WO2017139199A1 - Arginase inductible - Google Patents

Arginase inductible Download PDF

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Publication number
WO2017139199A1
WO2017139199A1 PCT/US2017/016484 US2017016484W WO2017139199A1 WO 2017139199 A1 WO2017139199 A1 WO 2017139199A1 US 2017016484 W US2017016484 W US 2017016484W WO 2017139199 A1 WO2017139199 A1 WO 2017139199A1
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cells
arginase
nfat
host cell
nucleic acid
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PCT/US2017/016484
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English (en)
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Udai S. Kammula
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The United States Of America, As Represented By The Secretary, Department Of Health And Human Services
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Publication of WO2017139199A1 publication Critical patent/WO2017139199A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/03—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amidines (3.5.3)
    • C12Y305/03001—Arginase (3.5.3.1)

Definitions

  • Adoptive cell therapy can be an effective treatment for conditions (e.g., cancer) in some patients.
  • conditions e.g., cancer
  • obstacles to the overall success of adoptive cell therapy still exist.
  • one or more of the in vivo persistence, survival, proliferation, and cytolytic activity of T cells can, in some cases, decrease following adoptive transfer.
  • the increased apoptosis of T cells can pose obstacles to the treatment of conditions.
  • 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).
  • NFAT nuclear factor of activated T-cells
  • 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).
  • PBL peripheral blood lymphocytes
  • 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).
  • GFP green fluorescent protein
  • LTR designates "long terminal repeat.”
  • PA2 designates a picornavirus 2A sequence.
  • 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.
  • 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.
  • 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.”
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Figure 7A is a graph showing the concentration (pg/mL) of lFN- ⁇ 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.
  • 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.
  • 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.
  • 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.
  • FIG. 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.
  • OCR oxygen consumption rate
  • 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.
  • SRC mitochondrial spare respiratory capacity
  • 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.
  • 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.
  • 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.
  • Figure 10D is a graph showing the tumor size (mm 2 ) 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.
  • 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.
  • 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).
  • inventive nucleic acids may provide many advantages including, for example, die inducible expression of arginase.
  • 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
  • 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.
  • 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.
  • the arginase encoded by the inventive nucleic acids may be either isoform of arginase, namely arginase I or arginase II.
  • the inventive nucleic acids encode arginase I, e.g., human arginase I.
  • human arginase I amino acid sequences include, but are not limited to, the amino acid sequences of GenBank
  • the full-length, wild-type human arginase I comprises the amino acid sequence of SEQ ID NO: 1.
  • the inventive nucleic acids encode arginase II, especially preferably human arginase II.
  • 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.
  • the full-length, wild-type human arginase II comprises the amino acid sequence of SEQ ID NO: 2.
  • 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).
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • an 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
  • the functional variants can comprise the amino acid sequence of the parent arginase with at least one non-conservative amino acid substitution.
  • the non-conservative amino acid substitution it is preferable for the non-conservative amino acid substitution to not interfere with or inhibit the biological activity of the functional variant.
  • 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.
  • 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.
  • the arginase can, for example, consist essentially of the amino acid sequence of SEQ ID NO: 1, 2, or 4.
  • 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.
  • the nucleotide sequence encoding arginase I may comprise any nucleotide sequence that encodes any of the arginase I amino acid sequences described herein.
  • 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,
  • nucleotide sequence encoding full-length wild-type, human arginase I comprises the nucleotide sequence of SEQ ID NO: 5.
  • the nucleotide sequence encoding arginase II may comprise any nucleotide sequence that encodes any of the arginase II amino acid sequences described herein.
  • nucleotide sequences encoding human arginase II include, but are not limited to, the nucleotide sequences of GenBank Accession Nos. NM 001172.3, AK312484.1 ,
  • nucleotide sequence encoding full- length wild-type, human arginase II comprises the nucleotide sequence of SEQ ID NO: 6.
  • nucleic acid and polynucleotide refer to a polymeric form of nucleotides of any length, either ribonucleotides (RNA) or
  • DNA deoxyribonucleotides
  • RNA deoxyribonucleotides
  • nucleotide 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
  • 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.
  • a codon-optimized nucleotide sequence encoding full-length, wild-type human arginase I may comprise the nucleotide sequence of SEQ ID NO: 7.
  • 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.
  • 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.
  • the NFAT-responsive promoter comprises six NFAT binding motifs.
  • the NFAT-responsive promoter comprises or consists of the nucleotide sequence of SEQ ID NO: 8.
  • 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.
  • NFAT is regulated by a calcium signaling pathway.
  • antigen-specific receptor stimulation by, e.g., an antigen
  • non-specific stimulation of the calcium signaling pathway of the cell by, e.g., antigen
  • 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.
  • 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.
  • 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.
  • 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.
  • WPRE woodchuck hepatitis post-transcriptional regulatory element
  • the nucleic acid comprises the reverse complement of any of the nucleic acids described herein.
  • 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.
  • 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.
  • a poly A tail e.g., SV40 polyA tail, BGH polyA tail, polyAl tail, poly A2 tail
  • the nucleic acid comprising the reverse complement of any of the nucleic acids described herein may provide one or more advantages.
  • the reverse complement may enhance arginase transcription efficiency as compared to the nucleic acid in "forward" orientation.
  • 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.
  • the nucleic acids of the invention are recombinant.
  • 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.
  • the replication can be in vitro replication or in vivo replication.
  • 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.
  • 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).
  • 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-
  • 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.
  • nucleic acids of the invention can be incorporated into a recombinant expression vector.
  • an embodiment of the invention provides recombinant expression vectors comprising any of the nucleic acids of the invention.
  • 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.
  • an embodiment of the invention provides a recombinant expression vector comprising (i) the inventive nucleic acid and (ii) a heterologous nucleic acid sequence.
  • inventive nucleic acid and a heterologous nucleic acid sequence.
  • heterologous nucleic acid sequence means a nucleic acid sequence that does not naturally occur in the species that expresses the arginase encoded by the vector.
  • the heterologous nucleic acid sequence in the vector may be any sequence that does not naturally occur in a mouse.
  • 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.
  • 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.
  • the non-naturally occurring or altered nucleotides or internucleotide linkages do not hinder the transcription or replication of the vector.
  • 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 ⁇ 149, also can be used.
  • plant expression vectors include pBlOl, pBI101.2, pBI101.3, pBI121 and pBIN19 (Clontech).
  • animal expression vectors include pEUK-Cl, pMAM and pMAMneo (Clontech).
  • 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.
  • 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.
  • LTR long terminal repeat
  • the reverse complement of the inventive nucleic acid is positioned in the vector reverse to the 5' LTR direction.
  • 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 ⁇ nucleotide sequences are positioned in the vector reverse to the 5' LTR. direction.
  • 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 ⁇ plasmid, ⁇ , SV40, bovine papilloma virus, and the like.
  • 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.
  • 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.
  • 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.
  • a host cell comprising any of the recombinant expression vectors described 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.
  • the host cell is preferably a prokaryotic cell, e.g., a DH5a cell.
  • the host cell is preferably a mammalian cell. Most preferably, Hie host cell is a human cell.
  • 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.
  • PBMC peripheral blood mononuclear cell
  • PBL peripheral blood lymphocyte
  • T cell e.g., a T cell or a natural killer (NK) cell. More preferably, the host cell is a T cell.
  • 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.
  • 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.
  • CD4+/CD8+ double positive T cells e.g., CD4+ helper T cells, e.g., Thl and Th2 ceils
  • CD8+ T cells e.g., cytotoxic T cells
  • TILs tumor infiltrating cells
  • memory T cells e.g., naive T cells, and die like.
  • the T cell is a CD8+ T cell or a CD4+ T cell.
  • inventive nucleic acids may be introduced into the host cell using any suitable method such as, for example, transfection, transduction, or elcctroporation.
  • 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.
  • viruses e.g., retrovirus or lentivirus
  • 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.
  • 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.
  • 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.
  • RT-PCR quantitative reverse transcription polymerase chain reaction
  • stem-loop quantitative RT-PCR stem-loop quantitative RT-PCR.
  • inventive host cells may provide any one or more of various advantages.
  • 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.
  • the inventive host cell may provide decreased apoptosis as compared to a negative control cell.
  • the host cell further comprises an antigen-specific receptor.
  • antigen-specific and antigenic specificity 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.
  • the antigen-specific receptor is a T-cell receptor (TCR).
  • TCR generally comprises two polypeptides (i.e., polypeptide chains), such as an a-chain of a TCR, a ⁇ -chain of a TCR, a ⁇ -chain of a TCR, a ⁇ -chain of a TCR, or a combination thereof.
  • 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.
  • the TCR can be an endogenous TCR, i.e., a TCR that is endogenous or native to (naturally-occurring on) the host cell.
  • 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.
  • the T cell is a primary T cell isolated from a mammal afflicted with cancer or a viral condition.
  • 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.
  • TIL tumor infiltrating lymphocyte
  • PBL peripheral blood lymphocyte
  • the mammal from which a cell is isolated is immunized with an antigen of, or specific for, a condition.
  • the mammal is immunized prior to obtaining the cell from the mammal.
  • 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.
  • 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.
  • 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.
  • 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.
  • exogenous antigen-specific receptors e.g., exogenous TCRs and chimeric antigen receptors (CARs) (described in more detail below)
  • CARs chimeric antigen receptors
  • 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 ⁇ -, ⁇ -, ⁇ -, and/or ⁇ -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.
  • 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;
  • the antigen-specific receptor is a CAR.
  • 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.
  • an antibody e.g., a single-chain variable fragment (scFv)
  • scFv single-chain variable fragment
  • 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.
  • any suitable nucleic acid encoding an antigen-specific receptor can be used.
  • 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).
  • the condition which is associated with or is characterized by the antigen recognized by the antigen-specific receptor can be any condition.
  • the condition can be a cancer or a viral condition, as discussed 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.
  • 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.
  • 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.
  • RSV respiratory syncytial virus
  • influenza virus herpes simplex virus
  • Epstein-Barr virus varicella virus
  • cytomegalovirus cytomegalovirus
  • hepatitis A virus hepatitis B virus
  • hepatitis C virus hepatitis C virus
  • human immunodeficiency virus HAV
  • human T-lymphotropic virus human T-lymphotropic virus
  • calicivirus calicivirus
  • adenovirus adenovirus
  • Arena virus Arena virus
  • 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.
  • influenza influenza
  • pneumonia herpes
  • hepatitis hepatitis A
  • hepatitis B hepatitis C
  • chronic fatigue syndrome hepatitis
  • SARS sudden acute respiratory syndrome
  • gastroenteritis enteritis
  • carditis carditis
  • encephalitis encephalitis
  • bronchiolitis respiratory papillomatosis
  • meningitis meningitis
  • H1V7AIDS 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.
  • the antigen-specific receptor has antigenic specificity for a cancer antigen (also termed a tumor antigen or a tumor-associated antigen).
  • cancer antigen 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.
  • normal, non-tumor, or non-cancerous cells are not as robust as the expression by tumor or cancer cells.
  • 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.
  • the cancer antigen can additionally be expressed by cells of a different state of development or maturation.
  • 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.
  • the cancer antigen can be additionally expressed by stem cells or precursor cells, which cells are not normally found in an adult host.
  • 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.
  • the cancer antigen may be a cancer antigen (e.g., may be characteristic) of more than one type of cancer or tumor.
  • 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 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.
  • 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, pancre
  • 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.
  • a host cell e.g., a T cell
  • 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
  • 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.
  • the population of cells is a clonal population comprising host cells comprising a recombinant expression vector as described herein.
  • the host cells and populations thereof may be isolated or purified.
  • isolated means having been removed from its natural environment.
  • purified 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.
  • inventive nucleic acids, recombinant expression vectors, and host cells can be formulated into a composition, such as a pharmaceutical composition.
  • a pharmaceutical composition comprising any of the inventive arginase materials described herein and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition comprises any of the inventive populations of cells described herein and a pharmaceutically acceptable carrier.
  • 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.
  • 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.
  • 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.
  • an administration route selected from the group consisting of intravenous, intratumoral, intraarterial, intramuscular, intraperitoneal, intrathecal, epidural, and subcutaneous administration routes.
  • 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.
  • an 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
  • the amount or dose of the inventive population of cells or pharmaceutical composition administered should be sufficient to effect, e.g., a therapeutic or prophylactic response, in the patient over a reasonable lime frame.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 10 2 or more, e.g., about 10 3 or more, about 10 4 or more, about 10 5 or more, about 10 8 or more, host cells of the invention. Alternatively, or additionally about 10 12 or less, e.g., about 10" or less, about 10 9 or less, about 10 7 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 10 2 to about 10 s , about 10 3 to about 10 7 , about 10 3 to about 10 9 , or about 10 5 to about 10 10 .
  • 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.
  • 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.
  • the condition is cancer.
  • inventive methods can provide any amount of any level of treatment or prevention of a condition in a patient.
  • 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.
  • treatment or prevention can include promoting the regression of a tumor.
  • prevention can encompass delaying the onset or recurrence of the condition, or a sign, symptom or condition thereof.
  • mammal refers to any mammal, including, but not limited to, mice, hamsters, rats, rabbits, cats, dogs, cows, pigs, horses, monkeys, apes, and humans.
  • the mammal is a human.
  • the inventive host cell or population thereof can be transferred into the same mammal from which cell(s) were obtained.
  • 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.
  • the host cell can be allogenically transferred into another mammal.
  • the host cell is autologous to the mammal in the inventive method of treating or preventing a condition in the mammal.
  • the mammal can be immunologically naive, immunized, diseased, or in another condition prior to isolation of the cell(s) from the mammal.
  • the method 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.
  • 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.
  • a mammal with a condition can be therapeutically immunized with an antigen from, or associated with, that condition, including immunization via a vaccine.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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).
  • 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.
  • Another embodiment of the invention provides a method of inducing arginase expression by peripheral blood lymphocytes (PBL).
  • PBL peripheral blood lymphocytes
  • 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.
  • an endogenous antigen-specific receptor e.g., an endogenous TCR
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • PBMC peripheral blood mononuclear cells
  • Hie NFAT-GFP Hie NFAT-GFP
  • NFAT-trunc Arg-2 NFAT-trunc Arg-2 vector of Example 1
  • Transduction efficiency based upon MSGV- GFP was -65% at Day 10.
  • OKT3 anti-CD3 antibody
  • Supernatant urea concentrations were determined without restimulation and 10 days after restimulation with OKT3. The results are 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.
  • 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.
  • 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.
  • DMSO dimethyl sulfoxide
  • This example demonstrates that transduction of PBMC with the NFAT-Arg2 vector of Example 1 decreases activation-induced cell death (AICD).
  • 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.
  • 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 (3x10 7 ) 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.
  • IL-2 50 cu/ml
  • anti- CD3 antibody 25 ng/ml
  • irradiated PBMCs 3x10 7
  • 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 (3xl0 7 ) for 10 days. 5x10 6 cells were adoptively transferred by tail vein injection into NOD scid gamma (NSG) mice. Five mice were in each treatment group.
  • NSG NOD scid gamma
  • 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.
  • CM complete medium
  • 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
  • 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).
  • 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.
  • 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.
  • lxlO 5 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.
  • 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.
  • mice 5x10 6 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 ⁇ g/dose, intraperitoneal) every alternative day (Chandran et al., Cancer Res., 75: 3216-3226 (2015); Wang et al., Blood, 117: 1888-1898 (201 1)).
  • mice were euthanized by C0 2 asphyxiation and spleens were harvested to quantitaie the persistence of the transferred T cells by flow cytometry.
  • melanoma xenografts were generated by subcutaneous inoculation of 2x10 6 526 Mel tumor cells on the right flank of NSG mice. After approximately 2 weeks, when rumors were established and palpable ( ⁇ 50 mm 2 in size), mice were randomized to receive by tail vein adoptive transfer of the indicated T cell populations (2xl0 7 cells) with concomitant human intraperitoneal) every alternative day. Tumor size was measured
  • 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.
  • NFAT-GFP NFAT-Arg2
  • 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.
  • 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.
  • 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
  • Arg2 arginase II
  • T cells were transduced with an inducible ⁇ -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)).
  • NFAT-Arg2 nuclear factor of activated T-cells
  • 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.
  • OCR oxygen consumption rate
  • SRC mitochondrial spare respiratory capacity
  • NFAT-Arg2 transduction significantly enhanced the persistence of both CD4+ and CD8+- cells in the absence of 4-1BB co-stimulation.
  • 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 (5xl0 6 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.
  • NFAT-Arg2 transduction significantly improved the anti-tumor activity of the cells against established melanoma xenografts in the absence of 4- 1BB co-stimulation.

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Abstract

La présente invention concerne un acide nucléique comprenant un facteur nucléaire de promoteur répondeur à des lymphocytes T activés (NFAT) fonctionnellement associé à une séquence nucléotidique codant pour (i) l'arginase, (ii) une partie fonctionnelle d'arginase, ou (iii) un variant fonctionnel de (i) ou (ii). L'invention concerne en outre des vecteurs d'expression recombinants, des cellules hôtes, des populations de cellules, et des compositions pharmaceutiques associés. L'invention concerne en outre des procédés de traitement ou de prévention d'une affection chez un mammifère et des procédés d'induction d'expression d'arginase par des lymphocytes sanguins périphériques (PBL).
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CN108575986A (zh) * 2018-04-25 2018-09-28 广州莱德尔生物科技有限公司 一种保存液组合物及其应用
WO2020260908A1 (fr) * 2019-06-27 2020-12-30 Cancer Research Technology Limited Protéines de fusion présentant une activité de l'arginase

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Publication number Priority date Publication date Assignee Title
CN108575986A (zh) * 2018-04-25 2018-09-28 广州莱德尔生物科技有限公司 一种保存液组合物及其应用
WO2020260908A1 (fr) * 2019-06-27 2020-12-30 Cancer Research Technology Limited Protéines de fusion présentant une activité de l'arginase
CN114585642A (zh) * 2019-06-27 2022-06-03 癌症研究技术有限公司 具有精氨酸酶活性的融合蛋白

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