WO2017079113A1 - Methods of producing t cell populations using prolyl hydroxylase domain-containing protein inhibitors - Google Patents

Abstract

Provided are methods of producing an isolated population of T cells for adoptive cell therapy, the method comprising culturing isolated T cells in vitro in the presence of one or both of (i) a prolyl hydroxylase domain containing protein (Phd) inhibitor and (ii) a low-oxygen atmosphere, wherein the T cells have antigenic specificity for a cancer antigen. Also provided are related isolated populations of T cells, pharmaceutical compositions, and methods of treating or preventing cancer in a mammal.

Description

METHODS OF PRODUCING T CELL POPULATIONS USING PROLYL HYDROXYLASE DOMAIN-CONTAINING PROTEIN INHIBITORS

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This patent application claims the benefit of U.S. Provisional Patent Application No. 62/249,645, filed November 2. 2015, which is incorporated by reference in its entirety herein.

INCORPORATIONS Y-REFEKENCE OF MATERIAL SUBMITTED

ELECTRONICALLY

[0002] Incorporated by reference in its entirety herein is a computer-readable

nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: One 54, 152 Byte ASCII (Text) file named "726756 ST25.TXT," dated November I, 2016.

BACKGROUND OF THE INVENTION

[0003] Adoptive cell therapy (ACT) using cancer reactive T ceils can produce positive clinical responses in cancer patients. Ne vertheless, several obstacles to the successful use of ACT for the treatment of cancer and other diseases remain. For example, regulatory T cells (Treg) may contribute to an immunosuppressive tumor microenvironment that may hamper the effectiveness of anti-cancer T celis. Accordingly, there is a need for methods of obtaining an improved isolated population of T cells for ACT.

BRIEF SUMMARY OF THE INVENTION

[0004] An embodiment of the invention provides a method of producing an isolated population of T cells for adoptive ceil therapy, the method comprising culturing isolated T ceils having antigenic specificity for a cancer antigen in vitro in the presence of a prolyl hydroxylase domain-containing protein (Phd) inhibitor.

[0005] Another embodiment of the invention provides a method of producing an isolated population of T cells for adoptive cell therapy, the method comprising culturing isolated T cells having antigenic specificity for a cancer antigen in vitro in the presence of an atmosphere, wherein the atmosphere has an oxygen content less than or equal to 5%, [0006] Further embodiments of the invention provide related isolated populations of T cells, pharmaceutical compositions., and methods of treating or preventing cancer in a mammal,

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

[0007] Figure 1 A is an image of an electrophoresis gel showing Foxp3₃ Tbet, and β-actin proteins detected in whole ceil i sate isolated from CD4^÷ T cells stimulated under ThO or iTreg conditions in the presence or absence of DMOG.

[0008] Figure IB is a graph showing the amount of interferon-gamtna (IFN-γ) measured in the supernatant of CD4^" T cells stimulated under ThO conditions in the presence or absence of DMOG for 72 hours. **** PO.0001 , Students T-test.

[0009] Figure 1 C shows the tumor size (area, mm") in mice treated with no ceils (circles) or 1x10" Trp-1 CD4+ T cells that were treated with control vehicle (squares) or DMOG (diamonds) at various time points (days) after transfer. Horizontal bars indicate means +/- SEM of individually evaluated mice, **P<0.001 (Wilcoxon rank sum),

} 001.0] Figure ID shows the survival (%) of tumor-bearing mice treated with no cells (grey dotted line) or 1 10° Trp-1 CD4+ T cells that were treated with control vehicle (black dotted line) or DMOG (solid line) at various time points (days) after transfer, Horizontal bars indicate means -⁽- - SEM of individually evaluated mice. **P<0.001 (Wilcoxon rank sum}.

[0011] Figure 2A is a graph showing the population distribution (%) of naive (N) (grey portion of bar), central memory (CM) (unshaded portion of bar) and effector memory (EM) (black portion of bar) cells in CD8⁺ Pmef transgenic splenocytes that were stimulated in the absence (VEH) or presence of the Phd inhibitor DMOG.

[0012] Figure 2B is a graph showing the percentages of ceils stimulated in the presence (shaded bars) or absence (unshaded bars) of DMOG having the indicated cytokine profile. ***<0.0001 , Student's T-test. S-not significant.

j0013j Figure 2C shows the tumor size (area, mm^"') in mice treated with no cells (circles) or 1 xl O⁶ Pmef CD8^÷ T ceils that were treated with vehicle (squares) or DMOG (diamonds) at various time points (days) after transfer, Horizontal bars indicate means /- SEM of individually evaluated mice. **P<0.001 (Wilcoxon rank sum).

[0014] Figure 2D shows the survival (%) of tumor-bearing mice treated with no cells (grey dotted line) or l xl O⁶ Pmef CD8⁺ T cells that were treated with vehicle (black dotted line) or DMOG (solid line) at various time points (days) after transfer. Horizontal bars indicate means +/- SEM of individually evaluated mice. ***p<0.G0I (Wiicoxon rank sum). 0015] Figures 3 A and 3B are graphs showing the percentage of cells expressing Foxp3 (A) or Tbet (B) when stimulated under ThO or iTreg conditions in the presence of 1 % or 20% oxygen. **p<0.001, Student's T-test.

[0016] Figure 4A is a representative ventral image of the gross anatomic examination of the lungs of wild type (WT) and Phd-tKO mice 21 days after injection with melanoma cells. The black spots on the WT kings are tumors.

[0017] Figure 4B is a representative dorsal image of the gross anatomic examination of the lungs of wild type (WT) and Phd-tKO mice 21 days after injection with melanoma cells. The black spots on the WT lungs are tumors.

[0018] Figure 4C is a graph showing the total tumor burden (number of tumors) in multiple WT or tKO mice 21 days after injection with melanoma cells. ** = p<0.001 , Student's T-tesi.

[00 ] Figure 5A is a photographic image of the livers of WT mice following intrasplenic tumor injection,

| 020] Figure 5B is a photographic image of the livers of PHD tKO mice following intrasplenic tumor injection,

[0021] Figure 5C is a graph showing the liver mass (g) measured in WT and PHD tKO mice following intrasplenic tumor injection.

[0022] Figure 5D is a graph showing the number of visible tumo nodules measured in the livers of WT and PFID tKO mice following intrasplenic tumor injection.

[0023] Figure 5E is a graph showing the number of rmcrometastatic lesions counted by histologic enumeration in the livers of WT and PFID tKO mice following intrasplenic tumor injection,

DETAILED DESCRIPTION OF THE INVENTION

[0024| It has been discovered that T cells that have been cultured in the presence of one or both of (i) a Phd inhibitor and (ii) a low-oxygen atmosphere have an improved capacity to target cancer antigen-expressing cells. CD4+ T ceils that have been cultured in the presence of one or both of (i) a Phd inhibitor and (ii) a low-oxygen atmosphere may, advantageously, adopt a Thl phenotype. Thl ceils can mediate anti-tumor immune responses at least in part through their capacity to produce the cytokine mterferon-gamma (IFN-γ), which has and- tumor properties. CD8+ T ceiis that have been cultured in the presence of one or both of (i) a Phd inhibitor and (ii) a low-oxygen atmosphere may, advantageously, express surface markers consistent with an effector memory phenotype. Effector memory cells may provide anti-tumor activity.

[0025] The prolyl hydroxylase domain containing (Phd) proteins are a family of enzymes that provide oxygen sensing machinery within T cells. The Phd family of proteins includes Phd-1, Phd-2, and Phd-3, The gene Eglnl encodes Phd2, the gene Egin2 encodes Phdl, and the gene EglnS encodes Phd3. The main protein substrates of the Phd enzymes are the transcription factors Hypoxia Inducible Factor 1 -alpha and Hypoxia inducible Factor 2-alpha (HIFla and H!F2a). in the presence of sufficient oxygen, the Phd proteins enzymaticaily hydroxylate HIFla and HIF2a, thus targeting these proteins for proteasome-mediated degradation. However, under very low oxygen conditions, the Phd proteins are unable to hydroxylate HIFla and HIF2a. As a result, the HIFl and HIF2a proteins are able to accumulate, translocate to the cell nucleus, and drive the expression of genes containing "hypoxia-response element" sequences in their genomic regulatory regions. Without being bound to a particular theory or mechanism, it is believed that HIFl and HIF2a may have roles in directing T cell metabolism and differentiation in both CD4^÷ and CDS⁺ T cells.

[0026] in tins regard, an embodiment of the invention provides a method of producing an isolated population oi^'T cells for adoptive ceil therapy, the method comprising culturing isolated T cells having antigenic specificity for a cancer antigen in vitro in the presence of a Phd inhibitor.

[0027] The method may comprise isolating T cells from a mammal by any suitable method known in the art. For example, the T cells can be obtained from the mammal by a blood draw or a leukapheresis. In an embodiment of the invention, the method comprises isolating peripheral blood lymphocytes (PBL) or a peripheral blood mononuclear cells (PBMC) from a mammal. Alternatively or additionally, the T cells can be obtained from a rumor sample taken from the mammal. In this regard, the T cells may be tumor infiltrating lymphocytes (TIL).

[0028] The population of T cells may include any type of T cells. For example, the T cells may be a cultured T cell, e.g., a primary T cell, or a T cell from a cultured T cell line, e.g.. Jurkat, SupTl , etc., or a T ceil obtained from a mammal, if obtained from a mammal, the T cell can be obtained from numerous sources, including but not limited to blood, bone marrow, lymph node, the thymus, tumor, or other tissues or fluids. T cells can also be enriched for or purified. The T cell may be a human T cell. The T cell can be any type of T cell and can be of any developmental stage, including but not limited to, CD4^'7CD8⁺ double positive T cells, CD4^'r helper T ceils, e.g., Thi and Tl¾ cells, CD8⁺ T ceils (e.g., cytotoxic T cells), tumor infiltrating lymphocytes (TIL), memory T cells, naive T cells, and the like. The T cell may be a CD8⁺ T cell or a CO4^÷ T cell. In a preferred embodiment, the T cells are TIL.

[0029] Unless stated otherwise, as used herein, the term ^"mammal" refers to any mammal including, but not limited to, mammals of the order Logomorpha, such as rabbits: the order Camivora, including Felines (cats) and Canines (dogs); the order Artiodactyla, including Bovines (cows) and S wines (pigs); or of the order Ferssodactyla, including Equines (horses). It is preferred that the mammals are non-human primates, e.g., of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes), in some embodiments, the mammal may be a mammal of the order Rodentia, such as mice and hamsters, in other embodiments, the mammal is not a mouse, Preferably, the mammal is a non-human primate or a human, An especially preferred mammal is the human,

|0030] The method comprising eulturing tumor fragments, isolated PBMC, or PBL (e.g., T cells) in vitro in the presence of one or more Phd inhibitors. The Phd inhibitor may be any agent that inhibits any one or more of Eg!nl-3 R A expression, Phd protein expression, and Phd biological activity (e.g., the ability of the Phd protein(s) to enzymatically hydroxylate one or both of HIFia and HIF2a). The Phd inhibitor may inhibit any one or more of the proteins in the Phd family and/or the corresponding mRNA, For example, the Phd inhibitor may inhibit any one of Phd-1, Phd-2, and Phd-3 and/or the corresponding mRNA , in another embodiment of the invention, the Phd inhibitor may inhibit any two Phd proteins, such as Phd-1 and Phd-2, Phd-1 and Phd-3, or Phd-2 and Phd-3, and/or the corresponding mRNA molecules. In still another embodiment of the invention, the Phd inhibitor may be a pan-Phd inhibitor thai inhibits all three members of the Phd family, i.e., Phd-1 , Phd-2, and Phd-3 and/or the corresponding mRNA molecules.

[0031] in an embodiment of the invention, the Phd inhibitor is a small molecule inhibitor of Phd. The small molecule Phd inhibitor may be an aliosteric inhibitor or a non-allosteric inhibitor of Phd. Examples of small molecule Phd inhibitors that may be useful in the inventive methods include, but are not limited to, dirnethyloxaiylglyeine, N- (methoxyoxoacetyl) -glycine methyl ester (DMOG), IOX2, desferoxamine. 3.4,dihydroxybenzoie acid (DHB), and HIF Phd Inhibitor 4. Preferably, the Phd inhibitor is DMOG.

|0032] In an embodiment of the invention, the Phd inhibitor is a small interfering RNA (siRNA), microRNA, antisense nucleic acid, or CRISPR-Cas9 guide RNA (gRNA). The Phd inhibitor can be a nucleic acid at least about 10 nucleotides in length that specifically binds to and is complementary to a target nucleic acid encoding Phd or a complement thereof. The Phd inhibitor may be introduced into the T cel ls, wherein the cells are capable of expressing Phd, in an effective amount for a time and under conditions sufficient to interfere with expression of Phd. In some embodiments, RNA interference (RNAi) is employed, In this regard, the Phd inhibitor may comprise an RNAi agent. In an embodiment, the RNAi agent may comprise a small interfering RNA (siRNA), a microRNA (miRNA), or an antisense nucleic acid. The RNAi agent, e.g., siRNA, miRNA, and/or antisense nucleic acid can comprise overhangs. That is, not all nucleotides need bind to the target sequence. RNA interference nucleic acids employed can be at least about 19, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about I SO, at least about 200, at least about 220, at least about 240, from about 19 to about 250, from about 40 to about 240, from about 60 to about 220, from about 80 to about 200, from about 60 to about 180, from about SO to about 160, and/or from about 100 to about 140 nucleotides in length.

[0033J The RNAi agent, e.g., siRNA or shRNA, can be encoded by a nucleotide sequence included in a cassette, e.g., a larger nucleic acid construct such as an appropriate vector. Examples of such vectors include leniivirai and adenoviral vectors, as well as other vectors described herein with respect to other aspects of the invention. An example of a suitable vector is described in Aagaard et al. Mol. Ther., 15(5): 938-45 (2007). When present as part of a larger nucleic acid construct, the resulting nucleic acid can be longer than the comprised RNAi nucleic acid, e.g., greater than about 70 nucleotides in length. In some embodiments, the RNAi agent employed cleaves the target mRNA. In other embodiments, the RNAi agent employed does not cleave the target mRNA.

[0034] Any type of suitable siRNA, miRNA, and/or antisense nucleic acid can be employed. In an embodiment, the antisense nucleic acid comprises a nucleotide sequence complementary to at least about 8, at least about 15, at least about 19, or from about 19 to about 22 nucleotides of a nucleic acid encoding Phd or a complement thereof In an embodiment, the siRNA may comprise, e.g., trans-acting siRNAs (tasiRNAs) and/or repeat- associated siRNAs (rasiRNAs). in another embodiment, the miRNA may comprise, e.g., a short hairpin miRNA (shMIR).

[0035] The Phd inhibitor may inhibit or downregulate to some degree the expression of the protein encoded by an Egln gene, e.g., at the D. , RNA, or other level, of regulation. In. this regard, a T cell comprising a Phd inhibitor expresses no Phd protein or lower levels of Phd protein as compared to a T cell that lacks a Phd inhibitor. Alternatively or additionally, a T cell comprising a Phd inhibitor expresses no Egln mRNA or lower levels of. Egln mRNA as compared to a T cell that lacks a Phd inhibitor. In accordance with an embodiment of the invention, the Phd inhibitor, such as an RNAi agent, such as a shMIR, can target a nucleotide sequence of an Egln gene or mRNA encoded by the same. In an embodiment, the Egln sequence is a human sequence. For example, human Egln2 mRN A sequences include Genbank Accession Nos. NM 053046.3 (SEQ ID NO: 1) and NM 080732.3 (SEQ ID NO: 2), with corresponding human Phd-1 protein sequences NP _444274, 1 (SEQ ID NO: 3) and NP_542770.2 (SEQ ID NO: 4), respectively. Human Egln I mRNA sequences include Genbank Accession Nos. NM_. 022051.2 (SEQ ID NO: 5), XM_ 005273167.3 (SEQ ID NO: 6), and XM 005273166.3 (SEQ ID NO: 7), with corresponding human Phd-2 protein sequences NP_071334.1 (SEQ ID NO: 8), XP_005273224.1 (SEQ ID NO: 9), and

XP_005273223, 1 (SEQ ID NO: 10), respectively. Human Egln-3 mRNA sequences include Genbank Accession Nos. NM_...001308103.1 (SEQ ID NO: 11) and NM_...022073.3 (SEQ ID NO: 12). Human Phd-3 protein sequences include ΝΡ_...001295032.1 (SEQ ID NO: 13) and NP_071356.1 (SEQ ID NO: 14). Other human sequences, as well as other Phd species can be employed in accordance with the invention.

[0036] In accordance with an embodiment of the invention, the Phd inhibitor, such as an RNAi agent, such as a shMIR, can target a nucleotide sequence selected from the group consisting of the 5^* untranslated region (5' UTR), the V untranslated region (3" UTR), and the coding sequence oiEgln, complements thereof, and any combination thereof. Any suitable Egln target sequence can be employed. RNAi agents can be designed against any appropriate Egln mRNA sequence,

[0037] In an embodiment of the invention, the Phd inhibitor may be an artificially engineered nuclease that inhibits expression of Phd. For example, Phd expression may be inhibited in a T cell using a genome editing technique. Genome editing techniques can modify gene expression in a target cell by inserting, replacing, or removing DNA in the genome using an artificially engineered nuclease. Examples of such nucleases may include zinc finger nucleases (ZF s) (Goniraans et a.l. J. Mo! Biol., 354(3): 507-519 (2005)), transcription activator-iike effector nucleases (TALENs) (Zhang et al., Nature Biotechnol, 29: 149-153 (201 1 )), the CRTSPR Cas system (Cheng et aL Cell Res., 23: 1163-71 (2013)), and engineered meganucleases (Riviere et al., Gene Ther., 21 (5): 529-32 (2014)). The nucleases create specific double-stranded breaks (DSBs) at targeted locations in the genome, and use endogenous mechanisms in the cell to repair the induced break by homologous recombination (HR) and nonhomologous end-joining (NHEJ). Such ieclmiques may be used to achieve inhibition of Phd in T ceils, in an embodiment of the invention, the Phd inhibitor is a CRISPR-Cas9 guide RNA (gRNA).

[0038] By "nucleic acid" as used herein includes "polynucleotide," "oligonucleotide," and "nucleic acid molecule," and generally means a polymer of DNA or RNA. which can be single- stranded or do ble- stranded, synthesized or obtained (e.g., isolated and/or purified) from natural sources, which can contain natural, non-natural or altered nucleotides (e.g., ribonucleic acid nucleotides as well as deoxyribonucleic acid nucleotides), and which can contain a natural, non-natural or altered internucieotide linkage, such as a phosphoroamidate linkage or a phosphorotliioatc linkage, instead of the phosphodiester found between the nucleotides of an unmodified oligonucleotide. When a nucleic acid is recked, it refers genetically to nucleic acids and includes DNA and RNA unless the recitation explicitly states that the nucleic acid is a specific one, e.g., DNA or RNA. If a nucleic acid refers to a sequence that contains thymine (1). that does not necessarily indicate that the nucleic acid is DNA; in some embodiments the nucleic acid is RNA and/cr DNA. Similarly, if a nucleic acid refers to a sequence that contains uracil (u), that does not necessarily indicate that the nucleic acid is RNA; in some embodiments, the nucleic acid is DNA and/or RNA. It is generally preferred that the nucleic acid does not comprise any insertions, deletions, inversions, and/or substitutions. However, it may be suitable in some instances tor the nucleic acid to comprise one or more insertions, deletions, inversions, and/or substitutions.

[0039] The nucleic acid molecules relevant to the invention can readily be obtained in a variety of ways, including, without limitation, chemical synthesis, cDNA or genomic library screening, expression library screening, and/or PGR amplification of cDNA. These methods and others useful for isolating such DNA are set forth, for example, in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4^th Ed.), Cold Spring Harbor Laboratory Press (2012). [0040] Chemical synthesis of a nucleic acid molecule can be accomplished using methods well known in the art. These methods include, inter aiia, the phosphotriester, phosphoramidite and H-phosphonate methods of nucleic acid synthesis. Nucleic acids larger than about 100 nucleotides in length can be synthesized as several fragments, each fragment being np to about 100 nucleotides in length. The fragments can then be ligated together to form a full length nucleic acid encoding the polypeptide. One method is polymer-supported synthesis using standard phosphoramidite chemistry.

[0041] Alternatively, the nucleic acid can be obtained by screening an appropriate cDNA library prepared from one or more tissue source(s) that express the polypeptide, or a genomic library from any subspecies. The source of the genomic library may be any tissue or tissues from a mammalian or other species believed to harbor a gene encoding a protein relevant to the invention (e.g., Phd). The library can be screened for the presence of a cDNA/gene using one or more nucleic acid probes (oligonucleotides. cDNA or genomic D A fragments that possess an acceptable level of homology to the gene or gene homologue cDNA or gene to be cloned) that will hybridize selectively with the gene or gene homologue cDNA(s) or gene(s) that is(are) present in the library. The probes preferably are complementary to or encode a small region of the DNA sequence from the same or a similar species as the species from which the library was prepared . Alternatively, the probes can be degenerate. After hybridization, the blot containing the library is washed at a suitable stringency, depending on several factors such as probe size, expected homology of probe to clone, type of library being screened, number of clones being screened, and the like. Stringent washing solutions can be low in ionic strength and are used at relatively high temperatures.

[0042] Another suitable method for obtaining a nucleic acid for use in accordance with the invention is the polymerase chain reac tion (PGR). n this method, poiy(A)÷RN A or total RKA is extracted from a tissue that expresses the gene product. cDNA is then prepared from the RJ A using the enzyme reverse transcriptase. Two primers typically complementary to two separate regions of the cDNA (oligonucleotides) are then added to the cD A along with a polymerase such as Taq polymerase, and the polymerase amplifies the cDNA region between the two primers.

[0043] An embodiment of the invention provides for the use of isolated, purified or enriched nucleic acid sequences of, for example, about 15 to about 500 nucleotides in length, about 1 5 to about 100 nucleotides in length, about 1 5 to about 50 nucleotides in length, and about 15 to about 30 nucleotides in length, which have a sequence that corresponds to a portion of one of the nucleotides described herein. The nucleic acid can be at least about 17, about 20, about 22, or about 25 nucleotides in length. The nucleic acid sequence can he about 30 to about 300 nucleotides in length, or about 45 to about 200 nucleotides in length, or about 45 to about 100 nucleotides i length. The nucleic acid can be at least about 5, about 6, about 7, about 8, about 9, about 10, about 12, bout 1 5, bout 17, about 20, about 22, bout 25, about 30, about 35, bout 40, about 50, about 100, about 1 0, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 1000, about 10,000, about 50,000, about 100,000 or more nucleotides in length, about 100,000, about 75,000, about 50,000, about 10,000, about 5,000, about 1000, about 750, about 500, about 250, about 200, about 100, about 50, about 40, about 30, about 25, about 22, about 20, about 17, about 15, about 12, about 10, about 9, about 8, about 7, about 6, about 5, or fewer nucleotides in length. The nucleic acid can have a length in a range from any one of the above lengths to any other of the above lengths including endpoints.

[0044] A nucleic acid or protein relevant to the invention can be at least, e.g., about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 82%, about 84%, about 86%, about 88%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to reference sequences provided herein.

[0045] Preferably, the nucleic acids described herein are recombinant. As used herein, the term "recombinant" refers to (i) molecules that are constructed outside living cells by joining natural or synthetic nucleic acid segments to nucleic acid molecules that can replicate in a living cell, or (ii) molecules that result from the replication of those described in (i) above. For purposes herein, the replication can be in vii.ro replication or in vivo replication.

[0046] Another embodiment of the invention provides a method of producing an isolated population of T cells for adoptive ceil therapy, the method comprising culturing isolated T cells having antigenic specificity fo a cancer antigen in vitro in a low-oxygen atmosphere, in an embodiment, the atmosphere has an oxygen content less than or equal to about 5%, less than or equal to about 4%, less than or equal to about 3%, less than or equal to about 2%, or less than or equal to about 1 %, or a r ange between any two of the foregoing values. The T cells may be cultured in a !ow-oxygen atmosphere in the presence or absence of a Phd inhibitor. [0047] The T cells may be cultured in ihe presence of one or both of (i) a Phd inhibitor and (ii) a low-oxygen atmosphere in any suitable manner. In an embodiment of the invention, the T cells are cultured in the presence of one or both of (i) a Phd inhibitor and (ii) a low-oxygen atmosphere with a cytokine such as, for example, interleukin-2 (IL-2), mterleukin-7 (I.L-7), mierleukin-15 (IL-15), interleukin-12 (IL-12) or a combination of two or more of the foregoing,

[0048] in an embodiment of the invention, the method further comprises introducing a nucleic acid encoding an exogenous T cell receptor (TCR) into the T cells under conditions sufficient to express the 'ICR by the T cells. The nucleic acid may be introduced into the T cells in ihe presence of one or both of (i) a Phd inhibitor and (ii) a low-oxygen atmosphere. By "exogenous" is meant that the TCR is not native to (naturally-occurring on) the T ceil. The exogenous TCR may be a recombinant TCR. A recombinant TCR is a TCR. which lias been generated through recombinant expression of one or more exogenous TCR α-, β-, γ-, and/or 8-ehain encoding genes. A recombinant TCR. can comprise polypeptide chains derived entirely from a single mammalian species, or the recombinant TCR can be a chimeric or hybrid TCR comprised of amino acid sequences derived from TCRs from two different mammalian species. For example, the antigen-specific TCR can comprise a variable region derived from a murine TCR, and a constant region, of a huma ICR such that Ihe TCR is "humanized.^'" Any exogenous TCR having antigenic specificity for a cancer antigen may be useful in the inventive methods. The TC generally comprises two polypeptides (i.e., polypeptide chains), such as an a-chain of a TCR, a β-chain of a TCR, a γ-chain of a TCR, a δ-chain of a TCR, or a combination thereof. Such polypeptide chains of TCRs are known in the art. The cancer antigen-specific TCR can comprise any amino acid sequence, provided that ihe TCR can specificaiiy bind to and immunologically recognize a cancer antigen or epitope thereof. Examples of exogenous TCRs that may be useful in the inventive methods include, but are not limited to, those disclosed in, for example. U.S. Patents 7,820,174;

7,915,036; 8,088,379; 8,216,565; 8,431 ,690; 8,613,932; 8,785,601 ; 9, 128,080; and

9,345,748; and U.S. Patent Application Publication Nos. 201 3/01. 16167; 2014/0378389; 2015/0141347; and 2015/0246959, each of which is incorporated herein by reference.

10049] A T cell comprising an endogenous cancer antigen-specific TCR can also be transformed, e.g., transduced or transfected, with one or more nucleic acids encoding an exogenous (e.g., recombinant) TCR or other recombinant chimeric receptor. Such exogenous chimeric receptors, e.g.. chimeric T CRs, can confer specificity for additional antigens to the transformed T celi beyond the antigens for which the endogenous TCR is naturally specific. This can, but need not, result in the production of T cell having dual antigen specificities, |0050] In an embodiment of the invention, the method further comprises introducing a nucleic acid encoding a chimeric antigen receptor (CAR) into the T cells under conditions to express the CAR by the T ceils. The nucleic acid may be introduced into the T ceils in the presence of one or both of (i) a Phd inhibitor and (ii) a low-oxygen atmosphere, Typically, a CAR. comprises the antigen binding domain of an antibody, e.g., a single-chain variable fragment (scFv), fused to ihe transmembrane and intracellular domains of a ^'ICR., Thus, the antigenic specificity of a TCR of the invention can be encoded by a scFv which specifically binds to the cancer antigen, or an epitope thereof Any CAR having antigenic specificity for a cancer antigen may be useful in the inventive methods. Examples of CARs that may he useful in the inventive methods include, but are not limited to, those disclosed in, for example, WO 201 5/187528; U.S. Patents 8,465,743; 9,266,960: and 9359,447; and U.S. Patent Application Publication Nos.2014/0274909 and 201 5/0051266, each of which is incorporated herein by reference.

[0051J In a preferred embodiment, the exogenous TCR or CAR has antigenic specificity for a cancer antigen. The phrases "antigen-specific" and "antigenic specificity,^"' as used herein, mean that the TCR or CAR can specifically bind to and immunologically recognize an antigen, or an epitope thereof, such that binding of the TCR or CAR to antigen, or the epitope thereof, elicits an immune response.

[0052] The nucleic acids described herein can be incorporated into a recombinant expression vector. For purposes herein, the term "recombinant expression vector" means a genetically-modified oligonucleotide or polynucleotide construct that permits the expression of an mRNA, protein, polypeptide, or peptide by a host cell, when the construct comprises a nucleotide sequence encoding the mRNA, protein, polypeptide, or peptide, and the vector is contacted with the cell under conditions sufficient to have the mRNA, protein, polypeptide, or peptide expressed within the cell. The vectors of the invention are not naturally-occurring as a whole. However, parts of the vectors can be natural !y-occurring. The inventive recombinant expression vectors can comprise any type of nucleotides, including, but not limited to DNA and RNA, which can be single-stranded or double-stranded, synthesized or obtained in part from natural sources, and which can contain natural, non-natural or altered nucleotides. The recombinant expression vectors can comprise naturaliy-occurring or non- naniralJy-occurring inlermicleotide linkages, or both types of linkages. Preferably, the non- naturally occurring or altered nucleotides or intermicleotide linkages do not hinder the transcription or replication of the vector. Examples of recombinant expression vectors that may be useful in the inventive methods include, but are not limited to, piasmids, viral vectors (retroviral vectors, gamma-retroviral vectors, or lentiviral vectors), and transposons. The vector may then, in turn, be introduced into the isolated popuiation of T cells by any suitable technique such as, e.g., gene editing, transfection, transformation, or transduction as described, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual (4^1,1 Ed,), Cold Spring Harbor Laboratory Press (2012). Many transfection techniques are known in the art and include, for example, calcium phosphate DMA co-precipitation; DEAE-dextran; eieetroporation; eationic liposome-mediated transfection; tungsten particle-facilitated micropariicle bombardment; and strontium phosphate DNA co-precipitation. Phage or viral vectors can be introduced into host ceils, after growth of infectious particles in suitable packaging cells, many of which are commercially available. Preferably, the vector is introduced into the isolated population of T ceils in the presence of one or both of (i) a Phd inhibitor and (ii) a low-oxygen atmosphere.

[0053] The isolated population of T cells, into which a vector encoding the exogenous TCR or CAR has been introduced, can be cultured ex vivo under conditions to express the exogenous TCR or CAR, and then directly transferred into a mammal (preferably a human) affected by cancer. Such a cell transfer method is referred to in the art as "adoptive ceil transfer" or "adoptive ceil therapy'¹ (ACT). Preferably, the T cells are cultured under conditions to express the exogenous TCR or CAR in the presence of one or both of (i) a Phd inhibitor and (ii) a low-oxygen atmosphere. n an embodiment of the invention, the Phd inhibitor is removed (e.g., washed) from the T cells prior to administering the cells to a patient, Jn another embodiment of the invention, the Phd inhibitor is not removed from the T cells prior to administering the cells to a patient.

[0054] The T cells administered to the mammal can be allogeneic or autologous to the mammal . In "autologous^"" administration methods, cells are removed from a mammal, stored (and optionally modified), and returned back to the same mammal. In "allogeneic" administration methods, a mammal receives ceils from a genetically similar, but not identical, donor. Preferably, the ceils are autologous to the mammal. Autologous cells may, advantageously, reduce or avoid the undesirable immune response that may target an allogeneic ceil such as, for example, graft-versus-host disease. [ 0SS] While the T cells may be cultured in the presence of one or both of (i) a Phd inhibitor and (ii) a low-oxygen atmosphere intermittently in vitro, in a preferred embodiment of the in vention, the T cells are cultured in the presence of one or both of (i) a Phd inhibitor and (ii) a low-oxygen atmosphere for the entire duration of in vitro culture, including during expansion of the numbers of ceils and during introduction of a nucleic acid encoding a CAR or an exogenous TCR into the cells.

[0056] The T cells may have antigenic specificity for a cancer antigen. The term "cancer antigen," as used herein, refers to any molecule (e.g., protein, polypeptide, peptide, lipid, carbohydrate, etc.) solely or predominantly expressed or over-expressed by a tumor cell or cancer cell, such that the antigen is associated with the tumor or cancer. The cancer antigen can additionally be expressed by normal, non-tumor, or non-cancerous cells. However, in such cases, the expression of the cancer antigen by normal, non-tumor, or non-cancerous cells is not as robust as the expression by tumor or cancer ceils, in this regard, the tumor or cancer cells can over-express the antigen or express the antigen at a significantly higher level, as compared to the expression of the antigen by normal, non-tumor, or non-cancerous cells. Also, the cancer antigen can additionally be expressed by ceils of a different state of development or maturation. For instance, the cancer antigen can be additionally expressed by cells of the embryonic or fetal stage, which cells are not normally found in an adult host. Alternatively, the cancer antigen can be additionally expressed by stem cells or precursor cells, which cells are not normally found in an adult host. Examples of cancer antigens include, but are not limited to, mesothelin, CD 19, CD22, CD30, CD70, CD276 (B7H3), gpiOO, MART-1 , Epidermal Growth Factor Receptor Variant III (EGFRVIII), Vascular Endothelial Growth Factor Receptor 2 (VEGFR-2), TRP-1, TRP-2, tyrosinase, human papillomavirus (HPV) 16 E6, HPV 16 E7, RAS. NY-BR-1 , NY-ESO-l (also known as CAG-3), SSX-2, SSX-3, SSX-4, SSX-5, SSX-9, SSX-10, MAGF.-A1 , MAGE-A2. BRCA, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE- A 10, MAGE-A1 1 , MAGE-A12, HER-2, etc. In an embodiment of the invention, the cancer antigen may be a mutated antigen that is expressed or overexpressed by tumor or cancer cells and which is not expressed by normal, non-tumor, or non-cancerous cells. T cells having antigenic specificity for a cancer antigen may, advantageously, reduce or avoid cross-reactivity with normal tissues such as, for example, that which may occur using T cells having antigenic specificity for minor histocompatability antigens. (0057] The cancer antigen can be an antigen expressed by any cell of any cancer or tumor, including the cancers and tumors described herein. The cancer antigen may be a cancer antigen of only one type of cancer or tumor, such that the cancer antigen is associated with or characteristic of only one type of cancer or tumor, Alternatively, the cancer antigen may be a cancer antigen (e.g., may be characteristic) of more than one type of cancer or tumor. For example, the cancer antigen may be expressed by both breast and prostate cancer cells and not expressed at all by normal, non-tumor, or non-cancer cells.

[0058] In an embodiment of the invention, the method further comprises expanding the number of T cells in the presence of one or more non-specific T cell stimuli, one or more cytokines, and. in the presence of one or both of (i) a Phd inhibitor and (ii) a low-oxygen atmosphere. Examples of non-specific T cell stimuli include, but are not limited to, one or more of irradiated allogeneic feeder cells, irradiated autologous feeder cells, anti-CD3 antibodies, anti-4-lBB antibodies, and anti~CD28 antibodies. In preferred embodiment, the non-specific T cell stimulus may be anti-CD3 antibodies and anti-CD28 antibodies conjugated to beads. Any one or more cytokines may be used in the inventive methods. Exemplary cytokines that may be useful for expanding the numbers of cells include interleukin (iL)-2, IL-7, LL-21 , and 1.1.-1.5, The Fhd inhibitor and low-oxygen atmosphere for expanding the numbers of cells may be as described herein with respect to other aspects of the invention.

[0059] Expansion, of the numbers of T cells can be accomplished by any of a number of methods as are known in the art as described in, for example, U.S. Patent 8,034,334; U.S. Patent 8,383,099; and U.S. Patent Application Publication No, 2012/024 133. in an embodiment of the invention, the numbers of T cells are expanded by physically contacting the T ceils with one or more non-specific T cell stimuli and one or more cytokines in the presence of one or both of (i) a Phd inhibitor and (ii) a low-oxygen atmosphere. For example, expansion of the numbers of T cells may be carried out by culturing the T cells with O T3 antibody, 1L-2, and feeder PBMC (e.g., irradiated allogeneic PBMC) in the presence of one or both of (i) a Phd inhibitor and (ii) a low-oxygen atmosphere, in an embodiment of the invention, expanding the number of T cells in the presence of one or both of (i) a Phd inhibitor and (ii) a low-oxygen atmosphere comprises culturing the cells for at least about 14 days in the presence of one or both of (i) a Phd inhibitor and (ii) a low-oxygen, atmosphere. |0060j The invention further provides an isolated or purified population of T cells produced by any of the inventive methods. The populations of T cells produced by the J 6 inventive methods may provide many advantages. For example, the populations of T cells produced by the inventive methods may, advantageously, demonstrate any one or more of greater persistence, proliferation, trafficking to tumor site(s), and antitumor activity upon in vivo transfer as compared to control T cells.

[0061] In an embodiment of the invention, the population of T cells cultured in the presence of the Phd inhibitor has an increased expression of one or both of interferon-gamma (IFN-γ) and tumor necrosis factor alpha (TNF-a) as compared to control T cells, wherein the control T celis are identical to the T cells that are cultured in the presence of a Phd inhibitor except that the control ceils have not been cultured in the presence of a Phd inhibitor. lFN-γ may, advantageously, inhibit tumor ceil proliferation,

[0062] In an embodiment of the invention, the population of T cells cultured in the presence of a low-oxygen atmosphere has an increased expression of one or both of in erferon-gamma (IFN-γ) and tumor necrosis factor alpha (TNF-a) as compared to control T cells, wherein the control T cells are identical to the T cells that are cultured in the presence of a low-oxygen atmosphere except that the control cells have not. been cultured in the presence of a low- oxygen atmosphere,

[0063 J in an embodiment of the invention, the population of T cells cultured in the presence of the Phd inhibitor are CD4H- and have an increased expression of Tbet as compared to control CD4+ T cells, wherein the control CD4+ T cells are identical to the T cells thai are cultured in the presence of a Phd inhibitor except that the control cells have not been cultured in the presence of a Phd inhibitor.

[0064] In an embodiment of the invention, the population of T cells cultured in the presence of a low oxygen atmosphere are CD4+ and have an increased expression of Tbet as compared to control CD4+ T cells, wherein the control CD4+ T cells are identical to the T ceils that are cultured in the presence of a low oxygen atmosphere except that the control cells have not been cultured in the presence of a low oxygen atmosphere.

[0065] in an embodiment of the invention, the population of T cells cultured in the presence of the Phd inhibitor are CD4+ and have a decreased expression of Foxp3 as compared to control CD4+ T cells, wherein the control CD4+ T cells are identical to the T ceils that are cultured in the presence of a Phd inhibitor except that the control cells have not been cultured in the presence of a Phd inhibitor.

[0066] In an embod iment of the invention, the population of T cells cultured in the presence of a low oxygen atmosphere are CD4+ and have a decreased expression of Foxp3 as compared to control CD4+ T cells, wherein the control CD4+ T cells are identical to the T cells that are cultured in the presence of a low oxygen atmosphere except that the control ceils have not been cultured in the presence of a low oxygen atmosphere,

[ΘΘ67] In an embodiment of the invention, the population of T cells cultured in the presence of the Phd inhibitor are CD8+ and have a decreased expression of CD62L as compared to control CD8+ T ceils, wherein the control CD8+ T cells are identical to the T cells that are cultured in the presence of a Phd inhibitor except that the control cells have not been cultured in the presence of a Phd inhibitor.

[0068 J In an embodiment of the invention, the population of T cells cultured in the presence of a low oxygen atmosphere are CD8+ and have a decreased expression of CD62L as compared to control CD8+ T cells, wherein the control CD8+ T cells are identical to the T ceils that are cultured in the presence of a low oxygen atmosphere except that the control cells have not been cultured in the presence of a low oxygen atmosphere.

0069] in an embodiment of the invention, the population of T ceils cultured in the presence of a Phd inhibitor provides greater antitumor acti vity upon in vivo transfer as compared to control T cells, wherei the control T cells are identical to the T cells that are cultured in the presence of a Phd inhibitor except that the control cells have not been cultured in the presence of a Phd inhibitor.

[0070] In. an embodiment of the invention, the population of T ceils cultured in the presence of a low-oxygen atmosphere provide greater antitumor activity upon in vivo transfer as compared to control T cells, wherein the control T cells are identical to the T cells that are cultured in the presence of a low-oxygen atmosphere except that the control cells have not been cultured in tire presence of a low-oxygen atmosphere.

[0071 ] The term "isolated," as used herein, means having been removed from, its natural environment. The term "purified," as used herein, means having been increased in punty, wherein "purity" is a relative term, and not to be necessarily construed as absolute purity. For example, the punty can be at least about 50%. can be greater than about 60%, about 70% or about 80%, about 90% or can be about 100%.

[0072] The population off cells produced by the inventive methods can be a

heterogeneous population comprising the T cells described herein, in addition to at. least one other cell, e.g., a cell other than a T cell, e.g., a B cell, a macrophage, a neutrophil, an erythrocyte, a hepatocyte, an endothelial cell, an epithelial cell, a muscle cell, a brain cell, etc. Alternatively, the population of T cells produced by the inventive methods can be a substantially homogeneous population, in which the population comprises mainly T cells described herein. The population also can be a clonal population of ceils, in which all cells of the population are clones of a single T cell. In one embodiment of the invention, the population of cells is a clonal population comprising T cells comprising a recombinant, expression vector encoding the exogenous ICR or CAR as described herein.

[0073] The inventive isolated or purified population of T cells produced according to any of the inventive methods described herein may be included in a composition, such as a pharmaceutical composition, in this regard, the invention provides a pharmaceutical composition comprising die isolated or purified population of T cells described herein and a pharmaceutically acceptable carrier.

[0074] Preferably, the earner is a phamiaeeutically acceptable carrier. With respect to pharmaceutical compositions, the carrier can be any of those conventionally used for the administration of cells. Such pharmaceutically acceptable carriers are well-known to those skilled in the art and are readily available to the public. It is preferred that the

pharmaceutically acceptable carrier be one which has no detrimental side effects or toxicity under the conditions of use.

[0075] The choice of carrier will be determined in part by the particular method used to administer the population of T cells. Accordingly, there are a variety of suitable formulations of the pharmaceutical composition of the invention. Suitable formulations may include any of those for parenteral, subcutaneous, intravenous, intramuscular, intraarterial, intrathecal, intra tumoral, or imerperitoneai administration. More than one route can be used to administer the population of T ceils, and in certain instances, a particular route can provide a more immediate and more effective response than another route.

[0076] Preferably, the population of T cells is administered by injection, e.g., intravenously. A suitable pharmaceutically acceptable carrier for the cells for injection may include any isotonic carrier such as, for example, normal saline (about 0.90% w/v of NaCl in water, about 300 mOsm/L NaCl in water, or about 9.0 g NaCl per liter of water),

NORMOSOL R electrolyte solution (Abbott, Chicago, IL), PLA SMA-LYTF. A (Baxter, Deeriield, IL), about 5% dextrose in water, or Ringer's lactate, in an embodiment, the pharmaceutically acceptable carrier is supplemented with human serum albumen.

[0077] For purposes of the invention, the dose, e.g., number of T cells administered should be sufficient to effect, e.g., a therapeutic or prophylactic response, in the mammal over a reasonable time frame. For example, the number of T cells administered should be sufficient to bind to a cancer antigen or treat or prevent cancer in a period of from about 2 hours or longer, e.g., 12 to 24 or more hours, from the time of administration. In certain embodiments, the time period could be even longer. The number of T cells administered will be determined by, e.g., the efficacy of the particular population of T ceils to be administered and the condition of the animal (e.g.. human), as well as the body weight of ihe animal (e.g., human) to be treated.

[0078] Many assays for determining an administered number of T cells are known in the art. For purposes of the invention, an assay, which comprises comparing the extent to which target, cells are iysed or one or more cytokines such as, e.g., IFN-v and 1L-2 is secreted upon administration of a given number of such T cells to a mammal among a set of mammals of which is each given a different number of the T cells, could be used to determine a starting number to be administered to a mammal. The extent to which target cells are lysed or cytokines such as, e.g., IFN-y and IL-2 are secreted upon administration of a certain number can be assayed by methods known in the art. Secretion of cytokines such as, e.g., IL-2, may also pro vide an indication of the quality (e.g., phenotype and/or effectiveness) of a T cell preparation.

[0079] The number of T ceils administered also will be determined by the existence, nature and extent of any adverse side effects that might accompany the administration of a particular population of T cells. Typically, the attending physician will decide the number of T cells with which to treat each individual patient, taking into consideration a variety of factors, such as age, body weight, general health, diet, sex, route of administration, and the severity of the condition being treated. By way of example and not intending to limit the invention, the number of T cells to be administered can be about 10 x 10^ό to about 10 x 1 CT ¹ cells per infusion, about 10 x 10^v cells to about 10 x 10^{: :} cells per infusion, or 10 x 10'^' to about 10 x 10'' ceils per infusion. While it may be possible to administer the cells together with the Phd inhibitor, in a preferred embodiment, the Fhd inhibitor is washed from the ceils prior to administering to a mammal.

[0080] !t is contemplated that the populations of T cells produced according to the mventive methods can be used in methods of treating or preventing cancer in a mammal In tins regard, the invention provides a method of treating or preventing cancer in a mammal, comprising administering to the mammal any of the pharmaceutical compositions or populations of T cells produced by any of the methods described herein in an amount effective to treat or prevent cancer in tire mammal. [0081] One or more additional therapeutic agents can be coadministered to the mammal. By "'coadministering" i.s meant administering one or more additional therapeutic agents and the isolated, population of T cells sufficiently close in time such that the isolated population of T ceils can enhance the effect of one or more additional therapeutic agents, or vice versa. In this regard, the isolated population of T cells can be administered first and the one or more additional therapeutic agents can be administered second, or vice versa. Alternatively, the isolated population of T cells and the one or more additional therapeutic agents can be administered simultaneously. Additional therapeutic agents that may enhance the function of the isolated population of T cells may include, for example, one or more cytokines or one or more antibodies (e.g., antibodies that inhibit PD-1 function). An exemplary therapeutic agent that can be co-administered with the isolated population of T cells is lL-2. Without being bound to a particular theory or mechanism, it is believed that IL-2 may enhance the therapeutic effect of the isolated population of T cells.

[0082] An embodiment of the invention further comprises lymphodepieting tire mammal prior to administering the isolated population of T cells. Examples of iymphodepletion include, but may not be limited to, nonmyeloablaiive lymphodepieting chemotherapy, myeloablative lymphodepleiing chemotherapy, total body irradiation, etc.

J0O83J The terms ^"treat," and "prevent" as well as words stemming therefrom, as used herein, do not necessarily imply 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. In this respect, the inventive methods can provide any amount of any level of treatment or prevention of cancer in a mammal.

Furthermore, the treatment or prevention provided by the inventive method can include treatment or prevention of one or more conditions or symptoms of the disease, e.g., cancer, being treated or prevented. Also, for purposes herein, "prevention" can encompass delaying the onset or recurrence of the disease, or a symptom or condition thereof.

1 084] For purposes of the inventive methods, wherein populations of T cells are administered, the T cells can be cells that are allogeneic or autologous to the host.

Preferably, the cells are autologous to the host.

[0085] With respect to the inventive methods, the cancer can be any cancer, including any of leukemia (e.g.. B cell leukemia), sarcomas (e.g., synovial sarcoma, osteogenic sarcoma, leiomyosarcoma uteri, and alveolar rhabdomyosarcoma), lymphomas (e.g.. Hodgkin lymphoma and non-Hodgkin lymphoma), hepatocellular carcinoma, glioma, head-neck cancer, acute lymphocytic cancer, acute myeloid leukemia, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, or anorectura, cancer of the eye, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancei of the oral cavity, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer (e.g., colon carcinoma), esophageal cancer, cervical cancer, gastrointestinal carcinoid tumor, hypopharynx cancer, larynx cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharynx cancer, ovarian cancer, pancreatic cancer, peritoneum, omentum, and mesentery cancer, pharynx cancer, prostate cancer, rectal cancer, renal cancer, small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, ureter cancer, and urinary bladder cancer.

[0086] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.

EXAMPLES 1 -16

[0087] The following materials and methods were employed for the experiments described in Examples 1 -16.

[0088] Expsrirnents were approved by the Institutional Animal Care and Use Committees of the NCI and performed in accordance with NIH guidelines. C57BL/6J, Ragl

(B6J29S7-Ragl ^{lm i Moi3l/J}), Ly5.1 +/+ (B6.SJL-Ptprc^a.Pepc^b BoyJ)_! TRP-1 (B6.Cg- Rag 1 ^{!m, Mom}Tyrp 1 ^BwTg(Tcra,Tcrb)9Rest/J), and Cd4-Cre (Tg(CD4-Cre) 1 Cwi/Bflu J) were purchased from The Jackson Laborator . The following mice have been described: Eglnl "'", Egln2^n/n, and Egm3^{n a} mice (Takeda et ai., Mol. Cell. Biol,. 26: 8336-8346 (2006)), Hifl a^{i f)} mice (Ryan et a!., Cancer Res., 60: 4010-4015 (2000)), Epasl '^ mice (Gruber et a! ., Proc. Natl. Acad. Sci. USA, 104: 2301 -2306 (2007)}. Deletion of /oxP-flanked genes in T cells was achieved by crossing to Cd4-Cre mice to obtain animals with homozygous loxP-flanked alleles without Cre or hemizygous for Cre. All mice were previously backcrossed over ten generations to the C57BL/6 background. For all in vivo studies (experimental metastasis, house dust mite challenge) age and sex matched WT and PHD-tKO mice were used with at least 5 mice per genotype. in Vitro T Cell Differentiation

[0089] CD4^! T cells from spleens and lymph nodes of 6-12-week-old mice were purified by negative magnetic selection (Miltenyi) followed by sorting of naive CD4^" CD62L¹ CD44^" CD25^" ceils using a FACSARiA II sorter (BD). Naive CD4⁺ T cells were activated with platebound anti-CD3 and soluble anti-CD28 (5 μ ml^'1 each; eBioscience) in media for 72h either under: ThO conditions (media alone); Thl conditions (IL-12 (10 ng ml^'1 , R&D

Systems), anti -11,-4 neutralizing antibodies (10 pg ml^"'): Thl 7 conditions (I.L-6 (20 ng ml^"1 , R&D Systems), human TGF-pl (0.2 ng ml^"', R&D Systems), anti-IFN-γ neutralizing antibodies (10 μκ ml^{" 1}) and anti-IL-4 neutralizing antibodies (10 ug ml^"'1); or iTreg conditions (human TGF-βΙ (0-2 ng ml.^"1, as indicated)). For human in vitro iTreg induction assays, naive CD4¾D45RA^÷CD45RQ^"CD62L^÷CCR7^÷ cells were FACS purified from three biologically independent healthy donor buffy coats and activated with plate-bound human anti-CD3 and soluble anti-CD28 (5 pg nil^"1 each; eBioscience) with huma TGF-βΙ (5 ng mL^"1 , R&D Systems) and recombinant human IL-2 (20 CU). Mouse and human cells were cultured under standard incubator conditions or 5% or 2% oxygen as indicated (Thermo Seientific-HERA CELL incubator equipped to replace oxygen with nitrogen) through the entire duration of culture. Where indicated, the FHD protein inhibitor DMQG (0.5 - 1.0 mM, Sigma-Aldrich), Rapamycin (50 nM. Sigma- Aldrich), or 2-deoxyglucose (1 μΜ, Sigma- Aldrich) was added for the duration of in vitro culture.

Histopathology

[0090 J Lungs were isolated from. WT and FHD-tKO mice and fixed m 10% formalin, embedded in methylacrylate, sectioned, and H&E stained. Sections were blindly evaluated by a veterinary pathologist. The presence of chronic, active inflammation, vasculitis, and hemorrhage in the alveoli was quantified on a standardized severity score system (0 least severe, 3 most severe).

House Dust Mite Airway Challenge

[0091] WT and PHD-tKO mice were sensitized with 25 fig of house dust mite (ΉΌΜ, D. Pteronyssinus: !ow-endotoxin, Greer) delivered to the retropharyngeal space in sterile PBS on days 0, 1 and 2, Animals were challenged with 5 .ug of HDM on days 15, 16, 17 and 18, and euthanized on day 19. Antibodies and Flow Cytometry

[0092] The following fluorescent dye-conjugated antibodies against surface and intracellular antigens were used: aiiti--FOXP3 (FJK-16 s), anti-IL-17A (eBiol 7B7), anti-lFN- g (XMGi .2), anti-Tbet (eBio4B10), atiti-Ror gamma (t) (B2D), anti-CD304 (Nrp-1,

3DS304M), antimouse TCR-beta (H57-597), anti-IL-13 (eBiol 3A), anti-Granzyme B ( GZB), and anti-Perforin (eBioOMAK-D) (eBioscience); anti-CD45.1 (A20), anti-CD4 (RM4-5), anti-CD25 (PC6i), anti-CD62L (MEL-14), anti-CD44 (ΪΜ7), anti--CD8a (53-6.7), anti-CD 1 52 (CTLA4, UC10-4F10-1 1 ), anti-GITR (DTA-1), anti-IL-4 (] 1 B1 1 ), anti-IL-S (TRFK5) (BD Biosciences); Anti-mouse IgG Fab2 (Ceil Signaling): Anti-mouse HIF1 a (Abeam). For the identification of NKT cells, a-ga actosyl ceramide loaded, PE-conjugated recombinant CD id tetramers were used (Proimmune). Cells were incubated with specific antibodies for 30 min on ice in the presence of 2.4G2 monoclonal antibody to block FcyR binding. All samples were acquired with a Fortessa or LSR flow cytometer (Becton

Dickinson) and analyzed using FLOWJO software (TreeStar). Intranuclear staining was carried out using the FOXP3 staining kit (eBioscience). To determine cytokine expression, cellular suspensions containing T cells were stimulated in media containing phorbol 12- rnyrisiate 33-acetate, ionomycin and brefeldin-A (Leukocyte activation cocktail with GOLGIPLUG protein transport inhibitor; BD biosciences) for 4 h. After stimulation, cells were stained an amine-reactive exclusion-based viability dye (Invitrogen) and with antibodies against cell-surface antigens, fixed and permeabiJized followed by intracellular staining with specific anti-cyiokine antibodies. Single-cell suspensions from lung tissues were prepared by mechanical disruption (GENTLEMAC8 dissociator, Miltenyi). COIJNTBRIGHT beads were spiked-in for the flow cytometric quantification of absolute cell number (Invitrogen).

RNA Sequencing and Analysis

10093] All RNA-Seq analyses were performed using R 2 biological replicates. Raw data from replicate measurements are pubiically available from the GEO repository. Total RNA was prepared from cells using the RNEASY PLUS Mini kit (QIAGEN). 200 ng of total RNA was subsequently used to prepare RNA-Seq library by using TRUSEQ RNA sample prep kit (Iliumina) according to manufacturer's instructions. Paired-end RNA sequencing was performed on a HiSEQ 2000 sequencing system (Iliumina). Sequenced reads were aligned to the mouse genome (NCBI37/mm9) with TOPHAT 2.0.1 1 software (Kim and Salzberg, Genome. Biol., 12 {201 1 )) and uniquely mapped reads were used to calculate gene expression. REFSEQ gene database (mm9) was downloaded from the UCSC genome browser for RNA-Seq analysis. RNA-Seq reads were mapped to mni9 (UCSC) using BOWTTE aligner. Gene expression, of annotated transcripts was calculated from mapped RNA-Seq reads using the CUFFDIFF program to obtain RPKM-normalized gene expression values (Trapnell et al., Nat. Protoc, 7: 562-578 (2012)). Two-tailed t tests were performed to identify different! l y expressed genes after applying the Benjamini-Hochberg correction for multiple testing. Gene set enrichment analysis was performed as previously described (Subrarnanian et al., Froc. Natl. Acad. ScL, 102: 15545-15550 (2005)).

Autoantibody Enzy e- Linked Immunosorbent Assay

[0094] For measurement of antinuclear (ANA) and anti-ds^'DNA autoantibodies, ELISA assays were performed on mouse serum according to manufacturer s instructions (Alpha Diagnostic international). ELISA quantification of IFN-γ in cell supematants collected after 72h or stimulation was performed according to manufacturer's instructions (Ebioscience),

Quantitative Reverse-Transcription Polymerase Chain Reaction

[0095] Cells were sorted or transferred into R ALATER solution (Ambion) and stored at -80 °C. Total RNA from pelleted cells was isolated using the RNEASY Plus mini kit (QIAGEN). First-strand cDNA synthesis was performed using random priming with the high-capacity cD A synthesis kit (Applied Biosystems) in the presence of SLiPERASEIN RNase inhibitor (Ambion). cDNA was used as a template for quantitative PGR reactions using TAQMAN primer-probes against specified mRNA transcripts (Applied Biosystems). Reactions were performed using Universal PGR Masiermix (Applied Biosystems). FAM channel intensity were normalized to ROX intensity, and C, values were calculated using automatically determined threshold values using SDS software (Applied Biosystems).

Immimoblot Analysis

[0096] Nuclear extracts were isolated (NE-PER kit; Pierce) and ~15 _,ug protein was loaded in each lane of a mini-protean precast TGX gel (Biorad). Protein was iransten'ed onto activated PVDF membrane (Biorad), blocked with 5% BSA in TBST at RT for Ih, and incubated in 1° antibody at 4 °C overnight. HiFl (NB 100-449, Novus) and loading control HDAC1 ( 10E2, Cell Signaling Technology) antibodies were used at 1 : 1000 dilution in TBST. Samples were washed 5x with TBST, incubated with 2° antibody at 1 : 5000 dilution in TBST for 2h, and washed 5x with TBST. Protein was visualized with enhanced chemiiuminescence (ThermoScieniific) and autoradiography film. Densitometry was calculated using IMAGEJ software,

Bone Marrow Chimeras

[0097] For bone marrow reconstitution experiments, Ragl^~'^~ mice were administered 1 ,000 Gy total-body-radiaiion from a ^{5 i7}Cs source before intravenous injection of BM ceils depleted of mature lineages from single-cell bone - marrow preparations using antibody coupled magnetic beads (Miltenyi), Bone marrow from 6-10-week-old donor mice were used. in Vivo iTreg induction

[00981 R ig '- mice were injected intravenously with 4 X 10^s CD4⁴ CD25^" CD45RB^liigii cells from wild-type or PHD-tKO mice. On day 21 to 23, transferred ceils were isolated and analyzed for FoxP3 expression by flow cytometry. hi Vivo Treg Stability

[0099] RagP'^' mice were injected intravenously with 4 X 10^'' CD4^' CD25* cells from wild-type or PHD-tKO mice. On day 7, transferred cells were isolated and analyzed for FoxP3 expression by flow cytometry.

In Vitro Treg Stability

[0100] CD4^*' CD25 ^' Treg cells were FACS purified from wild-type or PHD-tKO mice and stimulated in vitro with anti-CD 3 (1 pg mL^'!) in the presence of IL-2 (100 IU). Foxp3 expression was analyzed by flow cytometry 72 h post-stimulation.

In Vitro Suppression Assay

[0101] Naive CD4÷ T cells were FACS purified from Foxp3-GFP mice and stimulated i vitro in the presence of TGF-β ± DMOG for 72h. Foxp3-GFP+ iTreg cells were then FACS purified from VEH and DMOG treated cultures. VEH and DMOG iTreg cells were cultured in 96-well round-bottom plates with 5x10⁴ CFSE-labeled naive CD45.1 CD4+ CD25- (Tresp) cells along with I xt O⁴ CD! l e t dendritic cells used as antigen-presenting cells, isolated by immunomagnetic selection (Miltenyi). Cells were stimulated with anti-CD.^':? antibody (1 ^ig niL-i, BD Biosciences) for 72h at 37 and 5% CO_?.. Tresp cell proliferation was measured by CFSE dilution by flow cytometry.

Extracellular Acidification Rate and Glucose Uptake

0102] Extracellular acidification rates (ECAR) and oxygen consumption rates (OCR.) were measured ai 37 °C using an XF24 extracelkilar analyzer (Seahorse Bioscience). ECAR was measured in XF media (nonhuffered RPMI 1640 containing 25 mM glucose, 2 rnM L- glutariiine, and 1 mM sodium pyruvate) under basal conditions. To determine glucose uptake, T cells were incubated with 100 μΜ 2-NBDG (Invitrogen for 2 hr before measuring fluorescence by flow cytometry.

Foxp3 TSDR Methylation

[0103] 500 ng of sample DNA from WT and PHD-tKG FACS purified CD4⁺ CD25* Treg and CD4⁺ CD25^" non-Treg cells was bisulfite treated (Epigendx) and column purified (Zymogen). PCR amplification of the Foxp3 TSDR locus region (nucleotides -2369 to -2207 relative to Foxp3 start codon) was performed using biotinyiated PCR primer (Epigendx). PCR products were bound to S^'TREPT A VTDT SEPH AROSE HP medium (GE Healthcare Life Sciences), washed, and denatured with NaOH. PYROSEQUENC1NG primer was annealed to the purified single-stranded PCR product and sequencing was performed using the PYROSEQUE C1NG PSQ96 HS System according to manufacturer's instructions (QIAGEN). The methylation status of each locus was analyzed individually as a T/C S P using QCpG software (QIAGEN),

Experimental Metastasis

[0104] B16F1G (B 16) melanoma cell line was obtained from the NCI tumor repository and passaged in Dulbecco's Modified Eagle Medium (Invitrogen) supplemented with 10% fetal calf scrum. 2.5χΙ 0⁵ B 16 cells were injected subcutaneously in flanks and intravenously through the tail vein. Tumor implantation experiments were performed in littermate mice of 8-32 weeks of age. Subcutaneous tumors were measured at serial time points following implantation using digital calipers and the tumor area was calculated as the product of perpendicular diameter. Lung tumor nodules were enumerated by gross count of visible sites of disease. Micrometasiatic lesions were quantified following H&E stain of lung sections, and sections consistent with melanoma histology were circumscribed by a certified veterinarian. All subcutaneous and pulmonary tumor measurements were performed in a blinded manner. IFN-γ depletion was performed using intraperitoneal injection of 250 mg anti-IF -y (XM.G1.2, BioXcell) at indicated time points.

Adopiive Celt Transfer

[0105] Mice 6 to 12 weeks of age (n = 6-10 for all groups) were injected intravenously or subcutaneously with 2.5x10 ^" B 16 melanoma cells. 5-10 days later mice were treated with adoptively transferred TRP-1 specific CD4+ T cells derived from TCR transgenic splenocytes and stimulated in vitro in the presence or absence of the PHD inhibitor DMOG, At the time of ACT, mice received 500 Gy total body irradiation.

EXAMPLE 1

[0106] This example demonstrates thai Trp⁺ CD4⁺ T ceils adopt a Till phenotype when stiinuiaied in the presence of the small molecule pan-Phd inhibitor DMOG

(dimethyloxalylglycine, N-(methoxyoxoace†y!)-gJycine methyl ester).

[0107] The effects of the inhibition of T cell oxygen sensing on the efficacy of CD4^: based adoptive cell transfer (ACT) were evaluated using the established Trp⁺ TCR transgenic mouse model (Muranski et al.. Blood, i 12(2): 362-73 (2008)).

[0108] CD4* Tr l transgenic splenocytes were stimulated for 72 hours with Trp peptide and irradiated feeder cells under ThO or iTreg (0.2 ng/mL TGF-β) conditions in the absence (vehicle) or presence of the Ph.d inhibitor DMOG ( i rriM). Flow cytometric analysis of Foxp3 and Tbet were earned out. The results are shown in Table A (ThO cells) and Table B (iTreg cells). Tables A and B show the percentage of cells with the indicated phenotypes.

TABLE A

[0109] In a separate experiment, the cells were analyzed by Western blot for the presence Foxp3, Tbet, and β-actin proteins. Whole ceil protein lysate was isolated from open repertoire CD4 T cells stimulated with plate bound anti-CD3 (5 mg/mL) and anti-CD28 (5 mg/mL) under ThO or iTreg conditions in the presence or absence of DMOG for 72 hours. The results are shown Figure LA.

[0110] In a separate experiment, the supernatant of CD4 T cells stimulated with plate bound anti-CD3 (5 mg mL) and anti-CD28 (5 mg/mL) under ThO conditions in the presence or absence of DMOG for 72 hours was analyzed for the presence of IFN-γ by enzyme-linked immunosorbent assay (ELISA). The results are shown in Figure 1 B.

[0111] it was observed that the Trp^" CD4^÷ T cells adopted a Thl phenotype when stimulated in the presence of the small molecule pan-Phd inhibitor DMOG, even under conditions that would normally promote the development of immunosuppressive regulatory T cells {Tables A and B and Figures 1 A and IB).

EXAMPLE 2

[0112] This example demonstrates that tumor-specific CD4÷ T cells treated with a Phd inhibitor provide improved anti-tumor capacity as compared to T ceils that are not treated with a Phd inhibitor. [0113] Mice bearing 10-day established subcutaneous B 16 melanoma tumors were treated with no cells or 1 x10^b Trp+ CD4÷ T cells that were treated with vehicle or DMOG. Trp⁺ T cells recognize the tyrosinase melanocyte differentiation antigen expressed on many melanomas. Eight mice were in each treatment group. Tumor growth and overall survival of die mice were measured. The results are shown in Figiire I C (tumor growth) and Figure ID (survival). These data are representative of three independently performed tumor treatment experiments. All mice treated in Figures IC and ID received exogenous inierleiikin (IL)-2,

[0114] As shown in Figures IC and 1 D. when ihe numbers of Τφ T cells were expanded ex vivo and transferred into mice hearing established B16 melanoma, the cells that were generated in the presence of the Phd inhibitor had demonstrable stronger anti-tumor capacity, as indicated by delayed tumor progression and increased overall survival of treated mice.

[011.5] Consistent with their enhanced effector phenotype at the time of infusion, the DMOG-treated Trp⁺ T ceils also mediated superior clearance of mng metastases,

[0116] t was previously found that Thl cells are more effective than ThO cells at inducing tumor rejection (Muranski et ah, Blood, 1 12: 362-373 (2008). Augmented antitumor function in mice administered the DMOG-treated Trp⁺ T ceils is consistent with the induction of a Thl phenotype in these cells. Indeed, DMOG-treated Trp* T cells were as effective in mediating tumor regression as cells polarized under conventional Thl cell specifying conditions. Thus, inhibition of PHD proteins provides a viable strategy to enforce Thl cell differentiation under conditions used for ACT, thereby providing a potential means to improve the efficacy of ACT.

EXAMPLE 3

[0117] This example demonstrates that Phd inhibitor-treated Pmef^" CD8 ^r T cells express surface markers consistent with the acquisition of effector properties and have an increased capacity to produce the effector cytokines lFN-γ and TNF-cc as compared to PmeF CD8⁺ T cells thai are not treated with Phd inhibitor.

[01183 CD8^'r PmeF transgenic splenocytes were stimulated for 5 days with gplOO peptide and IL-2 (100 KJ) in the presence or absence of the Phd inhibitor DMOG (1 niM). The cells were analyzed for the expression of CD44 and CD62L by flow cytometry. Representative results are shown in Table C. Table C shows the percentage of cells with the indicated phenoiypes. Figure 2A shows the population distribution of nai e, central memory, and effector memory cells in the CD8* PmeF transgenic splenocyies ihat were stimulated in the absence or presence of the Phd inhibitor DMOG.

0119] Pme transgenic splenocyies were stimulated for 5 days with g lOO peptide and

IL-2 (100 iU) in the presence or absence of the Phd inhibitor DMOG (1 mM). IFN-γ and

TNF- were detected by flow cytometry Representative data are show in. ^'T able D. Table

D and Figure 2B show the pei -eeniage of cells with the indicated cytokine pro files.

TABLE D

[0120] To test the efficacy of oxygen sensing inhibition in CDS^"1" T cells, the pmel ^'ICR. transgenic rnonse system was utilized. In the pmel TCR transgenic mouse system, CD8^" T cells recognize the gpl OO melanocyte differentiation antigen present, on many melanomas (Overwijk et al., J, Exp. Med., 188(2): 277-86 (1998)).

[0121] As shown in Tables C-D and Figures 2A-2B, Pmel^4' CD8 T ceils expanded ex vivo in the presence of the Phd inhibitor DMOG expressed surface markers consistent with the acquisition of effector properties (Table C and Figure 2A) and had increased capacity to produce the effector cytokines IFN-γ and TNF-a (Table D and Figure 2B). EXAMPLE 4

[0122} This example demonstrates that tumor-specific CDS + T cells treated with a Phd inhibitor provide improved anti-tumor capacity as compared to T cells that are not treated with a Phd inhibitor.

[0123] Mice bearing 10-day established subcutaneous B16 melanoma tumors transduced with humanized gplOO antigen were treated with no cells or 1x106 PmeH- CD8+ T cells thai were treated with vehicle or DMOG. Eight mice were in each treatment group. Tumor growth and overall survival of the mice were measured. The results are shown in Figure 2C (tumor growth) and Figure 2D (.survival). All mice treated in Figures 2C and 2D received exogenous interleuk (JL)-2. and total body irradiation (600 Gy) prior to ceil transfer.

[0124] As shown in Figures 2C and 2D, CDS⁺ T cells can mediate dramatic tumor regression in adoptive cell transfer therapies. Upon transfer into mice bearing established B 16 melanoma, Pmef CD8⁺ T cells expanded ex vivo in the presence of DMOG were able to mediate stronger tumor regression leading to improved overall survival of the tumor bearing hosts.

EXAMPLE 5

[0125] This example demonstrates that low environmental oxygen inhibits iTreg induction and promotes Tbet expression.

[0126] CD4 T cells were stimulated for 72 hours with anti-CD3 antibody and anti-CD28 antibody (5 mg/tr.L each) under ThO or iTreg (0.2 ng/mL TGF-b) conditions in an ambient air incubator (20% i¾) or a hypoxic chamber incubator (1 % 0₂). The expression of Foxp3 and Tbet was analyzed by flow cytometry. The results are shown in ^'fable E (ThO) and Table F (iTreg). Tables E and F show the percentages of cells with the indicated phenotypes. The replicate values are shown in Figure 3 A.

[0127] As shown in Tables E and F and Figure 3 A, low environmental oxygen inhibited iTreg induction and promoted Tbet expression.

EXAMPLE 6

[0128] This example demonstrates that mice w th a T ceil specific deletion of all three Phd proteins are protected from hmg tumor colonization.

[0129] Mice with a T ceil specific deletion of all three Phd proteins {Phd-tKO) and wild- type (WT) littermate matched mice were injected intravenously with 2 I0⁵ B16 melanoma cells. The total number of Sung tumors formed was enumerated 21 days after injection. The results are shown in Figures 4A--4C.

[0130] As sho wn in Figures 4A-4C, mice with a T cell specific deletion of all three Phd proteins were protected from lung tumor colonization. It was also observed that lung resident P d-tKO CD4+ and CD8+ T ceils had an increased capacity to produce IF -γ as compared to WT T cells. Lung resident CD4+ T cells from Phd-tKO mice also demonstrated a reduced percentage of Nrpi-Lo iTreg as compared to hmg resident CD4+ T cells from WT mice.

[0131] This example demonstrates that T-celLmtrinsic expression of PHD proteins supports metastasis to the liver.

[0 32] B 16 melanoma cells were injected into the spleen of WT and PHD-tKO mice. Two weeks after injection, tumor metastatic in the liver was assessed,

[0133] The gross pheno ype of the livers in WT and PHD-tKO mice following intrasplenic tumor injection was assessed. The results are shown in Figures 5A-5B. As shown in Figures 5A-5B, PHD-tKO livers had a significantly reduced tumor burden as compared to WT livers. [0134] A histologic analysis of the tumor burden in livers from WT and PHD-tKO mice following intraspienic tumor injection was also carried out, The results of the histologic analysis showed that the PHD-tKO livers had a significantly reduced tumor burden as compared to WT livers,

[0135] The tumor burden in the livers of WT and PHD-tKO mice was also measured by liver weight (Fig. 5C), visible tumor nodules (Fig. 5D), and histologic enumeration (Fig. 5E), As shown in Figs, 5C-5.E, the tumor burden in the livers of PHD-tKO mice, as measured by liver weight, visible tumor nodules, and histologic enumeration, was significantly reduced as compared to WT livers.

EXAMPLE 8

[0136] This example demonstrates that PHD proteins function within T ceils to limit pulmonary effector responses,

[0137] Pulmonary tolerance requires suppression of inflammatory T cell responses under physiologic conditions, it was asked whether T-cell-mtrinsic expression of the oxygen- sensing PHD proteins contributes to site-specific tolerance in the lung. Mice harboring a T - cell-specific deletion of all three PHD proteins (henceforth PHD-tKO) were generated. Cd4- driven Cre recombmase expression resulted in significant reduction otEgln Egln2, and EglnS mR A transcripts, which encode PHD2, PHD1 , and PHD3 proteins respectively, in CD4+, CDS -K and KT T cells, but not in other lymphoid cell subsets. Upon gross evaluation, a patchy hemorrhagic appearance was observed within the lungs of PHD-tKO mice that was not present among wild-type (WT) littermales. Histologic examination revealed the presence of diffuse alveolar hemorrhage (DAH) of variable severity in PHD-tKO mice. PHD-tKO mice had increased serum autoantibodies, which can be elevated upon immune-mediated tissue damage. Pathology in PHD-tKO mice was only observed in the lung, and abnormal iiver and pancreas enzymes were not detected in the blood.

[0 38] PHD-tKO animals showed no defects in thymocyte number or phenotypic distribution. Similar numbers of CD4+and CD8÷ T lymphocytes were detected in peripheral blood and secondary lymphoid organs. In the bone marrow of PHD-tKO mice, there was a slight reduction in CD8+ T cells but similar numbers of CD + T cells. As the bone marrow is a reservoir of memory T cells, the differentiation state of T cells in WT and PHD-tKO mice was evaiulated. There was a similar distribution of naive and effector CD4+ T cells in WT and PHD-tKO mice. However, increased frequencies of terminally differentiated CD8+ T cells were observed in the peripheral blood, spleen, and bone marrow of PHD-tKO mice, Without being bound to a particular theory or meehaims, it is believed that this might explain the reduction of CD8+ T cells in the bone marrow of PHD-tKO mice and suggests a role for the PHD proteins in restraining terminal CD8+ T ceil differentiation.

[0139] T cells can drive immunopat ology in the lung. Increased numbers of pulmonary CD4+ and CD8+ T celis were observed in PHD-tKO mice. Additionally, pulmonary T cells in PHD-tKO mice had increased expression of the activation markers CD44, CD25, CTLA-4, and GiTR. In PHD-tKO mice, increased expression of IFN-g by CD4+ and CD8+ T c eils isolated from multiple lymphoid and non-lymphoid organs was measured, yet this was most pronounced in the lung. Elevated IFN-g production was more significant in the lung, including among pulmonary tissue resident memory (TRM) T cells, than in T ceils residing in mediastinal lymph nodes (meLN). These data snggest that the PHD proteins predominantly restrain T cell effector function in the lung parenchyma and airway-associated lymphoid tissue. Increased expression of cytolytic effector molecules tn pulmonary CD4+ and CD8÷ T celis in PHD-tKO mice was measured. Additionally, increased expression of IFN-g, but not other effector cytokines, was observed in pulmonary MKT ceils from PHDtKO mice. Thus, T-ceU-intrinsic expression of PHD proteins limits effector T cell function and prevents mild spontaneous immune pathology in the lung.

EXAMPLE 9

[0140] This example demonstrates that PHD proteins maintain pulmonary Nrp-l Lo Treg cells.

[0141] It was asked whether increased effector function observed in pulmonary T cells from PHD-tKO mice is secondary to a local deficit in Treg cells. Pulmonary Treg cell frequency and absolute number was similar in PHD-tKO mice compared to WT controls. The overall phenotypic profile thus revealed a significantly elevated lFN-g÷ Teff to Treg cell ratio in lungs of PHD-tKO mice,

[0142] The stability of Foxp3+ Treg cells is an important parameter in their

immunoregulatory function. Treg cell stability is proportional to the level of demetbylaiion within the Treg-specific-demethylated region (TSDR) of the Foxp3 locus (Floess et al., PLoS Bio!. , 5: e.38 (2007)). TSDR CpG methylation was similar in WT and PHD-tKO Treg celis. Consistently, the stability of Foxp3 expression was similar in WT and PHD-tKO Treg cells following restimulation in vitro or upon in vivo transfer into Ragl^"'^" hosts. [0143] A reduction in the frequency and number of Nrp-lLo Treg cells was detected in the lungs of PHD-tKO mice. Nrp-i expression can be induced on ^"freg cells in inflammatory environments (Weiss et al., J. Exp. Med., 209: 1723-42 (2012)). This raised the possibility that the observed reduction in pulmonary Nrp-lLo Treg cells in PHD-tKO mice is the result of induced rp-1 expression in the inflamed lung environment. To exclude this possibility, it was tested whether differences in Nrp-1 expression between WT and PHDtKO Treg cells exist when cells are exposed to an identical pulmonary environment. Lethally ablated Ragl- /- mice were reconstituted with bone marrow isolated from congemcally distinguishable WT or PHD-tKO mice in a 1 : 1 mixture. Pulmonary Treg cells were evaluated following reconsiitution, A reduced frequency of Nrp- lLo cells was observed within PHD-tKO pulmonary Treg cells. Thus, the reduced frequency of Nrp-lLo Treg cells in lungs of PHD- tKO mice is not secondary to the inflamed environment but instead is a cell-intrinsic phenomenon.

EXAMPLE 10

[0144] This example demonstrates that PHD proteins retrain Thl inflammation against innocuous foreign antigens,

[0145] It was hypothesized that the mild steady-state pathology observed in lungs of PHD-tKO mice was caused by excessive inflammatory responses against innocuous environmental antigens. To test this, the airway of WT and PHD-tKO mice was sensitized and challenged with house dust mite (HDM) extract.

[0146] HDM challenge promoted similar expansion of pulmonary CD4+ T cells in WT and PHD-tKO mice. HDM challenge induced a robust expansion of pulmonary Th2 cells in WT mice but failed to do so in PHD-tKO mice, instead, a dramatic increase in IFNg + Thl cells was observed in lungs of PHD-tKO mice upon HDM challenge. Additionally, HDM challenge induced accumulation of pulmonary Treg cells that was reduced in PHD-tKO mice compared to WT controls. PHD-tKO mice also exhibited greater expansion of CD8+ T cells and induction of IFN-g expression among these ceils upon HDM challenge. Consequently, PHD-tKO mice suffered from more severe inflammation following chronic HDM exposure that was consistent with Thl rather than Th2-driven tram unopathology including vasculitis and diffuse tissue damage leading to alveolar hemorrhage. Thus, the PHD proteins coordinate a T-cell -intrinsic program that restrains inflammatory T cell effector responses against innocuous foreign antigens in the lung. EXAMPLE 1 1

[0147] Ti ls example demonstrates that FHD proteins regulate reciprocal iTreg and Thl differentiation programs.

[0148] The decrease in steady-state pulmonary Nrp-lLo Treg cells and reduction in HDM-indueed Treg cell accumulation suggested that the PHD proteins might promote iTreg cell differentiation. To determine whether PHD proteins are required for iTreg cell differentiation, the gene expression profile of PHD-tKO and WT CD4+ T cells primed in vitro in conditions favoring iTreg cell generation was evaluated. RNA sequencing revealed widespread gene expression differences between PHD-tKO and WT cells (545 upregulated and 344 downreguiated genes in PHD-tKO). A significant reduction in Foxp3 expression and induction of genes encoding the Thl lineage specifying transcription factor T-bet (Tbx21) and effector cytokine IFN-g {Ifiig) was observed in PHD-tKO cells. By contrast, genes encoding Th2 and Thl 7 lineage specifying transcription factors and effector cytokines were expressed at similar levels in WT and PHD-tKO cells. Competitive antagonism of PHD protein enzymatic activity using a pan-PHD inhibitor, dimethyloxalylglycme (DMOG), caused similar changes in global gene expression as those observed in PHD-tKO T cells (Ifiig and Tbx2! were overexpressed, and Rorc and Foxp3 were underexpressed).

0149] To further evaluate the requirement for PliD proteins in iTreg cell differentiation, naive CD4+ T ceils isolated from WT and PHD-tKO mice were stimulated in the presence of titrated concenirations of TGF-b. PHD proteins were required for TGF-b-driven induction of Foxp3 and repression of T-bet. Consistently, higher amounts of IFN-g were detected in snpematants collected from PHD-tKO cell cultures. Naive PHD-tKO CD4+ T cells also demonstrated impaired iTreg conversion in vivo following transfer into Ragl -/- recipient animals. These results indicated that PHD proteins regulate reciprocal iTreg and Thl differentiation programs.

[0150J Foxp3-i- iTreg ceils derived from PHD-tKO cells or WT cells stimulated in the presence of DMOG expressed high levels of T-bet. T-bet expression can be induced in Treg cells in response to IFN-g (Koch et al., Immunity, 37: 501 -510 (2012)). Indeed, blockade of IFN-g limited T-bet expression in PHD-tKO Foxp3+ iTreg ceils. Foxp3+ T-bet+ Treg cells exert suppressive function, especially in inflammatory contexts (Koch et al,. Nat. Immunol.., 10: 595-602 (2009)). Consistently, fluorescence-activated cell sorting (FACS)-punfied Foxp3-GFP+ iTreg cells generated in the presence of DMOG, which express high levels of T-bet, were as suppressive in vitro as T-betLo iTreg cells.

[0151] Downstream substrates of PHD enzymatic activity including HlFia have been implicated in Thl 7 differentiation and CD8+ T cell effector function (Dang et ai., Cell, 146: 772-784 (201 1); Doedens et al, Nat. Immunol, 14: 1 173-82 (2013)). t was evaluated whether PHD proteins restrain differentiation and function of these lineages. IL-17A production was similar in CD4-S- T cells isolated from the limg and small intestine LP of WT and PHD-tKO mice. Moreo ver, no differences in RORgt expression or IL-17A production were detected between WT and PHD-tKO T cells stimulated under Thl 7 polarizing conditions in vitro. Consistent with the increased frequency of effector CD8+ T ceils in PHD-tKO mice, increased terminal differentiation and production of cytolytic moiecnles was observed among PHD-tKO CD8+ T cells stimulated in vitro.

[0152] Collectively, these results demonstrated that PHD proteins function within the CD4+ T cell lineage to promote iTreg cell differentiation while restraining the differentiation of Thl cells. Within the CD8+ T cell lineage, PHD proteins restrain the acquisition of effector cytokine and cytolytic functions.

EXAMPLE 12

[0153] T is example demonstrates that extracellular oxygen promotes iTreg cell differentiation in a PHD-dependent manner.

[0 54] it was asked whether extracellular oxygen availability influences the efficiency of iTreg and Thl cell differentiation. WT CD4+ T cells were stimulated in vitro under fixed extracellular oxygen concentrations. Cells stimulated under higher oxygen tensions demonstrated increased iTreg and reduced Thl cell differentiation, and this phenomenon was dependent on the oxygen-sensing PHD proteins. High extracellular oxygen also promoted human iTreg cell differentiation in a PHD-dependent manner. Thus, PHD protems enable T- cell-tntrinsic detection of environmental oxygen, which influences reciprocal iTreg and Thl cell differentiation efficiency.

EXAMPLE 13

[0155] This example demonstrates that PHD proteins are functionally redundant in T lymphocytes. jOI56j It was asked whether a single PHD protein is functionally predominant in T cells or if they function in a redundant manner. Mice were generated with T- cell-specific deletions of one, two, or all three PHD proteins. It was hypothesized that if any one PHD protein was functionally predominant, then removal of this factor should phenocopy the effect of deletion of all three. Deletion of one or two PHD proteins did not significantly affect IFN-g production or Nrp-ILo Treg cell frequency among pulmonary CD4 T ceils. Only loss of all three PHD proteins resulted in a dramatic increase in the pulmonary IFN-g÷ Teff to Nrp-ILo Treg cell ratio. Elevated IFN-g production in pulmonary CD8÷ T cells was detected only when all three PHD proteins were ablated. Deletion of one PHD protein or the combined removal of PHD1/.PHD2 or FHD1/PHD3 did not affect iTreg cell differentiation in vitro. PHD2/3 dKO T cells demonstrated a modest reduction in iTreg cell differentiation and reciprocal increase in Thl ceil differentiation. However, this defect was most pronounced in PHD-tKO cells. Collectively, these results provided evidence that PHD proteins function redundantly in T ceils.

EXAMPLE 14

[0157] This example demonstrates that PHD proteins repress HlF-driven glycolytic metabolism to mediate T cell fate specification.

[0158] It was next asked how PHD proteins mediate reciprocal iTreg and Thl cellular differentiation programs. Gene set enrichment analysis (GSEA) of the global transcriptional differences between PHD-tKO and WT CD4+ T cells activated in vitro revealed significant enrichment of genes involved in hypoxic responses and transcriptional targets of hypoxia- inducible factors in PHD-tKO cells. Nuclear accumulation of HIFa proteins correlates inversely with PHD hydroxylase activity (Kaelin and Raiclif e, Mol. Cell, 30: 393-402 (2008)}. Therefore, the dynamics of HIFla rnRNA and protein expression in WT and PHD- tKO T cells was measured. Naive and activated WT and PHD-tKO CD4÷ T cells had similar expression of Hifi a mRNA. HIFl a protein expression was similar in naive cells but significantly elevated in PHD-ίΚΟ T cells upon activation. Consistent with their functional redundancy, each PHD protein limited HIFl a accumulation in T cells, Furthermore, HIFla accumulation was suppressed in CD4+ T cells stimulated in high oxygen environments in a PHD-dependent manner.

[0159] it was asked whether PHD-mediated suppression of HIFl a accumulation is required for appropriate control of iTreg and Thl cell differentiation. HIFl a, Foxp3. and T- bet protein expression was analyzed in CD4-J- T cells isolated from each PHD KO genotype. HIFla accumulation inversely correlated with iTreg cell differentiation and positively correlated with Thl effector cell differentiation. Moreover, loss of HIFla and HIF2a partially rescued iTreg cell differentiation and reversed excessive Thl differentiation when the enzymatic activity of PHD proteins was inhibited using DMOG. Loss of HIFla alone only partially reversed the phenotype induced by DMOG, while loss of HIF2a had little effect on the phenotype. Thus, PHD proteins regulate CD4÷ T cell differentiation in part by controlling the expression of H!Fa proteins.

[0160] HIF transcriptional activity drives expression of genes involved in multiple cellular programs including glycolysis. Expression of components of the glycolytic machinery was elevated in PHDtKO and DMOG-treated VVT T cells in a HIFa-dependent manner. T ceils stimulated in the presence of TGF-b demonstrated a PflD-dependent reduction in extracellular acidification rate (ECAR), a measure of glycolytic activity. PHDtKO CD4÷ T ceils demonstrated increased glucose uptake and adopted an anaerobic metabolic signature. PHD proteins also restrained glycolysis in CD8+ T cells,

[0161 [ It was asked whether PHD-rnediated repression of glycolysis is required for appropriate iTreg and Thl ceil specification . Pharmacological blockade of mTOR-driven glycolytic programs or glycolysis with rapamycin and 2-deoxyglueose, respectively, completely abrogated spontaneous Thl differentiation and IFN-g production and partially restored iTreg cell differentiation in PHD-tKO T cells. Collectively, these findings suggest that PHD proteins coordinate a transcriptional and metabolic program that regulates the reciprocal differentiation of Thl and iTreg cells.

EXAMPLE 15

[0162] This example demonstrates that T-ce!l intrinsic expression of PHD proteins licenses tumor colonization of the lung.

[01 3] it. was found that PHD proteins coordinate a T-cell-inirinsie imrnunoreg-ulatcry program to sustain tolerance against harmless environmental antigens in the lung. It was reasoned that infiltrating pre-metastatic cancer ceils might resemble innocuous foreign antigens through their expression of mutated neoantigenic epitopes. It was therefore hypothesized that PHD proteins limit effector responses against infiltrating tumor cells in the lung. The ability of circulating tumor cells to colonize lungs of VVT and PHD-tKO mice was evaluated. As a control, subcutaneous tumor growth was also evaluated. WT and PHD-tKO mice were injected with B 16 melanoma tumors subcuianeously in the flank and intravenously (i.v.) through the tail vein, to introduce rumor cells at each site within the same animal. While subcutaneous tumor growth was similar in WT and PHDiKO mice, PHD-tKO mice were significantly protected from tumor colonization in the lung. PHD-tKO animals had fewer detectable lung tumors upon gross and microscopic evaluation, resulting in a significant reduction in total pulmonary tumor burden.

[0164] The immune phenotype of CD4+ T cells in WT and PHD-tKO mice that were injected i.v. with B i o melanoma was next analyzed. The frequency of splenic Treg cells was similar in WT and PHD-tKO mice. However, an increase in the frequency of pulmonary Treg ceils following i.v. tumor administration was observed that was absent in PHD-iKO mice. Additionally, increased frequencies of IFN-g-expressing pulmonary CD4+ T cells were detected in PHD-tKO mice compared to WT controls upon tumor coloniz tion.

[0165] It was asked whether PHD proteins support pulmonary umor colonization through suppression of IFN-g-mediaied anti-tumor immunity. PHD-tKO mice were injected intravenously with B1.6 melanoma and received IFN-g neutralizing or control antibodies at serial time points following tumor implantation. The protection from lung tumor colonization observed in PHD-tKO mice was abrogated by IFN-g n utralization. Thus, PHD proteins limit IFN-g-mediated tumor clearance in the lung.

[0166] These results indicated that T~eell-mtrmsic expression of PHD proteins restrains anti-tumor immunity in the lung, thus creating favorable conditions for metastatic tumor colonization. 0167] All references, including publications, patent applications, and patents, Cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set. forth in its entirety herein.

[0168] The use of the terms "a" and "an" and "the" and "at least one" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term "at least one" followed by a list of one or more items {for example, "at least one of A and B") is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items {A and B). unless otherwise indicated herein or clearly contradicted by context. The terras "comprising," "^'having,^'" "'including," and ''containing" are to be construed as open-ended terms (i.e., meaning "'including, but not limited to,") unless otherwise noted. Recitation, of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it. were individually recited herein. All methods described herein can be perfonned in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g.. "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0169] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herem. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

CLAIM(S); ί , A method of producing an isolaied population of T cells for adoptive cell therapy, the method comprising cul taring isolated T ceils having antigenic specificity for a cancer antigen in vitro in the presence of a prolyl hydroxylase domain-containing protein (Phd) inhibitor.

2. The method according to claim 1 , wherein the Phd inhibitor is a small molecule.

3. The method according to claim 1 , wherein the Phd inhibitor is

dimethyloxalylglyerae, N-(methoxyoxoacetyl)-glyerae methyl ester (DMOG).

4. The method according to claim 1 , wherein the Phd inhibitor is small interfering RNA (siRNA), microRNA, antisense nucleic acid, or CRTSPR-Cas9 guide RNA fgRNA).

5. The method according to any one of claims 1-4, wherein the population of T cells produced according to the method has an increased expression of one or both of interferon- gamma and tumor necrosis factor alpha as compared to control T cells, wherein the control T cells are identical to the T cells that are cultured in the presence of an Phd inhibitor except that the control cells have not been cultured in the presence of an Phd inhibitor.

6. The method according to any one of claims 1 -4, wherein the population of T cells produced according to the method are CD4+ and have an increased expression of Tbet as compared to control CD4+ T cells, wherein the control CD4+ T cells are identical to the T cells that are cultured in the presence of an Phd inhibitor except that the control cells have not been cultured in the presence of an Phd inhibitor.

7. The method according to any one of claims 1-4, wherein the population of T ceils produced according to the method are CD4+ and have a decreased expression of Foxp3 as compared to control CD4+ T cells, wherein the control CD4+ T cells are identical to the T cells that are cultured in the presence of an Phd inhibitor except that the control cells have not been cultured in the presence of an Phd inhibitor.

8. The method according io any one of claims 1 -4, wherein the population of T cells produced according to the method are CD8+ and have a decreased expression of CD62L as compared to control CD8+ T ceils, wherein the control CD8+ T cells are identical to the T cells that are cultured in the presence of an Phd inhibitor except that the control cells have not been cultured in the presence of an Phd inhibitor,

9. The method according to any one of claims 1-8, wherein the population of T cells produced according to the method provides greater antitumor activity upon in vivo transfer as compared to control T cells, wherein the control T ceils are identical to the T cells that are cultured i the presence of an Phd inhibitor except that the control cells have not been cultured in the presence of an Phd inhibitor.

10. The method according to any one of claims 1-9, further comprising expanding the number of T cells in the presence of one or more non-specific T cell stimuli, one or more cytokines, and a Phd inhibitor.

1 1. A method of producing an isolated population of T cells for adoptive cell therapy, the method comprising culturir.g isolated T cells having antigenic specificity for a cancer antigen in vitro in the presence of an atmosphere having an oxygen content less than or equal to 5%.

12. The method according to any one of claims 1-11 , further comprising introducing a nucleic acid encoding a chimeric antigen receptor (CAR) into the T cells under conditions sufficient to express the CAR by the T cells.

13. The method according to any one of claims 1 -1 1 , further comprising introducing a nucleic acid encoding an exogenous T cell receptor (TCR) into the T cells under conditions sufficient to express the TCR by the T cells.

14. The method according to any one of claims 1 - 13, wherein the T cells are tumor infiltrating lymphocytes (TIL).

15. An isolated population of T cells produced by the method according to any one of claims 1-14.

16. A pharmaceuiical composition comprising the isolated population of T cells of claim 15 and a pharmaceutically acceptable carrier.

17. The isolated population of T cells of claim 15 for use in treating or preventing cancer in a mammal,

18. The isolated population of T cells for the use of claim 17, wherei the T cells are autologous to the mammal