WO2017139199A1 - Inducible arginase - Google Patents

Abstract

The invention provides a nucleic acid comprising a nuclear factor of activated T-cells (NFAT)-responsive promoter operatively associated with a nucleotide sequence encoding (i) arginase, (ii) a functional portion of arginase, or (iii) a functional variant of (i) or (ii). Also provided are related recombinant expression vectors, host cells, populations of cells, and pharmaceutical compositions. The invention further provides methods of treating or preventing a condition in a mammal and methods of inducing arginase expression by peripheral blood lymphocytes (PBL).

Description

INDUCIBLE ARGINASE

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This patent application claims the benefit of U.S. Provisional Patent Application No. 62''293.513, filed February 10, 2016, which is incorporated by reference in its entirety herein.

INCORPORATION-BY-REFERENCE 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 25,840 Byte ASCII (Text) file named "727420... ST25.txt," dated January 31, 2017.

BACKGROUND OF THE INVENTION

[0003] Adoptive cell therapy can be an effective treatment for conditions (e.g., cancer) in some patients. However, obstacles to the overall success of adoptive cell therapy still exist. For example, one or more of the in vivo persistence, survival, proliferation, and cytolytic activity of T cells can, in some cases, decrease following adoptive transfer. Alternatively or additionally, in some cases, the increased apoptosis of T cells can pose obstacles to the treatment of conditions.

[0004] In spite of considerable research into methods of producing cells for adoptive cell therapy and treatments for cancer and viral conditions, there still exists a need for improved methods for producing cells for adoptive cell therapy and treating and/or preventing cancer and viral conditions.

BRIEF SUMMARY OF THE INVENTION

[0005] An embodiment of the invention provides a nucleic acid comprising a nuclear factor of activated T-cells (NFAT)-responsive promoter operatively associated with a nucleotide sequence encoding (i) arginase, (ii) a functional portion of arginase, or (iii) a functional variant of (i) or (ii). [0006] Further embodiments of the invention provide related recombinant expression vectors, host cells, populations of cells, pharmaceutical compositions, methods of treating a condition in a mammal, and methods of inducing arginase expression by peripheral blood lymphocytes (PBL).

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

[0007] Figure 1 A is schematic showing the structure of a retroviral vector comprising the reverse complement of a nucleic acid comprising a NFAT-responsive promoter operatively associated with a nucleotide sequence encoding green fluorescent protein (GFP). "LTR" designates "long terminal repeat." "PA2" designates a picornavirus 2A sequence.

[0008] Figure IB is schematic showing the structure of a retroviral vector comprising the reverse complement of a nucleic acid comprising a NFAT-responsive promoter operatively associated with a nucleotide sequence encoding truncated arginase II.

[0009] Figure 1C is schematic showing the structure of a retroviral vector comprising the reverse complement of a nucleic acid comprising a NFAT-responsive promoter operatively associated with a nucleotide sequence encoding arginase II.

[0010] Figure 2 is a graph showing the concentration (mM) of urea secreted into the supernatant by cells transduced with a NFAT-GFP, NFAT-trunc-Arg-2, or NFAT-Arg2 vector with (+) or without (-) restimulation with anti-CD3 antibody. Data are mean + standard error of the mean (SEM) of triplicate cultures and are representative of two independent experiments. ** P< 0.01, *** P< 0.001 , ns>0.05. "ns" designates "not significant."

[0011] Figure 3A is a graph showing the concentration (mM) of urea secreted into the supernatant by DMF5 anti-MART TCR-transduced cells which were further transduced with a NFAT-GFP, NFAT-trunc-Arg-2, or NFAT-Arg2 vector after re-stimulaiion with T2 target cells pulsed with MART peptide or vehicle (dimethyl sulfoxide (DMSO)). Data are mean + SEM of triplicate cultures. ** P< 0.01 , ns>0.05.

[0012] Figure 3B is a graph showing the concentration (optical density (O.D.)) of interferon (IFN)-y secreted into the supernatant by DMF5 anti-MART TCR-transduced cells which were further transduced with a NFAT-GFP, NFAT-trunc-Arg-2, or NFAT-Arg2 vector after re-stimulation with T2 target cells pulsed with MART peptide or vehicle (DMSO). Data are mean + SEM of triplicate cultures. ns>0.05. [0013] Figure 4 is a graph showing the percentage (%) of NFAT-GFP, NFAT-lrunc-Arg- 2, or NFAT-Arg2 vector-transduced PBMC staining positive for Annexin V following re- exposure of the transduced cells to plate-bound anti-CD3. Data shown are the mean * SEM of triplicate cultures assayed and are representative of three independent experiments. ** P< 0.01, ns>0.05.

[0014] Figure 5 is a graph showing the fold proliferation of PBMC transduced with one of the NFAT-GFP, NTAT-trunc-Arg-2, or NFAT-Arg2 vector following expansion of the numbers of cells using IL-2, anti-CD3 antibody, and irradiated PBMC (rapid expansion protocol (REP)). Data shown are the mean fold proliferation + SEM of triplicate cultures and are representative of 3 independent experiments. *P< 0.05, ** P< 0.01, ns>0.05.

[0015] Figure 6 A is a graph showing the percentage of human CD3-i- cells persisting in mice following adoptive transfer of human PBMC transduced with one of the NFAT-GFP, NFAT-trunc-Arg-2, or NFAT-Arg2 vector. Bar depicts mean. Persistence data are representative of 2 independent adoptive transfer experiments. *P< 0.05, ** P< 0.01, ns>0.05.

[0016] Figure 6B is a graph showing the percentage of human CD8+ cells persisting in mice following adoptive transfer of human PBMC transduced with one of the NFAT-GFP, NFAT-trunc-Arg-2, or NFAT-Arg2 vector. Bar depicts mean. Persistence data are representative of 2 independent adoptive transfer experiments. ** P< 0.01, ns>0.05.

[0017] Figure 6C is a graph showing the percentage of human CD4+ cells persisting in mice following adoptive transfer of human PBMC transduced with one of the NFAT-GFP, NFAT-trunc-Arg-2, or NFAT-Arg2 vector. Bar depicts mean. Persistence data are representative of 2 independent adoptive transfer experiments. *P< 0.05, ** P< 0.01, ns>0.05.

[0018] Figure 7A is a graph showing the concentration (pg/mL) of lFN-γ secreted into the supernatant by MART specific CD8+ clones which were further transduced with a NFAT-GFP (1), NFAT-Argl (2), NFAT-trunc-Arg-2 (3), or NFAT-Arg2 (4) vector after re- stimulation with T2 target cells pulsed with MART peptide or vehicle (DMSO). Data shown are the mean + SEM of triplicate cultures assayed and are representative of three independent experiments performed with three unique clones. ns>0.05 by two tailed t-test.

[0019] Figure 7B is a graph showing the concentration (mM) of urea secreted into the supernatant by MART specific CD8+ clones which were further transduced with a NFAT- GFP (1), NFAT-Argl (2), NFAT-trunc-Arg-2 (3), or NFAT-Arg2 (4) vector after re- stimulation with T2 target cells pulsed with MART peptide or vehicle (DMSO). Data shown are the mean + SEM of triplicate cultures assayed and are representative of three independent experiments performed with three unique clones. ***P<0.001, ns>0.05 by two tailed t-test.

[0020] Figure 7C is a graph showing the percentage (%) of NFAT-GFP (1), NFAT-Argl (2), NFAT-trunc-Arg-2 (3), or NFAT-Arg2 (4) vector-transduced MART specific CD8+ clones staining positive for Annexin V following re-exposure of the transduced cells to plate- bound anti-CD3. Data shown are the mean + SEM of triplicate cultures assayed and are representative of three independent experiments performed with three unique clones. ** P< 0.01, ns>0.05 by two tailed t-test.

[0021] Figure 8A is a graph showing the extracellular acidification rate (ECAR) of (mM/pH) NFAT-GFP (1), NFAT-Argl (2), NFAT-trunc-Arg-2 (3), or NFAT-Arg2 (4) vector-transduced MART specific CD8+ clones following re-exposure of the transduced cells to plate-bound anti-CD3. Data shown are the mean + SEM of triplicate cultures assayed and are representative of three independent experiments performed with three unique clones. ** P< 0.01, ns>0.05 by two tailed t-test.

[0022] Figure 8B is a graph showing the oxygen consumption rate (OCR) (pMoles/min) of (mM/pH) NFAT-GFP (1), NFAT-Argl (2), NFAT-trunc-Arg-2 (3), or NFAT-Arg2 (4) vector-transduced MART specific CD8+ clones under basal conditions and following treatment with the mitochondrial inhibitors oligomycin (oligo), FCCP (carbonyl cyanide-p- trifluoromethoxy-phenylhydrazone), rotenone (R), and antimycin A (A). Data shown are the mean + SEM of triplicate cultures assayed and are representative of three independent experiments performed with three unique clones.

[0023] Figure 8C is a graph showing the mitochondrial spare respiratory capacity (SRC) of NFAT-GFP (1), NFAT-Argl (2), NFAT-trunc-Arg-2 (3), or NFAT-Arg2 (4) vector- transduced MART specific CD8+ clones following re-exposure of the transduced cells to plate-bound anti-CD3. Data shown are the mean + SEM of triplicate cultures assayed and are representative of three independent experiments performed with three unique clones. *** P< 0.001, ns>0.05 by two tailed t-test.

[0024] Figure 8D is a graph showing the fold proliferation of NFAT-GFP (1), NFAT- Argl (2), NFAT-trunc-Arg-2 (3), or NFAT-Arg2 (4) vector-transduced MART specific CD8+ clones following re-exposure of the transduced cells to plate-bound anti-CD3 over three weeks. Data shown are the mean + SEM of triplicate cultures assayed and are representative of three independent experiments performed with three unique clones. *** P< 0.001, ns>0.05 by two-way ANOVA.

[002S| Figures 9A-9E are lymphocyte gated flow cytometry dot plots of a representative spleen harvested at day 21 from mice receiving saline (with no T cells) (sham transfer) (A) or cells transduced with the NFAT-GFP (B), NFAT-Argl (C), NFAT-trunc-Arg-2 (D), or NFAT-Arg2 (E) vector. Numbers in dot plots indicate the frequency of human CD3+CD8+ and CD3+CD4+ T cells. Adoptive transfer data are representative of three independent experiments.

[0026] Figures 1 OA- 1 OC are graphs showing the percentages of total CD3+ (A), CD8+ (B), and CD4+ (C) T cells persisting (on day 21 after transfer) in spleens harvested from each of the individual mice (represented by dot) receiving cells transduced with the NFAT-GFP (1), NFAT-Argl (2), NFAT-trunc-Arg-2 (3), or NFAT-Arg2 (4) vector. Adoptive transfer data are representative of three independent experiments. ** P< 0.01, *P< 0.05, ns>0.05 by two tailed t-test.

[0027] Figure 10D is a graph showing the tumor size (mm²) measured in tumor-bearing mice receiving no treatment (1) or adoptive transfer of cells transduced with the NFAT-trunc- Arg-2 vector (unshaded squares) (2) or NFAT-Arg2 (3) vector. Adoptive transfer data are representative of three independent experiments. *** P< 0.001, by Wilcoxon rank sum test.

DETAILED DESCRIFriON OF THE INVENTION

[0028] Arginase is an enzyme that catalyzes the hydrolysis of 1-arginine to 1-ornithine and urea. At least two isoforms of arginase are expressed in mammals: types I and II. Arginase I and II differ with respect to tissue distribution, subcellular localization, and physiological function. Arginase II is located in the mitochondria and is expressed in extra-hepatic tissues, particularly the kidney. Arginase I is located in the cytosol and is mainly expressed in the liver. As used herein, the term "arginase" collectively refers to arginase I and 11, functional portions of arginase I and II, and functional variants of arginase I and II, unless specified otherwise. Without being bound to a particular theory or mechanism, it is believed thai the 1- omithine produced by arginase can be further metabolized to produce one or both of polyamines and proline, which may facilitate cell proliferation.

[0029] An embodiment of the invention provides a nucleic acid comprising a NFAT- responsive promoter operatively associated with a nucleotide sequence encoding (i) arginase, (ii) a functional portion of arginase, or (iii) a functional variant of (i) or (ii). The inventive nucleic acids may provide many advantages including, for example, die inducible expression of arginase. In this regard, the inventive nucleic acids may make it possible to control the expression of arginase to enhance one or more of persistence, survival, proliferation, and cytolytic activity of cells following adoptive transfer. Cells comprising the inventive nucleic acids may express arginase only when the cell (e.g., an antigen-specific receptor expressed by the cell) is specifically stimulated by antigen and/or the cell (e.g., the calcium signaling pathway of the cell) is non-specifically stimulated by, e.g., phorbol myristate acetate

(PMA)Aonomycin. Accordingly, the expression of arginase may be controlled to occur only when and where it is needed, e.g., in the presence of cancer, a virally-infected cell, or at a tumor site. It is believed that little or no arginase is released outside of the presence of a virally-infected cell, cancer, or at a tumor site. Therefore, the production of unnecessary and/or excess arginase can be reduced or eliminated, which may, advantageously, decrease or avoid arginase toxicity.

[0030] The arginase encoded by the inventive nucleic acids may be any suitable mammalian arginase, e.g., human arginase or mouse arginase. In a preferred embodiment, the inventive nucleic acids encode human arginase.

[0031] The arginase encoded by the inventive nucleic acids may be either isoform of arginase, namely arginase I or arginase II. In an embodiment of the invention, the inventive nucleic acids encode arginase I, e.g., human arginase I. Examples of human arginase I amino acid sequences include, but are not limited to, the amino acid sequences of GenBank

Accession Nos. NP_001231367.1 (isoform 1 of argainse I), NP_000036.2 (isoform 2 of arginase I), XP_011534103.1, EAW48053.1, EAW48054.1, EAW48055.1, EAW48056.1, AAL71547.1, AAH05321.1, AAH20653.1, AAP35387.1 , AAA51776.1, ABM82965.1, and ABM86156.1. In a preferred embodiment, the full-length, wild-type human arginase I comprises the amino acid sequence of SEQ ID NO: 1.

[0032] In a preferred embodiment, the inventive nucleic acids encode arginase II, especially preferably human arginase II. Examples of human arginase II amino acid sequences include, but are not limited to, the amino acid sequences of GenBank Accession Nos. NP..001163.1, EAW80943.1, EAW80944.1 , BAG35387.1, AAL71548.1, AAH01350.1, AAH08464.1, AAH29050.1, CAG38787.1 , BAA13158.1, AAB39855.1, and AAC51664.1. In a preferred embodiment, the full-length, wild-type human arginase II comprises the amino acid sequence of SEQ ID NO: 2. [0033] In an embodiment of the invention, the nucleotide sequence encodes a functional portion of arginase. The term "functional portion" refers to any part or fragment of the arginase, which part or fragment retains the biological activity of the arginase of which it is a part (the parent arginase). In reference to the parent arginase, the functional portion can comprise, for instance, about 10%, about 25%, about 30%, about 50%, about 68%, about 80%, about 90%, about 95%, or more, of the parent arginase. Examples of functional portions include, but are not limited to, full-length, wild-type human arginase which lacks a leader sequence. In full-length arginase, the leader sequence may be positioned at the amino terminus. An example of a leader sequence includes, but is not limited to, the human arginase II leader amino acid sequence of SEQ ID NO: 3. Accordingly, in an embodiment of the invention, the functional portion comprises the amino acid sequence of SEQ ID NO: 4 (full-length, wild-type human arginase II without a leader sequence).

[0034] In an embodiment of the invention, the nucleotide sequence encodes a functional variant of arginase. The term "functional variant," as used herein, refers to arginase having substantial or significant sequence identity or similarity to a parent arginase, which functional variant retains the biological activity of the arginase of which it is a variant. In reference to the parent arginase, the functional variant can, tor instance, be at least about 30%, about 50%, about 75%, about 80%, about 90%, about 98% or more identical in amino acid sequence to the parent arginase. The functional variant can, for example, comprise the amino acid sequence of the parent arginase with at least one conservative amino acid substitution.

Conservative amino acid substitutions are known in the art, and include amino acid substitutions in which one amino acid having certain physical and/or chemical properties is exchanged for another amino acid that has the same chemical or physical properties. For instance, the conservative amino acid substitution can be an acidic amino acid substituted for another acidic amino acid (e.g., Asp or Glu), an amino acid with a nonpolar side chain substituted for another amino acid with a nonpolar side chain (e.g., Ala, Gly, Val, He, Leu, Met, Phe, Pro, Trp, Val, etc.), a basic amino acid substituted for another basic amino acid (Lys, Arg, etc.), an amino acid with a polar side chain substituted for another amino acid with a polar side chain (Asn, Cys, Gin, Ser, Thr, Tyr, etc.), etc.

[0035] Alternatively or additionally, the functional variants can comprise the amino acid sequence of the parent arginase with at least one non-conservative amino acid substitution. In this case, it is preferable for the non-conservative amino acid substitution to not interfere with or inhibit the biological activity of the functional variant. Preferably, the non- conservative amino acid substitution enhances the biological activity of the functional variant, such thai the biological activity of the functional variant is increased as compared to the parent arginase.

[0036] The arginase can consist essentially of the specified amino acid sequence or sequences described herein, such that other components of the functional variant, e.g., other amino acids, do not materially change the biological activity of the functional variant. In this regard, the arginase can, for example, consist essentially of the amino acid sequence of SEQ ID NO: 1, 2, or 4.

[0037] Functional portions and functional variants encompass, for example, those portions and variants, respectively, of arginase that retain the ability to catalyze the hydrolysis of 1-arginine to l-ornithine and urea; increase one or more of the persistence, survival, proliferation, and cytolytic activity of T cells following adoptive transfer; decrease apoptosis of T cells, or treat or prevent a condition, to a similar extent, the same extent, or to a higher extent, as the parent arginase.

[0038] The nucleotide sequence encoding arginase I may comprise any nucleotide sequence that encodes any of the arginase I amino acid sequences described herein.

Examples of nucleotide sequences encoding human arginase I include, but are not limited to, the nucleotide sequences of GenBank Accession Nos. NM_001244438.1 (arginase 1 isoform 1), NM_000045.3 (arginase T isoform 2), XM_011535801.1, AL121575.24,

AMYH02014494.1, CH471051.2, EAW48054.1, EAW48055.1, EAW48056.1, X12662.1, X12663.1, X12664.1, X12665.1, X12666.1, X12667.1, X12668.1, X12669.1, AK128314.1, AW236349.1 , AY074488.1, BC005321.1, BC020653.1, BG217880.1, BG542163.1, BT006741.1, M14502.1, DQ892039.2, and DQ895230.2. Preferably, the nucleotide sequence encoding full-length wild-type, human arginase I comprises the nucleotide sequence of SEQ ID NO: 5.

[0039] The nucleotide sequence encoding arginase II may comprise any nucleotide sequence that encodes any of the arginase II amino acid sequences described herein.

Examples of nucleotide sequences encoding human arginase II include, but are not limited to, the nucleotide sequences of GenBank Accession Nos. NM 001172.3, AK312484.1 ,

AL135439.1, AY074489.1, BC001350.1, BC008464.1 , BC029050.1 , CR536550.1,

D86724.1 , U75667.1 , and U82256.1. Preferably, the nucleotide sequence encoding full- length wild-type, human arginase II comprises the nucleotide sequence of SEQ ID NO: 6. [0040] The terms "nucleic acid" and "polynucleotide," as used herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides (RNA) or

deoxyribonucleotides (DNA). These terms refer to the primary structure of the molecule, and thus include double- and single-stranded DNA, double- and single-stranded RNA, and double-stranded DNA-RNA hybrids. The terms include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs and modified polynucleotides such as, though not limited to, methylated and/or capped polynucleotides. In an embodiment of the invention, the nucleic acid is complementary DNA (cDNA).

[0041] The term "nucleotide" as used herein refers to a monomelic subunit of a polynucleotide that consists of a heterocyclic base, a sugar, and one or more phosphate groups. The naturally occurring bases (guanine (G), adenine (A), cytosine (C), thymine (T), and uracil 0-0) ^are typically derivatives of purine or pyrimidine, though the invention includes the use of naturally and non-naturally occurring base analogs. The naturally occurring sugar is the pentose (five-carbon sugar) deoxyribose (which forms DNA) or ribose (which forms RNA), though the invention includes the use of naturally and non-naturally occurring sugar analogs. Nucleic acids are typically linked via phosphate bonds to form nucleic acids or polynucleotides, though many other linkages are known in the art (e.g., phosphorothioates, boranophosphates, and the like). Methods of preparing polynucleotides are within the ordinary skill in the art (Green and Sambrook, Molecular Cloning: A

Laboratory Manual, (4th Ed.) Cold Spring Harbor Laboratory Press, New York (2012)).

[0042] In an embodiment of the invention, the nucleotide sequence encoding arginase is codon optimized. Without being bound to a particular theory or mechanism, it is believed that codon optimization of the nucleotide sequence increases the translation efficiency of the mRNA transcripts. Codon optimization of the nucleotide sequence may involve substituting a native codon for another codon that encodes the same amino acid, but can be translated by tRNA that is more readily available within a cell, thus increasing translation efficiency. Codon optimization of the nucleotide sequence may also reduce secondary mRNA structures that would interfere with translation, thus increasing translation efficiency. For example, a codon-optimized nucleotide sequence encoding full-length, wild-type human arginase I may comprise the nucleotide sequence of SEQ ID NO: 7.

[0043] The nucleic acid of the invention may comprise any suitable NFAT-responsive promoter. NFAT is a family of transcription factors including four calcium-responsive proteins NFAT1, NFAT2, NFAT3, and NFAT 4. "NFAT-responsive promoter," as used herein, encompasses any one or more NFAT-responsive elements linked to a minimal promoter of any gene expressed by T-cells. Preferably, the minimal promoter of a gene expressed by T-cells is a minimal human IL-2 promoter. The NFAT-responsive elements may comprise, e.g., any one or more of NFAT1, NFAT2, NFAT3, and NFAT4-responsive elements. The NFAT-responsive promoter may comprise any number of binding motifs, e.g., at least two, at least three, at least four, at least five, or at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, or up to twelve binding motifs. In a preferred embodiment, the NFAT-responsive promoter comprises six NFAT binding motifs. In an especially preferred embodiment, the NFAT-responsive promoter comprises or consists of the nucleotide sequence of SEQ ID NO: 8.

[0044] The NFAT-responsive promoter is operatively associated with the nucleotide sequence encoding arginase. "Operatively associated with" means that the nucleotide sequence encoding arginase is transcribed into arginase mRNA when the NFAT protein binds to the NFAT-responsive promoter sequence. Without being bound to a particular theory or mechanism, it is believed that NFAT is regulated by a calcium signaling pathway. In particular, it is believed that antigen-specific receptor stimulation (by, e.g., an antigen) and/or non-specific stimulation of the calcium signaling pathway of the cell (by, e.g.,

PMA-lonomycin) increases intracellular calcium concentration and activates calcium channels. It is believed that the NFAT protein is then dephosporylated by calmoduin and translocates to the nucleus where it binds with the NFAT-responsive promoter sequence and activates downstream gene expression. By providing a NFAT-responsive promoter that is operatively associated with the nucleotide sequence encoding arginase, the nucleic acids of the invention advantageously make it possible to express arginase only when the host cell including the nucleic acid is stimulated by, e.g., PMA-'lonomycin and/or an antigen.

[0045] In an embodiment of the invention, the nucleic acid comprises the NFAT- responsive promoter and the nucleotide sequence encoding argainase in a "forward" orientation, i.e., a 5' to 3' orientation. A nucleic acid is in "forward" orientation when the nucleic acid (i) provides one or more NFAT binding motifs and (ii) encodes the arginase amino acid sequence when the nucleic acid strand is read in a 5' to 3' direction. Moreover, the nucleic acid in "forward" orientation may comprise the NFAT-responsive promoter positioned 5' of the arginase nucleotide sequence and the arginase nucleotide sequence positioned 3 ' of the NFAT-responsive promoter. Furthermore, the nucleic acid in "forward" orientation may comprise the NFAT-responsive promoter positioned 5' of both the arginase nucleotide sequence and any post-transcriptional regulatory element, (e.g., woodchuck hepatitis post-transcriptional regulatory element (WPRE)) and the arginase nucleotide sequence positioned 3' of the NFAT promoter and 5' of the post-transcriptional regulatory element. The nucleotide sequences disclosed herein are in "forward" orientation unless specified otherwise.

[0046] In an embodiment of the invention, the nucleic acid comprises the reverse complement of any of the nucleic acids described herein. Examples of reverse complements may include, but are not limited to, the nucleotide sequences of SEQ ID NO: 9 (reverse complement of the nucleotide sequence encoding human arginase II) and SEQ ID NO: 10 (the reverse complement of a NFAT-responsive promoter). The reverse complement of the NFAT-responsive promoter may be positioned 3' of the reverse complement of the arginase nucleotide sequence and the reverse complement of the arginase nucleotide sequence may be positioned 5' of the reverse complement of the NFAT-responsive promoter when the nucleic acid strand is read from the 5' to 3' direction. Furthermore, the reverse complement of the NFAT-responsive promoter may be positioned 3^* of both the reverse complement of the arginase nucleotide sequence and the reverse complement of any post-transcriptional regulatory element, (e.g., a poly A tail (e.g., SV40 polyA tail, BGH polyA tail, polyAl tail, poly A2 tail)), and the reverse complement of the arginase nucleotide sequence may be positioned 5' of the reverse complement of the NFAT promoter-responsive and 3 ' of the reverse complement of the post-transcriptional regulatory element when the nucleic acid strand is read from the 5' to 3' direction.

[0047] The nucleic acid comprising the reverse complement of any of the nucleic acids described herein may provide one or more advantages. For example, the reverse complement may enhance arginase transcription efficiency as compared to the nucleic acid in "forward" orientation. Alternatively or additionally, the reverse complement may reduce or avoid expression of arginase until the nucleic acid is incorporated into the host cell and the host cell is stimulated by, e.g., PMA/Ionomycin and/or an antigen. Accordingly, the premature expression of arginase may be reduced or eliminated.

[0048] Preferably, the nucleic acids of the invention 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 vitro replication or in vivo replication. [0049] The nucleic acids can be constructed based on chemical synthesis and/or enzymatic ligation reactions using procedures known in the art. See, for instance, Green and Sambrook, supra. For example, a nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed upon hybridization (e.g., phosphorothioate derivatives and acridine substituted nucleotides).

Examples of modified nucleotides that can be used to generate the nucleic acids include, but are not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydn)xymethyl) uracil, 5-carboxymethylaminomethyl- 2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-substituted adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-memoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, S'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio- N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2- thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5- oxyacetic acid methylester, 3-(3-amino-3-N-2-carboxypropyl) uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleic acids of the invention can be purchased from companies, such as Macromolecular Resources (Fort Collins, CO) and Synthegen (Houston, TX).

[0050] In an embodiment of the invention, the nucleic acid comprises a nucleotide sequence which is at least about 75%, e.g., at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to any of the nucleotide sequences described herein.

[0051] The nucleic acids of the invention can be incorporated into a recombinant expression vector. In this regard, an embodiment of the invention provides recombinant expression vectors comprising any of the nucleic acids of the invention. 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 naUw¾lly-occuning. Accordingly, an embodiment of the invention provides a recombinant expression vector comprising (i) the inventive nucleic acid and (ii) a heterologous nucleic acid sequence. The phrase "heterologous nucleic acid sequence," as used herein, means a nucleic acid sequence that does not naturally occur in the species that expresses the arginase encoded by the vector. For example, if the arginase encoded by the vector is mouse arginase, the heterologous nucleic acid sequence in the vector may be any sequence that does not naturally occur in a mouse. In an embodiment in which the arginase encoded by the vector is human arginase, the heterologous nucleic acid sequence in the vector may be any sequence that does not naturally occur in a human. The heterologous nucleic acid sequence may be a nucleic acid sequence from any species other than the species that expresses the arginase encoded by the vector.

[0052] The inventive recombinant expression vectors can comprise any type of nucleotide, 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 naturally-occurring, non-naturally-occurring internucleotide linkages, or both types of linkages. Preferably, the non-naturally occurring or altered nucleotides or internucleotide linkages do not hinder the transcription or replication of the vector.

[0053] The recombinant expression vector of the invention can be any suitable recombinant expression vector, and can be used to transform or transduce any suitable host. Suitable vectors include those designed for propagation and expansion or for expression or both, such as plasmids and viruses. The vector can be selected from the group consisting of the pUC series (Fermentas Life Sciences), the pBluescript series (Stratagene, LaJolla, CA), the pET series (Novagen, Madison, WI), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, CA). Bacteriophage vectors, such as XGTIO, XGTl 1, XZapII (Stratagene), XEMBL4, and λΝΜΙ 149, also can be used. Examples of plant expression vectors include pBlOl, pBI101.2, pBI101.3, pBI121 and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-Cl, pMAM and pMAMneo (Clontech). Preferably, the recombinant expression vector is a viral vector (e.g., adenoviral vector, adeno-associated viral (AAV) vector, herpes viral vector, retroviral vector, or lentiviral vector) or a transposon vector, and preferably has a native or engineered capacity to transform T cells. [0054] The inventive nucleic acid may be positioned in the recombinant expression vector in any suitable orientation. In an embodiment in which the inventive nucleic acid is in a "forward" orientation, the inventive nucleic acid is positioned in the vector consistent with the 5' to 3' direction of the long terminal repeat (LTR) of the vector. In another embodiment of the invention, the reverse complement of the inventive nucleic acid is positioned in the vector reverse to the 5' LTR direction. For example, the recombinant vector may comprise the nucleotide sequence of SEQ ID NO: 11, which comprises the reverse complement of a nucleotide sequence comprising an NF AT -responsive promoter operatively associated with a nucleotide sequence encoding arginase II, wherein the NFAT and arginase Π nucleotide sequences are positioned in the vector reverse to the 5' LTR. direction.

[00SS] The recombinant expression vectors of the invention can be prepared using standard recombinant DNA techniques described in, for example, Green and Sambrook, supra. Constructs of expression vectors, which are circular or linear, can be prepared to contain a replication system functional in a prokaryotic or eukaryotic host cell. Replication systems can be derived, e.g., from ColEl, 2 μ plasmid, λ, SV40, bovine papilloma virus, and the like.

[0056] Desirably, the recombinant expression vector comprises regulatory sequences, such as transcription and translation initiation and termination codons, which are specific to the type of host cell (e.g., bacterium, fungus, plant, or animal) into which the vector is to be introduced, as appropriate and taking into consideration whether the vector is DNA- or RNA- based.

[0057] The recombinant expression vector can include one or more marker genes, which allow for selection of transformed or transduced hosts. Marker genes include biocide resistance, e.g., resistance to antibiotics, heavy metals, etc., complementation in an auxotrophic host to provide prototrophy, and the like. Suitable marker genes for the inventive expression vectors include, for instance, neomycm/G418 resistance genes, hygromycin resistance genes, histidinol resistance genes, tetracycline resistance genes, and ampicillin resistance genes.

[0058] Another embodiment of the invention provides a host cell comprising any of the recombinant expression vectors described herein. As used herein, the term "host cell" refers to any type of cell that can contain the inventive recombinant expression vector. The host cell can be a eukaryotic cell, e.g., plant, animal, fungi, or algae, or can be a prokaryotic cell, e.g., bacteria or protozoa. The host cell can be a cultured cell or a primary cell, i.e., isolated directly from an organism, e.g., a human. The host cell can be an adherent cell or a suspended cell, i.e., a cell that grows in suspension. Suitable host cells are known in the art and include, for instance, DH5a E. coli cells, Chinese hamster ovarian cells, monkey VERO cells, COS cells, HEK293 cells, and the like. For purposes of amplifying or replicating the recombinant expression vector, the host cell is preferably a prokaryotic cell, e.g., a DH5a cell. For purposes of producing arginase, the host cell is preferably a mammalian cell. Most preferably, Hie host cell is a human cell. While the host cell can be of any cell type, can originate from any type of tissue, and can be of any developmental stage, the host cell preferably is a peripheral blood mononuclear cell (PBMC) or a peripheral blood lymphocyte (PBL), e.g., a T cell or a natural killer (NK) cell. More preferably, the host cell is a T cell.

[0059] The T cell can be any T cell, such as 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 cell 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, or other tissues or fluids. T cells can also be enriched for or purified. Preferably, the T cell is a human T cell. More preferably, the T cell is a T cell isolated from a human. The T cell can be any type of T cell and can be of any developmental stage, including but not limited to, CD4+/CD8+ double positive T cells, CD4+ helper T cells, e.g., Thl and Th2 ceils, CD8+ T cells (e.g., cytotoxic T cells), tumor infiltrating cells (TILs), memory T cells, naive T cells, and die like. Preferably, the T cell is a CD8+ T cell or a CD4+ T cell.

[0060] The inventive nucleic acids may be introduced into the host cell using any suitable method such as, for example, transfection, transduction, or elcctroporation. For example, host cells can be transduced with viral vectors using viruses (e.g., retrovirus or lentivirus) and host cells can be transduced with transposon vectors using eleciroporation.

[0061] The inventive host cell overexpresses arginase mRNA, polypeptide, or protein as compared to a negative control cell. The term "negative control cell," as used herein, refers to a cell that is identical to the inventive host cell except that the negative control cell does not comprise the inventive nucleic acid. For example, without limiting the invention, a host cell comprising the inventive nucleic acid expresses an amount of arginase (mRNA, protein, or polypeptide) that is 1.5-fold higher or more, e.g., 2-fold higher or more, 3-fold higher or more, 5-fold higher or more, 10-fold higher or more, 20-fold higher or more, or 50-fold higher or more, than the amount of arginase present in a negative control cell. The arginase can be present in a host cell in an amount bounded by any two of the above endpoints. For example, the host cell comprising the inventive nucleic acid can contain an amount of arginasc (mRNA, protein, or polypeptide) that is about 1.5-fold to about 20-fold higher, about 2-fold to about 5-fold higher, about 3-fold to about 50-fold higher, or about 20-fold to about 50-fold higher, than the amount of arginase present in a negative control cell. Any suitable method known in the art can be utilized to determine the amount of arginase mRNA, protein, or polypeptide present in a host cell or a population thereof, such as quantitative reverse transcription polymerase chain reaction (RT-PCR) or stem-loop quantitative RT-PCR.

[0062] The inventive host cells may provide any one or more of various advantages. For example, the inventive host cell may provide one or more of increased persistence, survival, proliferation, and cytolytic activity following adoptive transfer as compared to a negative control cell. Alternatively or additionally, the inventive host cell may provide decreased apoptosis as compared to a negative control cell.

[0063] In an embodiment of the invention, the host cell further comprises an antigen- specific receptor. The phrases "antigen-specific" and "antigenic specificity," as used herein, mean that the antigen-specific receptor can specifically bind to and immunologically recognize an antigen, or an epitope thereof, such that binding of the antigen-specific receptor to antigen, or the epitope thereof, elicits an immune response.

[0064] In an embodiment of the invention, the antigen-specific receptor is a T-cell receptor (TCR). A TCR 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 antigen- specific TCR can comprise any amino acid sequence, provided that the TCR can specifically bind to and immunologically recognize an antigen, such as a condition-specific antigen or epitope thereof.

[0065] The TCR can be an endogenous TCR, i.e., a TCR that is endogenous or native to (naturally-occurring on) the host cell. In such a case, the host cell comprising the

endogenous TCR can be a T cell that was isolated from a mammal which is known to express the particular condition-specific antigen. In certain embodiments, the T cell is a primary T cell isolated from a mammal afflicted with cancer or a viral condition. In some

embodiments, the cell is a tumor infiltrating lymphocyte (TIL) or a peripheral blood lymphocyte (PBL) isolated from a human cancer patient or a human patient with a viral condition. [0066] In some embodiments, the mammal from which a cell is isolated is immunized with an antigen of, or specific for, a condition. Desirably, the mammal is immunized prior to obtaining the cell from the mammal. In this way, the isolated cells can include T cells induced to have specificity for the condition to be treated, or can include a higher proportion of cells specific for the condition.

[0067] Alternatively, a T cell comprising an endogenous antigen-specific TCR can be a T cell within a mixed population of cells isolated from a mammal, and the mixed population can be exposed to the antigen which is recognized by the endogenous TCR while being cultured in vitro. In this manner, the T cell which comprises the TCR that recognizes the condition-specific antigen expands or proliferates in vitro, thereby increasing the number of T cells having the endogenous antigen-specific TCR.

[0068] The inventive host cell comprising an endogenous antigen- specific TCR can also be modified to express one or more nucleic acids encoding an exogenous (e.g., recombinant) antigen-specific receptor. Such exogenous antigen-specific receptors, e.g., exogenous TCRs and chimeric antigen receptors (CARs) (described in more detail below), can confer specificity for additional antigens to the modified host cell beyond the antigens for which the endogenous TCR is naturally specific. This can, but need not, result in the production of a T cell having dual antigen specificities.

[0069] In an embodiment of the invention, the antigen-specific receptor is an exogenous (e.g., recombinant) TCR, i.e., an antigen-specific TCR that is not native to (not naturally- occurring on) the host cell. A recombinant TCR is a TCR which has been generated through recombinant expression of one or more exogenous TCR α-, β-, γ-, and/or δ-chain encoding genes. A recombinant TCR can comprise polypeptide chains derived entirely from a single mammalian species, or the antigen-specific TCR can be a chimeric or hybrid TCR comprised of amino acid sequences derived from TCRs from two different mammalian species. For example, the exogenous antigen-specific TCR can comprise a variable region derived from a murine TCR and a constant region of a human TCR such that the TCR is "humanized." Recombinant TCRs and methods of making them are known in the art. See, for example, U.S. Patent Nos. 7,820,174; 7,915,036; 8,088,379; 8,216,565; 8,785,601 ; 9,345,748;

9,487,573; U.S. Patent Application Publication Nos. 2014/0378389; 2015/0246959;

2016/0152681 ; 2016/0333422; and WO 2015/184228.

[0070] In an embodiment of the invention, the antigen-specific receptor is a CAR.

Typically, a CAR comprises the antigen binding domain of an antibody, e.g., a single-chain variable fragment (scFv), fused to the transmembrane and intracellular domains of a TCR. Thus, the antigenic specificity of a TCR of the invention can be encoded by a scFv which specifically binds to the antigen, or an epitope thereof. CARs, and methods of making them, are known in the art. See, for example, U.S. Patent No. 8,465,743; 9,266,960; 9,359,447; U.S. Patent Application Publication Nos. 2014/0274909; 2015/0299317; 2015/0051266; 2016/0053017; and 2016/0333422.

[0071] Any suitable nucleic acid encoding an antigen-specific receptor can be used. In these embodiments, introducing a nucleic acid comprising an NFAT-responsive promoter opcratively associated with a nucleotide sequence encoding arginase, as discussed herein, can occur before, after, or simultaneously with, introducing a nucleic acid encoding an antigen- specific receptor. The antigen-specific receptor encoded by the nucleic acid can be of any suitable form including for example, a single-chain TCR, a single chain CAR, or a fusion with other proteins or polypeptides (e.g., without limitation co-stimulatory molecules).

[0072] The condition which is associated with or is characterized by the antigen recognized by the antigen-specific receptor can be any condition. For instance, the condition can be a cancer or a viral condition, as discussed herein.

[0073] For purposes herein, "viral condition" means a condition that can be transmitted from person to person or from organism to organism, and is caused by a virus. In an embodiment of the invention, the viral condition is caused by a virus selected from the group consisting of herpes viruses, pox vimses, hepadnaviruses, papilloma viruses, adenoviruses, corono viruses, orthomyxoviruses, paramyxoviruses, flaviviruses, and caliciviruses. For example, the viral disease may be caused by a virus selected from the group consisting of respiratory syncytial virus (RSV), influenza virus, herpes simplex virus, Epstein-Barr virus, varicella virus, cytomegalovirus, hepatitis A virus, hepatitis B virus, hepatitis C virus, human immunodeficiency virus (HIV), human T-lymphotropic virus, calicivirus, adenovirus, and Arena virus.

[0074] The viral condition may be, for example, influenza, pneumonia, herpes, hepatitis, hepatitis A, hepatitis B, hepatitis C, chronic fatigue syndrome, sudden acute respiratory syndrome (SARS), gastroenteritis, enteritis, carditis, encephalitis, bronchiolitis, respiratory papillomatosis, meningitis, H1V7AIDS, and mononucleosis. Viral antigens are known in the art and include, for example, any viral protein, e.g., env, gag, pol, gpl20, thymidine kinase, an HIV antigen, an influenza antigen, a Herpes virus antigen, a malaria antigen, and the like. [0075] Preferably, the antigen-specific receptor has antigenic specificity for a cancer antigen (also termed a tumor antigen or a tumor-associated 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 cells. 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 cells 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.

[0076] 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. Cancer antigens are known in the art and include, for instance, mesothelin, CD19, CD22, CD276 (B7H3), gplOO, MART-1, Epidermal Growth Factor Receptor Variant III (EGFRVIII), TRP-1, TRP-2, tyrosinase, NY-ESO-1 (also known as CAG-3), MAGE-1, MAGE-3, etc.

[0077] The cancer may be any cancer, including any of acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, or anorectum, 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, cancer of the oral cavity, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumor, Hodgkin lymphoma, hypopharynx cancer, kidney cancer, larynx cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharynx cancer, non-Hodgkin lymphoma, ovarian cancer, pancreatic cancer, peritoneum, omentum, and mesentery cancer, pharynx cancer, prostate cancer, rectal cancer, renal cancer (e.g., renal cell carcinoma (RCC)), small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, ureter cancer, and urinary bladder cancer. In certain preferred embodiments, the antigen-specific receptor has specificity for a melanoma antigen.

[0078] Also provided by an embodiment of the invention is a population of cells comprising at least one host cell described herein. The population of cells can be a heterogeneous population comprising the host cell comprising any of the recombinant expression vectors described herein, in addition to at least one other cell, e.g., a host cell (e.g., a T cell), which does not comprise any of the recombinant expression vectors, or 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 cells, a muscle cell, a brain cell, etc. Alternatively, the population of cells can be a substantially homogeneous population, in which the population comprises mainly of (e.g., consisting essentially of) host cells comprising the recombinant expression vector. The population also can be a clonal population of cells, in which all cells of the population are clones of a single host cell comprising a recombinant expression vector, such that all cells of the population comprise the recombinant expression vector. In one embodiment of the invention, the population of cells is a clonal population comprising host cells comprising a recombinant expression vector as described herein.

[0079] The host cells and populations thereof may be isolated or purified. 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 purity, wherein "purity" is a relative term, and not to be necessarily construed as absolute purity. A "purified" T cell refers to a T cell which has been separated from other natural components, such as tissues, cells, proteins, nucleic acids, etc.

[0080] The inventive nucleic acids, recombinant expression vectors, and host cells (including populations thereof), all of which are collectively referred to as "inventive arginase materials" hereinafter, can be formulated into a composition, such as a pharmaceutical composition. In this regard, an embodiment of the invention provides a pharmaceutical composition comprising any of the inventive arginase materials described herein and a pharmaceutically acceptable carrier. In a preferred embodiment, the pharmaceutical composition comprises any of the inventive populations of cells described herein and a pharmaceutically acceptable carrier.

[0081] The pharmaceutically acceptable carriers described herein, for example, vehicles, adjuvants, excipients, and diluents, are well-known and readily available to those skilled in the art. Preferably, the pharmaceutically acceptable carrier is chemically inert to the active agent(s), e.g., inventive arginase material(s), and does not elicit any detrimental side effects or toxicity under the conditions of use.

[0082] The composition can be formulated for administration by any suitable route, such as, for example, an administration route selected from the group consisting of intravenous, intratumoral, intraarterial, intramuscular, intraperitoneal, intrathecal, epidural, and subcutaneous administration routes. Preferably, the composition is formulated for a parenteral route of administration. An exemplary pharmaceutically acceptable carrier for 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), PLASMA-LYTE A (Baxter, Deerfield, IL), about 5% dextrose in water, or Ringer's lactate, hi an embodiment, the pharmaceutically acceptable carrier is supplemented with human serum albumin.

[0083] For purposes of the invention, the amount or dose of the inventive population of cells or pharmaceutical composition administered (e.g., numbers of cells when the inventive population of cells is administered) should be sufficient to effect, e.g., a therapeutic or prophylactic response, in the patient over a reasonable lime frame. For example, the dose of the inventive population of cells or pharmaceutical composition should be sufficient to treat or prevent cancer or a viral condition 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 dose will be determined by the efficacy of the particular inventive population of cells or pharmaceutical composition administered and the condition of the patient, as well as the body weight of the patient to be treated.

[0084] Many assays for determining an administered dose are known in the art. For purposes of the invention, an assay, which comprises comparing the extent to which target cells are lysed upon administration of a given dose of such T cells to a mammal among a set of mammals of which is each given a different dose of die cells, could be used to determine a starting dose to be administered to a patient. The extent to which target cells are lysed upon administration of a certain dose can be assayed by methods known in the art. [0085] The dose of the inventive population of cells or pharmaceutical composition also will be determined by the existence, nature and extent of any adverse side effects that might accompany the administration of a particular inventive population of cells or pharmaceutical composition. Typically, the attending physician will decide the dosage of the population of cells or pharmaceutical composition with which to treat each individual patient, taking into consideration a variety of factors, such as age, body weight, general health, diet, sex, inventive population of cells or pharmaceutical composition to be administered, route of administration, and the severity of the condition being treated.

[0086] Any suitable number of host cells of the invention can be administered to a mammal. While a single host cell of the invention theoretically is capable of expanding and providing a therapeutic benefit, it is preferable to administer about 10² or more, e.g., about 10³ or more, about 10⁴ or more, about 10⁵ or more, about 10⁸ or more, host cells of the invention. Alternatively, or additionally about 10¹² or less, e.g., about 10" or less, about 10⁹ or less, about 10⁷ or less, or about 10^s or less, host cells of the invention can be administered to a mammal. The number of host cells of the invention can be administered to a mammal in an amount bounded by any two of the above endpoints, e.g., about 10² to about 10^s, about 10³ to about 10⁷, about 10³ to about 10⁹, or about 10⁵ to about 10¹⁰.

[0087] It is contemplated that the inventive pharmaceutical compositions, nucleic acids, recombinant expression vectors, host cells, and populations of cells can be used in methods of treating or preventing a condition. In this regard, an embodiment of the invention provides a method of treating or preventing a condition in a mammal, comprising administering to the mammal any of the pharmaceutical compositions, nucleic acids, recombinant expression vectors, host cells, or populations of cells described herein, in an amount effective to treat or prevent the condition in the mammal.

[0088] In an embodiment of the invention, the condition is cancer or a viral condition. The cancer and viral condition may be any of the cancers and viral conditions described herein with respect to other aspects of the invention. In a preferred embodiment, the condition is cancer.

[0089] 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 a condition in a patient. Furthermore, the treatment or prevention provided by the inventive method can include treatment or prevention of one or more conditions, signs, or symptoms of the condition being treated or prevented. For example, treatment or prevention can include promoting the regression of a tumor. Also, for purposes herein, "prevention" can encompass delaying the onset or recurrence of the condition, or a sign, symptom or condition thereof.

[0090] The term "mammal" as used herein refers to any mammal, including, but not limited to, mice, hamsters, rats, rabbits, cats, dogs, cows, pigs, horses, monkeys, apes, and humans. Preferably, the mammal is a human.

[0091] In the treatment or prevention of a condition in a mammal, the inventive host cell or population thereof can be transferred into the same mammal from which cell(s) were obtained. In other words, the host cell(s) used in the inventive method of treating or preventing a condition can be an autologous cell, i.e., can be obtained from the mammal in which the condition is treated or prevented. Alternatively, the host cell can be allogenically transferred into another mammal. Preferably, the host cell is autologous to the mammal in the inventive method of treating or preventing a condition in the mammal.

[0092] In the instance that the host cell(s) are autologous to the mammal, the mammal can be immunologically naive, immunized, diseased, or in another condition prior to isolation of the cell(s) from the mammal. In some instances, it is preferable for the method to comprise immunizing the mammal with an antigen of the condition prior to isolating the cell(s) from the mammal, introducing the inventive nucleic acid into the host cell(s), and the administering of the cell(s) or composition thereof. As discussed herein, immunization of the mammal with the antigen of the condition will allow a population of T cells having an endogenous TCR reactive with the condition-specific antigen to increase in numbers, which will increase the likelihood that a T cell obtained for being modified to comprise the inventive nucleic acid will have a desired antigen-specific TCR.

[0093] In accordance with an embodiment of the invention, a mammal with a condition can be therapeutically immunized with an antigen from, or associated with, that condition, including immunization via a vaccine. While not desiring to be bound by any particular theory or mechanism, the vaccine or immunogen is provided to enhance the mammal's immune response to the condition-associated antigen present in or on the infectious agent or diseased tissue. Such a therapeutic immunization includes, but is not limited to, the use of recombinant or natural condition-associated proteins, peptides, or analogs thereof, or modified condition peptides, or analogs thereof that can be used as a vaccine therapeutically as part of adoptive immunotherapy. The vaccine or immunogen, can be a cell, cell lysate (e.g., from cells transfected with a recombinant expression vector), a recombinant expression vector, or antigenic protein or polypeptide. Alternatively, the vaccine, or immunogen, can be a partially or substantially purified recombinant condition protein, polypeptide, peptide or analog thereof, or modified proteins, polypeptides, peptides or analogs thereof. The protein, polypeptide, or peptide may be conjugated with lipoprotein or administered in liposomal form or with adjuvant. Preferably, the vaccine comprises one or more of (i) the condition- associated antigen for which the antigen-specific receptor of the host cell of the invention has antigenic specificity, (ii) an epitope of the antigen, and (iii) a vector encoding the antigen or the epitope.

[0094] The inventive method of treating or preventing a condition in a mammal can comprise additional steps. For instance, a variety of procedures, as discussed below, can be performed on the host cells prior to, substantially simultaneously with, or after their isolation from a mammal. Similarly, a variety of procedures can be performed on the host cells prior to, substantially simultaneously with, or after introducing the inventive nucleic acids into the host cell(s).

[0095] In an embodiment of the invention, the host cells are expanded in vitro after introducing the inventive nucleic acids into the host cell(s), but prior to the administration to a mammal. Expansion of the numbers of host 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. 2012/0244133. For example, expansion of the numbers of host cells may be carried out by culturing the host cells with OKT3 antibody, TL-2, and feeder PBMC (e.g., irradiated allogeneic PBMC). In another embodiment of the invention, the host cells are not expanded in vitro after introducing the inventive nucleic acids into the host cell(s) and prior to the administration to a mammal.

[0096] Another embodiment of the invention provides a method of inducing arginase expression by peripheral blood lymphocytes (PBL). The inventive nucleic acid or the inventive recombinant expression vector may be introduced into isolated PBL which already comprise an endogenous antigen-specific receptor (e.g., an endogenous TCR), as described herein with respect to other aspects of the invention. Accordingly, an embodiment of the invention provides a method comprising: (a) introducing the inventive nucleic acid or the inventive recombinant expression vector into isolated PBL to obtain modified PBL, wherein the PBL further comprise an endogenous antigen-specific receptor, (b) administering the modified PBL to a mammal; and (c) stimulating the antigen-specific receptor to induce arginase expression by the modified PBL.

[0097] Another embodiment of the invention provides a method comprising: (a) introducing the inventive nucleic acid or the inventive recombinant expression vector into isolated PBL to obtain modified PBL, wherein the PBL further comprise an endogenous antigen-specific receptor and (b) stimulating the antigen-specific receptor to induce arginase expression by the modified PBL.

[0098] In another embodiment of the invention, the method of inducing arginase expression by PBL further comprises introducing a nucleic acid (or recombinant expression vector) encoding any of the antigen-specific receptors described herein. In this regard, an embodiment of the invention provides (a) introducing a nucleic acid encoding an antigen- specific receptor into isolated PBL; (b) introducing the inventive nucleic acid or the inventive recombinant expression vector into the isolated PBL; (c) administering the PBL to a mammal, wherein the administered PBL comprise (i) the nucleic acid of (a) and (ii) the nucleic acid or recombinant expression vector of (b); and (d) stimulating the antigen-specific receptor to induce arginase expression by the modified PBL.

[0099] In another embodiment of the invention, the method of inducing arginase expression by PBL further comprises introducing a nucleic acid (or recombinant expression vector) encoding any of the antigen-specific receptors described herein. In this regard, an embodiment of the invention provides (a) introducing a nucleic acid encoding an antigen- specific receptor into isolated PBL; (b) introducing the inventive nucleic acid or the inventive recombinant expression vector into the isolated PBL; and (c) stimulating the antigen-specific receptor to induce arginase expression by the modified PBL.

[0100] The nucleic acid encoding an antigen-specific receptor may be introduced into isolated PBL before, during, or after introducing the nucleic acid encoding an antigen- specific receptor into isolated PBL. The nucleic acids may be introduced into the PBL, the PBL may be administered to the mammal, and the antigen-specific receptor may be stimulated to induce arginase expression as described herein with respect to other aspects of the invention.

[0101] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope. EXAMPLE 1

[0102] This example demonstrates the construction of a retroviral vector comprising a nucleic acid comprising a NFAT-responsive promoter operatively associated with a nucleotide sequence encoding arginase II.

[0103] The reverse complement of a nucleic acid comprising a NFAT-responsive promoter operatively associated with a nucleotide sequence encoding arginase II was inserted into a MSGV-1 vector in a position reverse to the 5' LTR direction of the vector (Figure 1 C) (SEQ ID NO: 11) (NFAT-Arg-2). As a first control, the reverse complement of a nucleic acid comprising a NFAT-responsive promoter operatively associated with a nucleotide sequence encoding green fluorescent protein (GFP) was inserted into a MSGV-1 vector in a position reverse to the 5' LTR direction of the vector (Figure 1A) (NFAT-GFP). As a second control, the reverse complement of a nucleic acid comprising a NFAT-responsive promoter operatively associated with a nucleotide sequence encoding truncated arginase II was inserted into a MSGV-1 vector in a position reverse to the 5' LTR direction of the vector (Figure IB) (NFAT-Trunc-Arg-2). Trunc-Arg-2 encoded arginase II protein without the mitochondrial targeting sequence that allows translocation of the enzyme from the cytosol to the mitochondria. Thus, this form of the non-translocated enzyme does not have physiologic enzymatic activity.

EXAMPLE 2

[0104] This example demonstrates transduction of cells with the NFAT-Arg-2 vector of Example 1.

[0105] PBMC or CD8+ clones were stimulated with 25 ng/ml OKT3 in the presence of 50 CU 1L-2 in a T25 flask for 2 days. On day 2, cells were harvested for retroviral transduction on vector-preloaded RETRONECTIN recombinant human fibronectin fragment (Takara, Otsu, Japan)-coated non-tissue culture 6-well plates (Becton Dickinson). After transduction, the cells were cultured in complete medium containing 5% human serum and 50 CU IL-2 for an additional 10 days.

EXAMPLE 3

[0106] This example demonstrates that PBMC transduced with the NFAT-Arg2 vector of Example 1 secrete more urea into the supernatant as compared to cells transduced with NFAT-GFP or NFAT-trunc-Arg-2 vector when the transduced cells are restimulated with anti-CD3 antibody.

[0107] PBMC were stably transduced with one of Hie NFAT-GFP, NFAT-trunc Arg-2, or NFAT-Arg-2 vector of Example 1 on Day 0. Transduction efficiency based upon MSGV- GFP was -65% at Day 10. To induce the expression of the NFAT-driven transgene, the transduced cells underwent restimulation with 25 ng/ml OKT3 (anti-CD3 antibody) in the presence of 50 CIJ IL-2 on Day 10. Supernatant urea concentrations were determined without restimulation and 10 days after restimulation with OKT3. The results are shown in Figure 2. As shown in Figure 2, cells transduced with the NFAT-Arg2 vector secreted more urea into the supernatant as compared to cells transduced with the NFAT-GFP or NFAT- trunc-Arg-2 vector when the transduced cells were restimulated with anti-CD3 antibody.

EXAMPLE 4

[0108] This example demonstrates that transduction of DMF5 TCR-transduced cells with the NFAT-Arg-2 vector of Example 1 enhances arginase activity but does not alter antigen reactivity.

[0109] PBMC were stably transduced with the DMF5 MART-specific TCR (MART tetramerf frequency -75%). These cells were additionally transduced with one of the NFAT- GFP, NFAT-trunc Arg-2, or NFAT-Arg-2 vector of Example 1 on Day 0. Transduction efficiency based on MSGV-GFP was -70% at day 10. To induce the expression of the NFAT driven transgene, the transduced cells underwent re-stimulation with T2 target cells pulsed with MART peptide or vehicle (dimethyl sulfoxide (DMSO)). Supernatant urea and interferon (IFN)-y concentrations were determined 3 days post co-culture with target cells. The results are shown in Figures 3A-3B. As shown in Figures 3A-3B. transduction of DMF5 TCR-transduced cells with the NFAT-Arg-2 vector of Example 1 enhanced arginase activity but did not alter antigen reactivity.

EXAMPLE 5

[0110] This example demonstrates that transduction of PBMC with the NFAT-Arg2 vector of Example 1 decreases activation-induced cell death (AICD).

[0111] PBMC were stably transduced with one of the NFAT-GFP, NFAT-trunc Arg-2, or NFAT-Arg-2 vector of Example 1 on Day 0. Transduction efficiency based upon MSGV- GFP was -65% at Day 10. AICD was assessed by Annexin V staining of transduced T cells after re-exposure to plate bound anti-CD3 antibody (1 .ug/ml) for 48 hours (hrs). The results are shown in Figure 4. As shown in Figure 4, NFAT-Arg2 vector-transduced PBMC demonstrated decreased AICD as compared to NFAT-GFP or NFAT-trunc-Arg-2 vector- transduced PBMC.

EXAMPLE 6

[0112] This example demonstrates that transduction of PBMC with the NFAT-Arg2 vector of Example 1 enhances in vitro T cell proliferation.

[0113] PBMC were stably transduced with one of the NFAT-GFP, NFAT-trunc Arg-2, or NFAT-Arg-2 vector of Example 1 on Day 0. Transduction efficiency based upon MSGV- GFP was ~65% at Day 10. T cells were expanded in media containing IL-2 (50 cu/ml), anti- CD3 antibody (25 ng/ml) and irradiated PBMCs (3x10⁷) in sets of triplicate cultures for 21 days (rapid expansion protocol (REP)). Cell proliferation was measured. The results are shown in Figure 5. As shown in Figure 5, transduction of PBMC with the NFAT-Arg2 vector of Example 1 enhanced in vitro T cell proliferation as compared to transduction of PBMC with the NFAT-GFP or NFAT-rrunc-Arg 2 vector.

EXAMPLE 7

[0114] This example demonstrates that transduction of PBMC with the NFAT-Arg2 vector of Example 1 enhances the in vivo engraftment of human T cells following adoptive transfer into immunodeficient mice.

[0115] Human PBMC were stably transduced with one of the NFAT-GFP, NFAT-trunc Arg-2. or NFAT-Arg-2 vector of Example 1 on Day 0. Transduction efficiency based upon MSGV-GFP was ~65% at Day 10. Transduced cells were then expanded in media containing IL-2 (50 cu/ml), anti-CD3 antibody (25 ng/ml) and irradiated PBMCs (3xl0⁷) for 10 days. 5x10⁶ cells were adoptively transferred by tail vein injection into NOD scid gamma (NSG) mice. Five mice were in each treatment group. Spleens were harvested at day 21 to assess the persistence of the adoptively transferred cells by measuring the frequency of human CD3+CD8+ and human CD3+CD4+ T cells by fluorescence activated cell sorting (FACS). The results are shown in Figures 6A-6C. As shown in Figures 6A-6C, NFAT-Arg-2 vector- iransduced PBMC demonstrated enhanced in vivo engraftment following adoptive transfer into immunodeficient mice as compared to NFAT-GFP or NFAT-trunc-Arg-2 vector- transduced PBMC. EXAMPLE 8-12

[0116] The following materials and methods were employed in the experiments described in Examples 8-12.

Cell Culture

[0117] Human cultured T2 cell line (HLA-A2+ peptide transporter-associated protein deficient T-B hybrid) was cultured in complete medium (CM) consisting of RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine (Invitrogen, Waltham, MA), 50 units/mL penicillin (Invitrogen), 50 ug/mL streptomycin (Invitrogen), 50 gentamicin (Invitrogen), 10 mM Hepes (Invitrogen), and 250 ng-'mL Amphotericin B

(Invitrogen). Human melanoma specific CD8+ T cell clones were isolated as described in (Wang et al., Sci. Transl. Med., 4: 149ral20 (2012)) . Human T cell clones were cultured in CM with 10% heat-inactivated human AB serum (Gemini Bio-Products, Broderick, CA).

Peptides, tetramers, antibodies, and flow cytometric immunofluorescence analysis

[0118] The MART-I27-33 (AAGIG1LTV) (SEQ ID NO: 12) peptide was produced to GMP grade by solid phase synthesis techniques by Multiple Peptide Systems (San Diego, CA). The purity of the peptide was confinned by mass spectrometry, and the peptide was re- suspended in DMSO to 1 mg/ml for in vitro use. Anti-human CD8+ and CD3

fluorochromes- conjugated monoclonal antibodies were obtained from BD Biosciences (Franklin Lakes, NJ). Immunofluorescence, analyzed as the relative log fluorescence of live cells, was measured using a FACSCANTO II flow cytometer with FACSDIVA software (BD Biosciences) and FLOWJO software (Tree Star, Inc., Ashland, OR). For proliferation assays, 1x10^s T cells from were loaded with 2.5 uM CFSE (Life Technologies, Carlsbad, CA) for 5 minutes at room temperature, and then washed thoroughly. CFSE labeled T cells were cultured for 3 days and analyzed for CFSE dilution by flow cytometry. For apoptosis assay, lxlO⁵ T cells were exposed to plate-bound anti-CD3 (^g/ml) for 48 hrs and stained for Annexin V (eBioscience, San Diego, CA). Cytokine staining was assessed on CD3+CD8+ gated cells. Mice and xenograft model for cell persistence and tumor treatment:

[0119] NOD.Cg-PrkdcscidI12rgtml Wjl/SzJ (NSG) immunodeficient mice were purchased from Jackson Laboratories (Bar Harbor, ME) and housed at the animal facility at the NCI (Bethesda) in pathogen-free conditions. Mouse experiments were approved by the NCI Animal Care and Use Committee (ACUC) and performed in accordance with N1H guidelines. For T cell persistence studies, 5x10⁶ of the indicated T cells were adoptively transferred by tail vein injection into randomized NSG mice (5 animals per group) with concomitant human IL-15 (1 μg/dose, intraperitoneal) every alternative day (Chandran et al., Cancer Res., 75: 3216-3226 (2015); Wang et al., Blood, 117: 1888-1898 (201 1)). At day 21 post transfer, mice were euthanized by C0₂ asphyxiation and spleens were harvested to quantitaie the persistence of the transferred T cells by flow cytometry. For tumor therapy experiments, melanoma xenografts were generated by subcutaneous inoculation of 2x10⁶ 526 Mel tumor cells on the right flank of NSG mice. After approximately 2 weeks, when rumors were established and palpable (~50 mm² in size), mice were randomized to receive by tail vein adoptive transfer of the indicated T cell populations (2xl0⁷ cells) with concomitant human intraperitoneal) every alternative day. Tumor size was measured

with calipers by a blinded investigator.

Retroviral vectors and transduction

[0120] The NFAT-responsive promoter-containing six repeats of NFAT-binding motif were obtained from the SIN-(NFAT)₆-GFP piasmid (Zhang et al., Mol. Ther., 19: 751-759 (2011)) and then subcloned into a pMSGV-1 vector downstream of 3-LTR using Sail and Ncol to create the pMSGVl-NFAT vector. Codon-optimized genes encoding GFP, human arginase-1 (Argl ), human arginase-2 (Arg2), and truncated arginase-2 (Arg2 -mito leader; also referred to herein as "trunc Arg-2"), which lacks the NH-2-terminal mitochondrial targeting sequence, were synthesized (Integrated DNA Technologies, Coralville, IA). The respective gene products were individually cloned into the pMSGVl-NFAT vector immediately downstream of the NFAT promoter using Ncol and Noll site, followed by insertion of the PA2, polyA signaling sequence. The NFAT-GFP, NFAT-Arg2 (SFiQ ID NO: 11), and NFAT-trunc Arg-2 vectors employed in the experiments described in Examples 8-12 are the same as those described in Example 1 (Figs. 1A-1C). All piasmid constructs were confirmed by sequencing and restriction enzyme analysis. For retroviral transduction experiments, CD8+ T cell clones were stimulated with anti-CD3 antibody (25 ng/ml) and IL- 2 (300 lU/ml) for two days, followed by retroviral transduction on vector-preloaded

RETRONECTIN recombinant human fibronectin fragment (Takara, Otsu, Japan)-coated non- lissue culture 6-well plates (Becton Dickinson, Downers Grove, 1L). After transduction, the cells were cultured in complete medium (CM) for an additional 10 days. To induce the expression of the NFAT driven transgene, the transduced cells were re-stimulated with anti- CD3 antibody (25 ng/ml). Transgene expression was confirmed by gene specific reverse transcription polymerase chain reaction (RT-PCR) and efficiency of transduction was assessed by flow cytometric assessment of GFP expression. To endow open repertoire CD8+ and CD4+ T cells with MART-1 specificity, the cells were additionally transduced with the DMF5 MART-1 specific TCR.

Cellular Metabolic Assays

[0121] Functional metabolic parameters were assessed using an extracellular flux analyzer (XF24 analyzer, Seahorse Bioscience, Santa Clara, CA) as described in (Kawaiekar et al., Immunity, 44: 380-390 (2016)). ECAR was measured under basal conditions while cellular OCRs were measured both under basal conditions and following treatment with 1.5 μΜ oligomycin, 1.5 uM FCCP (carbonyi cyanide-p-trifluoromethoxy-phenylhydrazone), and 40 nM rotenone, and luM antimycin A (XF CELL MiTO stress kit, Seahorse Bioscience). Measurement of the OCR following serial additions of oligomycin (an inhibitor of ATP synthesis), FCCP (an uncoupling ionophore), and rotenone with antimycin A (blocking agents for complexes 1 and III of the electron transport chain, respectively) was performed to discern the relative contributions of mitochondrial and non-mitochondrial mechanism of oxygen consumption (van der Windt et al., Immunity, 36: 68-78 (2012); van der Windt et al., PNAS, 110: 14336-14341 (2013)). Mitochondrial spare respiratory capacity (SRC) was calculated from the difference between maximum OCR after FCCP treatment and basal OCR.

ELISA and western blot assays

[0122] T cell supernatants from replicate cultures were harvested at specified times and commercially available ELISA kits with standards were used to determine the concentrations of IFN-^y (Endogen, Wobum, MA) and urea (QUANTICHROM urea assay kit; BioAssay Systems, Ilayward, CA). Statistical Analysis

[0123] Paired and unpaired t test were used for group comparisons. Linear regression was used to quantify the relationship between parameters and was presented as R2 values with their significance (P) level. When comparing multiple groups, data were analyzed by two-way ANOVA followed by Tukey's multiple comparisons test. Tumor growth curves were compared with the Wilcoxon rank sum test. Log rank test was used to evaluate survival differences in murine treatment experiments. All P values are two-sided and considered significant at the 0.05 level. EXCEL and GRAPHPAD PRISM (v. 6.01) software were used for analyses.

EXAMPLE 8

[0124] This example demonstrates that transduction of MART specific CD8+ clones with the NFAT-Arg-2 vector enhances arginase activity but does not alter antigen reactivity.

[0125] It was sought to directly evaluate the independent role of arginase II (Arg2) upon T cell fate and anti-tumor activity by overexpressing the enzyme in human T cells without the need for 4-lBB co-stimulation. To mimic the induction of Arg2 seen after co- stimulation, T cells were transduced with an inducible γ -retroviral vector in which Arg2 expression was under the control of the nuclear factor of activated T-cells (NFAT) responsive promoter (referred as NFAT-Arg2) using methods described in (Hooijberg et al, Blood, 96: 459-466 (2000); Zhang et al., Mol. Ther., 19: 751-759 (2011)). Transduction of T cells with this inducible vector enabled expression of Arg2 upon TCR engagement (without the need for 4- 1BB co-stimulation) but prevented its constitutive expression in the basal (non-activated) state.

[0126] As a control, a parallel vector that expressed a truncated form of Arg2 which lacked the NH2-terminal leader sequence that is responsible for mitochondrial targeting and import (Morris et al.. Gene, 193: 157-161 (1997); Gotoh et al., FEBSLetL, 395: 119-122 (1996))) (that is, without the mitochondrial targeting sequence that allows translocation of the enzyme from the cytosol to the mitochondria); referred to as NFAT-Trunc-Arg-2 (or NFAT- Arg2 (-mito leader)). Additional control vectors encoded for Argl (NFAT-Argl) and GFP (NFAT-GFP).

[0127] These four NFAT inducible vectors were independently transduced into a common MART specific CD8+ clone (average transduction efficiency -67%). These gene- modified T cells were co-cultured overnight with T2 cells pulsed with MART peptide or vehicle (DMSO). Culture supematants were assessed for IFN-γ and urea. The results are shown in Figures 7A-7B. As shown in Figure 7 A, equivalent peptide reactivity was measured for each of the transduced clone populations. However, me NFAT-Arg2 transduced T cells demonstrated increased urea production, indicating the successful induction of Arg2 enzymatic activity after TCR stimulation (Figure 7B). Neither the NFAT- Argl nor the NFAT-Trunc-Arg-2-transduced cells demonstrated a change in urea production after antigen stimulation, supporting the involvement of the type 2 variant of the enzyme and its mitochondrial translocation.

EXAMPLE 9

[0128] This example demonstrates that transduction of MART specific CD8+ clones with the NFAT-Arg-2 vector decreases apoptosis.

[0129] MART specific CD8+ clones were independently transduced with one of the four NFAT inducible vectors as described in Example 8. Apoptosis (% annexin V) was assessed after re-exposure of transduced T cells to plate bound anti-CD3 for 48 hours (hrs) (without 4- 1BBL co-stimulation). The results are shown in Figure 7C.

[0130] As shown in Figure 7C, the NFAT-Arg2 transduced cells demonstrated decreased apoptosis after anti-CD3 antibody stimulation.

EXAMPLE 10

[0131] This example demonstrates that transduction of MART specific CD8+ clones with the NFAT-Arg-2 vector enhances mitochondrial bioenergetics.

[0132] MART specific CD8+ clones were independently transduced with one of the four NFAT inducible vectors as described in Example 8. After re-exposure of transduced T cells to plate bound anti-CD3 for 48 hrs (without 4-1BBL co-stimulation), cellular bioenergetics were assessed by measurements of (i) extracellular acidification rate (ECAR) (Figure 8A);

[11] oxygen consumption rate (OCR) (Figure 8B) under basal conditions and in response to mitochondrial inhibitors; (iii) mitochondrial spare respiratory capacity (SRC) (Figure 8C); and (iv) proliferation kinetics over three weeks (Figure 8D).

[0133] The results showed that, after anu-CD3 antibody stimulation, the NFAT-Arg2 transduced cells demonstrated increased ECAR (Fig. 8A), increased basal and maximum OCR (Fig. 8B), increased SRC (Fig. 8C), and increased in vitro proliferative capacity (Fig. 8D).

EXAMPLE 11

[0134] This example demonstrates that transduction of DMF5 TCR-transduced cells with the NFAT-Arg-2 vector significantly enhances the persistence of the cells after adoptive transfer into immunodeficient NSG mice.

[0135] It was postulated that if Arg2 was a lineage independent and conserved regulator of T cell metabolism, its induced expression should improve the in vivo fate of both CD8+ and CD4+ cells. For these experiments, a mixture of CD8+ and CD4+ cells were transduced with the DMF5 MART-1 specific TCR and one of the four NFAT inducible vectors described in Example 8. The stably transduced cells (5x10⁶ cells) were adoptively transferred by tail vein injection into immunodeficient NSG mice (n=5 mice per group) with concomitant IL-15. The frequencies of human CD3+CD8+ and CD3+CD4+ T cells persisting 21 days after adoptive transfer were measured. The results are shown in Figures 9A-9E.

[0136] The persistence (on day 21) of total CD3+, CD8+, and CD4+- T cells in spleens harvested from each of the individual mice for each transfer group was also measured. The results are shown in Figures 1 OA- IOC.

[0137] As shown in Figures 9A-9E and Figures 1 OA-10C, NFAT-Arg2 transduction significantly enhanced the persistence of both CD4+ and CD8+- cells in the absence of 4-1BB co-stimulation.

EXAMPLE 12

[0138] This example demonstrates that transduction of DMF5 TCR-transduced cells with the NFAT-Arg-2 vector significantly improves the anti-tumor activity of the cells against established melanoma xenografts.

[0139] A mixture of CD8+ and CD4+ cells were transduced with the DMF5 MART-1 specific TCR and one of the four NFAT inducible vectors, as described in Example 11. The stably transduced cells (5xl0⁶ cells) were adoptively transferred by tail vein injection into immunodeficient NSG mice with established 526 Mel tumor xenografts (a~-5 mice per group) with concomitant IL-15 on Day 0. The tumor size was measured for 45 days after adoptive cell transfer. The results are shown in Figure 10D. [0140] As shown in Figure 10D, NFAT-Arg2 transduction significantly improved the anti-tumor activity of the cells against established melanoma xenografts in the absence of 4- 1BB co-stimulation.

[0141] 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.

[0142] 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 terms "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. AH methods described herein can be performed 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 belter 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.

[0143] 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 herein. 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);

1. A nucleic acid comprising a nuclear factor of activated T-cells (NFAT)-responsive promoter operatively associated with a nucleotide sequence encoding (i) arginase, (ii) a functional portion of arginase, or (iii) a functional variant of (i) or (ii).

2. The nucleic acid according to claim 1, wherein the arginase is arginase II.

3. A nucleic acid comprising the reverse complement of the nucleic acid of claim 1 or

2.

4. A recombinant expression vector comprising the nucleic acid of any one of claims

1-3,

5. A host cell comprising the recombinant expression vector of claim 4.

6. The host cell according to claim 5, wherein the host cell comprises an exogenous T-cell receptor (TCR).

7. The host cell according to claim S, wherein the host cell comprises an endogenous

TCR.

8. The host cell according to claim 5, wherein the host cell comprises a chimeric antigen receptor (CAR).

9. The host cell according to claim 6 or 7, wherein the TCR has antigenic specificity for a cancer antigen.

10. The host cell according to claim 6 or 7, wherein the TCR has antigenic specificity for a viral antigen.

11. The host cell according to claim 8, wherein the CAR has antigenic specificity for a cancer antigen.

12. The host cell according to claim 8, wherein the CAR has antigenic specificity for a viral antigen.

13. A population of cells comprising the host cell according to any one of claims 5-

12;:

14. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the nucleic acid of any one of claims 1-3, the recombinant expression vector of claim 4, the host cell according to any one of claims 5-12, or the population of cells of claim 13.

15. The nucleic acid of any one of claims 1-3, the recombinant expression vector of claim 4, the host cell according to any one of claims 5-12, the population of cells of claim 13, or the pharmaceutical composition of claim 14, for use in the treatment or prevention of a condition in a mammal.

16. The nucleic acid, recombinant expression vector, host cell, population of cells, or pharmaceutical composition for the use according to claim 15, wherein the condition is cancer.

17. The nucleic acid, recombinant expression vector, host cell, population of cells, or pharmaceutical composition for the use according to claim 15, wherein the condition is a viral condition.

18. A method of inducing arginase expression by peripheral blood lymphocytes (PBL), the method comprising:

(a) introducing the nucleic acid according to any one of claims 1-3 or the recombinant expression vector according to claim 4 into isolated PBL to obtain modified PBL, wherein the PBL further comprise an antigen-specific receptor; and

(b) stimulating the antigen-specific receptor to induce arginase expression by the modified PBL.

19. A method of inducing arginase expression by peripheral blood lymphocytes (PBL), the method comprising:

(a) introducing a nucleic acid encoding an antigen-specific receptor into isolated PBL;

(b) introducing the nucleic acid according to any one of claims 1-3 or the recombinant expression vector according to claim 4 into the isolated PBL; and

(c) stimulating the antigen-specific receptor to induce arginase expression by the modified PBL.