and human tumors show a dramatic loss of mitochondrial mass and dependence of glycolytic metabolism, rendering them unable to carry out critical cellular functions in the glucose-poor tumor microenvironment.

Example 3

Loss of mitochondrial function is specific T cell responses

in the tumor microenvironment

To determine whether this mitochondrial dysfunction was specific to the anti-tumor response or if it occurred in other types of robust effector responses, an adoptive transfer system of naive, congenically mismatched OT-I T cells into mice bearing OVA-expressing B 16 tumors or mice infected with OVA-expressing Vaccinia virus (VV ^OVA ) for 6 days was utilized. This experiment compared the chronic activation seen in cancer to a robust, acute in vivo response in which antigen is effectively cleared (Pollizzi et al., 2015).

Consistent with the data generated in polyclonal populations from mouse and human tumors, OT-I T cells infiltrating tumors showed dramatically decreased MitoTracker FM staining relative to their LN-resident counterparts (FIGS. 3A, 3B). In stark contrast, OT-I T cells responding in the spleen to VV ^OVA infection had increased mitochondrial mass compared to splenic OT-I T cells adoptively transferred into mock infected mice, as evidenced by increased

MitoTracker FM and intracellular VDAC staining (FIGS. 3A, 4A). T cells responding to OVA in the context of VV ^OVA also increased basal OXPHOS and spare respiratory capacity (FIG. 3B). VV ^OVA -responsive T cells also displayed heightened glycolytic function, even compared to OT-I T cells isolated from tumors (FIG. 3B). Comparisons of ATP reserves from OT-I T cells responding in B16 ^OVA tumors compared to VV ^OVA -infected spleens revealed TIL show an inability to maintain ATP reserves, while this pool is dramatically increase in cells responding to viral infection (FIG. 3C). Functionally led to differential patterns of cytokine production upon peptide or PMA/ionomycin restimulation (FIG. 4B). Importantly the phenotype of mitochondrial insufficiency observed in tumor- infiltrating lymphocytes was quite stable; OT-I T cells isolated from B 16 ^OVA tumors retained a phenotype of low mitochondrial mass, even when isolated and transferred into a new, VV ^OVA infected mouse for 7 days (FIG. 3D). Thus, T cell dysfunction associated with loss of mitochondria occurs specifically within the chronic activation and microenvironment of cancer.

Example 4

Mitochondria are depolarized as T cells respond in the tumor microenvironment

This adoptive transfer model was used to explore the metabolic consequences of activated T cells as they enter the tumor microenvironment versus responding to acute infection. Naive. OT-I Thyl.l ^"1" T cells were labeled with CellTrace Violet to monitor their proliferation and then transferred into B 16 ^OVA -bearing or VV ^OVA -infected C57/BL6 (Thy 1.2 ⁺ ) mice for 72h. This resulted in robust proliferation in both scenarios, with OT-I T cells undergoing as many as seven cell divisions during this time (FIG. 5A). Loss of mitochondrial mass was observed as T cells enter the tumor microenvironment, as observed with longer incubations (6 days, FIG. 3A). Experiments employing DCFDA, a ROS indicator, and TMRE, a mitochondrial membrane potential-sensitive dye (FIG. 1A), indicate that T cells responding to antigen in the tumor microenvironment show mitochondrial depolarization as well as a loss of ROS production (FIGS. 5B, 5C).

As mitochondrial depolarization can lead to autophagy, it was determined whether mitophagy may be mediating the loss of mitochondria in tumor-infiltrating T cells. No significant increases were observed in LC3b staining in permeabilized OT-I T cells, suggesting that autophagic processes are likely not overtly deregulated in these cells (FIG. 5D). Tumor-bearing mice were treated 24 h after adoptive transfer with the mitophagy and mitochondrial fission inhibitor m-divi- 1 (Cui et al., 2010); this also failed to improve mitochondrial staining in tumor infiltrating T cells (FIG. 5E). It was observed that response of naive or previously activated OT-I T cells to B 16 ^OVA tumor cells in vitro did not result in mitochondrial mass loss (FIGS. 6A, 6B).

Thus, T cells responding to cancer lose oxidative metabolism relatively rapidly, but this requires signals that are present specifically in the tumor microenvironment.

Example 5

Loss of mitochondrial mass correlates with upregulation of co-inhibitory molecules

As loss of mitochondrial function was progressive and specific to the tumor

microenvironment, the relationship between the loss of mitochondria seen in tumor-infiltrating T cells and the expression of molecular markers for dysfunctional, 'exhausted' T cells was determined. B16 melanoma is highly enriched for dysfunctional T cells expressing high levels of PD-1, LAG-3 and Tim-3 (FIG. 7A). Indeed, mitochondrial loss in the polyclonal T cell response appeared to be progressive, as T cells expressing more co-inhibitory molecules had decreased mitochondrial mass, as evidenced by MitoTracker FM staining as well as staining for VDAC (FIGS. 7B, 7C, and FIG. 8A). While mitochondrial mass was inversely correlated with upregulation of coinhibitory molecules, glucose competency was consistently depressed in tumor- infiltrating T cells and did not specifically correlate with these markers (FIG. 7D), in agreement with previous reports (Chang et al., 2015; Ho et al., 2015). This resulted in a failure to maintain a sufficient reserve of ATP (as measured directly ex vivo) (FIG. 7E).

Similar results were observed in MC38 and LLC (FIGS. 8B, 8C). Notably, LLC does not induce similar sustained co-inhibitory molecule expression in CD8 ⁺ T cells (compared to the other two models) (FIG. 8C) but still exhibited a significant mitochondrial defect (FIGS. 2B, 1A-1E). CD8 ⁺ T cells infiltrating head-and-neck cancers exhibit decreased MitoTracker staining compared to PBL T cells (FIG. 2D) and high levels of coinhibitory molecule expression (FIG. 7F) that correlated with mitochondrial loss (FIG. 7G).

To determine whether, directly, lower MitoTracker FM staining correlated with poor cytokine production, tumor-infiltrating T cells were sorted based on MitoTracker FM staining and then stimulated 16 h to monitor cytokine production. Consistent with their 'exhausted' phenotype, T cells having the lowest mitochondrial staining have the lowest cytokine production (FIG. 8D). Thus, T cells infiltrating solid tumors show a progressive loss of mitochondrial mass and function that appears to be progressive, such that the most 'exhausted' cells show the lowest mitochondrial mass.

Example 6

Loss of oxidative metabolism in cancer is largely independent of PD-1 signaling and regulatory T cells

PD- 1 blockade can reverse tumor-induced T cell dysfunction and lead to heightened antitumor immunity and cancer regression (La-Beck et al., 2015). To determine if PD-1 blockade might rescue loss of mitochondrial function in tumor-infiltrating T cells, B16 was used, in which PD-1 therapy is not effective, despite the presence of large numbers of PD-1 ⁺ T cells, as well as MC38, which is sensitive to PD-1 monotherapy (Woo et al., 2012). Mice were inoculated with B 16 or MC38 tumors and received anti-PD-1 therapy (200 μg, thrice weekly) or its isotype control when palpable tumors were present (l x l mm). However, regardless of treatment or tumor type, tumor-infiltrating T cells showed similar decreases in mitochondrial mass (FIGS. 9A, 9B). The PD-1 blockade strategy was therapeutically effective, resulting in 40% regression in MC38 bearing mice (FIG. 9C).

To determine whether PD- 1 signaling might impact the mitochondrial sufficiency of recent entrants into the tumor, dye-labeled OT-I T cells were transferred into mice bearing established B 16 ^OVA tumors under the cover of PD-1 blockade or its control for 72h. In this situation, a temporary and incomplete recovery of MitoTracker FM staining in later cell divisions was observed (FIG. 9D). However, these changes were statistically significant only when analyzed as broken down by cell division, and could not be sustained or detected past 72 h. Thus, while PD-1 may play a role in modulation of metabolism, blockade of PD- 1 is not sufficient to reverse

mitochondrial insufficiency observed in tumor-infiltrating T cells.

Regulatory T cells also represent a major immunosuppressive player in the tumor microenvironment (Liu et al., 2016). Thus, it was determined whether T _reg cells mediate metabolic insufficiency in the tumor microenvironment by examining CD8 ⁺ TIL from Foxp3 ^{DTR GFP} mice treated with diphtheria toxin (Kim et al., 2007a). This results in near complete depletion of tumor- infiltrating T _reg cells (FIG. 10A) but no significant increases in CD8 ⁺ T cell MitoTracker FM staining (FIG. 10B). In agreement with these in vivo findings, CD8 ⁺ T cells suppressed in vitro by purified T _reg cells also maintain mitochondrial sufficiency (FIG. IOC).

Thus, metabolic insufficiency in CD8 ⁺ TIL appears to be driven in a manner independent of 'classic' immunosuppressive mechanisms in the tumor microenvironment.

Example 7

PGCla-mediated mitochondrial biogenesis is repressed

by Akt in tumor -infiltrating T cells

Having found that PD-1 and T _reg cells did not appear to outright cause the mitochondrial dysfunction seen in tumor- infiltrating T cells, the molecular mechanism for this metabolic phenotype was determined. Kinetic analyses showed that these T cells divided extremely rapidly in response to tumor antigen in the LNs, so it was hypothesized that T cells failed to properly program mitochondrial biogenesis during rapid cell division upon entry into the tumor microenvironment. Mitochondrial replication is programmed in part by the transcription factor TFAM and regulated by the transcriptional coactivator PGCla (encoded by Ppargcla) (Finck and Kelly, 2006; Spiegelman, 2007). Intracellular staining and qPCR analysis revealed that tumor infiltrating CD8 ⁺ T cells have dramatically lower levels of PGCla (FIGS. 11A and 12A). Kinetic analysis of dye labeled, naive OT-I cells injected into B16 ^OVA -bearing mice showed PGCla downregulation occurred

concomitant with cell division specifically in the tumor microenvironment, suggesting microenvironment-derived signals promoted a downregulation of mitochondrial biogenesis during T cell proliferation (FIG. 11B). Repression of PGCla occurred even in the presence of PD-1 blockade, suggesting that another dominant signal present in the tumor microenvironment suppresses PGCla expression (FIG. 12B).

Analysis of PGC la-deficient T cells (Ppargclaf ^/f Cd4 ^Cre mice) revealed progressive losses of mitochondrial mass in vitro after activation, resulting in decreased OCR (FIGS. 12C, 12D). Comparison of cytokine production in LN- and TIL-resident OT-I T cells responding to cognate peptide revealed that the small proportion of PGCla ⁺ cells in tumor-infiltrating compartments marked the T cells that were competent to produce cytokines, indicating that this pathway is important for intratumoral T cell function (FIG.l 1C). In agreement with this observation, T cells showing repressed PGCla staining also show decreased T-bet and Ki-67 staining, consistent with a model in which PGCla repression is concomitant with a terminally exhausted phenotype (FIGS. 12E, 12F).

PGCla is dynamically regulated by a number of signaling pathways relevant to T cell activation, but a prominent repressive pathway is mediated by Akt (Fernandez-Marcos and

Auwerx, 2011). Akt has been shown to upregulate glycolytic metabolism through a variety of mechanisms, but it also can actively repressive oxidative programs, particularly through the phosphorylation and consequent inactivation of Foxo family transcription factors, which have been previously shown to promote PGCla expression (Borniquel et al., 2010; Olmos et al., 2009). Thus, the Akt status of tumor-infiltrating T cells was determined, with the hypothesis that the strong, chronic activation signals mediated by persistent antigen in cancer might promote Akt activation and repress the oxidative phenotype programmed by Foxo.

CD8 ⁺ T cells infiltrating B16 tumors show increased Akt activation and Akt-mediated inhibitory Foxo phosphorylation compared to LN (where the vast majority of T cells are resting) (FIG. 11D). Comparison of Akt activation to PD-1 status revealed that Akt is highest in tumor- infiltrating T cells that are PD-l ^mid as well as those expressing very high levels of PD-1 (as well as LAG-3), suggesting that Akt is persistent in newly activated T cells as well as those receiving chronic stimulation and differentiating toward terminal exhaustion (FIG. 12G). Intratumoral cells that have high pAkt are particularly low in PGCla protein levels (FIG. HE).

Early (3 d) and late (6 d) responses of T cells responding to B 16 ^OVA or VV ^OVA were examined, and it was observed that while Akt activation in acute viral infection is transient, T cells activated in the tumor microenvironment appear to show chronic Akt signaling, persisting 6 days after adoptive transfer (FIG. 1 IF). Short-term treatment (72 h) of B 16-bearing mice with a potent Akt kinase inhibitor revealed the Akt, in part, mediates losses in PGCla and mitochondrial sufficiency, such that treatment with Akt inhibitor results in partial rescue of the metabolically suppressive phenotype (FIGS. 11G, 11H).

Thus, T cells responding in tumor microenvironments repress mitochondrial biogenesis through repression of PGCla, driven, in part, by chronic Akt activation and consequent repression of Foxo activity.

Example 8

Metabolic reprogramming of tumor -specific T cells results in

increased antitumor immunity

Having demonstrated that PGCla acts as a key node of dysregulation for mitochondrial sufficiency in tumor- specific T cells, it was determined whether reprogramming T cells to favor mitochondrial biogenesis would result in increased intratumoral T cell persistence and function. To this end, retroviral vectors were used to overexpress PGCla and transduced OT-I T cells. PGCla overexpression significantly increased mitochondrial levels early (48 and 96 h) after transduction in in vitro culture, although during the expansion phase, empty- vector expressing cells initiate mitochondrial biogenesis and eventually reach mitochondrial levels equivalent to their

reprogrammed counterparts (FIG. 13A); however, even at this later stage, PGCla-reprogrammed T cells show significantly increased OXPHOS (FIG. 13 A).

In addition, PGCla-overexpressing T cells showed a significant upregulation of spare respiratory capacity (FIG. 13A), indicating high mitochondrial reserve and that mitochondrial biogenesis was primed in these reprogrammed cells. No significant increases in aerobic glycolysis (ECAR) was observed in these cells, although a trend was observed in some experiments (FIG. 13A). It was determined if any particular carbon source dominated this increase in SRC. FCCP- uncoupled T cells were treated with inhibitors of pyruvate, fatty acid, or glutamine oxidation, revealing that the increased respiratory capacity did not preferentially apply to a particular carbon source, suggesting that mitochondrial capacity was improved generally when PGCla expression was enforced (FIG. 14A). In vitro, these T cells exhibit similar effector function as their control counterparts, suggesting that in this environment where nutrients are not limiting, T cells are operating more or less at maximal capacity (FIG. 14B).

OT-I T cells overexpressing PGCla transferred into mice with established B 16 ^OVA tumors were resistant to loss of mitochondrial sufficiency and highly enriched in the tumor

microenvironment (FIG. 13B). Restimulation with OVA peptide showed that these metabolically reprogrammed T cells were superior at producing type 1 cytokines, compared to their wild-type counterparts (FIG. 13C). Notably, these reprogrammed T cells expressed (at even higher levels than EV) co-inhibitory molecules, suggesting retention of mitochondrial function promoted further activation and upregulation of these checkpoint molecules (FIG. 14C).

Having observed that PGCla-reprogrammed T cells display increased effector function, it was determined whether these T cells had better therapeutic efficacy. Mice bearing small (2-6mm ² ) B16 ^OVA tumors received an adoptive transfer of 250,000 (if tumor was < 4mm ² ) or 500,000 (if starting tumor was > 4mm ² ) PGCla or empty- vector transduced OT-I T cells, and tumor growth was measured over time. PGCla-reprogrammed T cells show enhanced antitumor efficacy resulting in significantly prolonged survival a higher incidence of complete regressions (20%) in this highly aggressive tumor model (FIGS. 13D, 13E).

Thus, reprogramming tumor- specific T cells to favor mitochondrial biogenesis protects them from the loss of function observed in the tumor microenvironment.

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In view of the many possible embodiments to which the principles of the disclosure may be applied, it should be recognized that the illustrated embodiments are only examples of the disclosure and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. I therefore claim as my invention all that comes within the scope and spirit of these claims.