WO2017079113A1 - Procédés de production de populations de lymphocytes t à l'aide d'inhibiteurs de protéines contenant un domaine prolyl hydroxylase - Google Patents

Procédés de production de populations de lymphocytes t à l'aide d'inhibiteurs de protéines contenant un domaine prolyl hydroxylase Download PDF

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WO2017079113A1
WO2017079113A1 PCT/US2016/059834 US2016059834W WO2017079113A1 WO 2017079113 A1 WO2017079113 A1 WO 2017079113A1 US 2016059834 W US2016059834 W US 2016059834W WO 2017079113 A1 WO2017079113 A1 WO 2017079113A1
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cells
phd
cultured
inhibitor
population
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David Cameron CLEVER
Nicholas P. Restifo
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The United States Of America, As Represented By The Secretary, Department Of Healh And Human Services
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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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4621—Cellular immunotherapy characterized by the effect or the function of the cells immunosuppressive or immunotolerising
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/46449—Melanoma antigens
    • A61K39/464492—Glycoprotein 100 [Gp100]
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C12N5/0637—Immunosuppressive T lymphocytes, e.g. regulatory T cells or Treg
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/57—Skin; melanoma
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/02—Atmosphere, e.g. low oxygen conditions
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/998—Proteins not provided for elsewhere
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/999—Small molecules not provided for elsewhere

Definitions

  • nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: One 54, 152 Byte ASCII (Text) file named "726756 ST25.TXT,” dated November I, 2016.
  • Adoptive cell therapy (ACT) using cancer reactive T ceils can produce positive clinical responses in cancer patients.
  • ACT adoptive cell therapy
  • Treg regulatory T cells
  • Reg may contribute to an immunosuppressive tumor microenvironment that may hamper the effectiveness of anti-cancer T celis. Accordingly, there is a need for methods of obtaining an improved isolated population of T cells for ACT.
  • An embodiment of the invention provides a method of producing an isolated population of T cells for adoptive ceil therapy, the method comprising culturing isolated T ceils having antigenic specificity for a cancer antigen in vitro in the presence of a prolyl hydroxylase domain-containing protein (Phd) inhibitor.
  • a prolyl hydroxylase domain-containing protein (Phd) inhibitor a prolyl hydroxylase domain-containing protein (Phd) inhibitor.
  • Another embodiment of the invention provides a method of producing an isolated population of T cells for adoptive cell therapy, the method comprising culturing isolated T cells having antigenic specificity for a cancer antigen in vitro in the presence of an atmosphere, wherein the atmosphere has an oxygen content less than or equal to 5%, [0006] Further embodiments of the invention provide related isolated populations of T cells, pharmaceutical compositions., and methods of treating or preventing cancer in a mammal,
  • Figure 1 A is an image of an electrophoresis gel showing Foxp3 3 Tbet, and ⁇ -actin proteins detected in whole ceil i sate isolated from CD4 ⁇ T cells stimulated under ThO or iTreg conditions in the presence or absence of DMOG.
  • Figure IB is a graph showing the amount of interferon-gamtna (IFN- ⁇ ) measured in the supernatant of CD4 " T cells stimulated under ThO conditions in the presence or absence of DMOG for 72 hours. **** PO.0001 , Students T-test.
  • Figure 1 C shows the tumor size (area, mm") in mice treated with no ceils (circles) or 1x10" Trp-1 CD4+ T cells that were treated with control vehicle (squares) or DMOG (diamonds) at various time points (days) after transfer. Horizontal bars indicate means +/- SEM of individually evaluated mice, **P ⁇ 0.001 (Wilcoxon rank sum),
  • Figure ID shows the survival (%) of tumor-bearing mice treated with no cells (grey dotted line) or 1 10° Trp-1 CD4+ T cells that were treated with control vehicle (black dotted line) or DMOG (solid line) at various time points (days) after transfer, Horizontal bars indicate means - ( - - SEM of individually evaluated mice. **P ⁇ 0.001 (Wilcoxon rank sum ⁇ .
  • Figure 2A is a graph showing the population distribution (%) of naive (N) (grey portion of bar), central memory (CM) (unshaded portion of bar) and effector memory (EM) (black portion of bar) cells in CD8 + Pmef transgenic splenocytes that were stimulated in the absence (VEH) or presence of the Phd inhibitor DMOG.
  • Figure 2B is a graph showing the percentages of ceils stimulated in the presence (shaded bars) or absence (unshaded bars) of DMOG having the indicated cytokine profile. *** ⁇ 0.0001 , Student's T-test. S-not significant.
  • FIG. 2C shows the tumor size (area, mm "' ) in mice treated with no cells (circles) or 1 xl O 6 Pmef CD8 ⁇ T ceils that were treated with vehicle (squares) or DMOG (diamonds) at various time points (days) after transfer, Horizontal bars indicate means /- SEM of individually evaluated mice. **P ⁇ 0.001 (Wilcoxon rank sum).
  • Figure 2D shows the survival (%) of tumor-bearing mice treated with no cells (grey dotted line) or l xl O 6 Pmef CD8 + T cells that were treated with vehicle (black dotted line) or DMOG (solid line) at various time points (days) after transfer. Horizontal bars indicate means +/- SEM of individually evaluated mice. ***p ⁇ 0.G0I (Wiicoxon rank sum).
  • Figures 3 A and 3B are graphs showing the percentage of cells expressing Foxp3 (A) or Tbet (B) when stimulated under ThO or iTreg conditions in the presence of 1 % or 20% oxygen. **p ⁇ 0.001, Student's T-test.
  • Figure 4A is a representative ventral image of the gross anatomic examination of the lungs of wild type (WT) and Phd-tKO mice 21 days after injection with melanoma cells.
  • the black spots on the WT kings are tumors.
  • Figure 4B is a representative dorsal image of the gross anatomic examination of the lungs of wild type (WT) and Phd-tKO mice 21 days after injection with melanoma cells.
  • the black spots on the WT lungs are tumors.
  • Figure 5A is a photographic image of the livers of WT mice following intrasplenic tumor injection
  • Figure 5B is a photographic image of the livers of PHD tKO mice following intrasplenic tumor injection
  • Figure 5C is a graph showing the liver mass (g) measured in WT and PHD tKO mice following intrasplenic tumor injection.
  • Figure 5D is a graph showing the number of visible tumo nodules measured in the livers of WT and PFID tKO mice following intrasplenic tumor injection.
  • Figure 5E is a graph showing the number of rmcrometastatic lesions counted by histologic enumeration in the livers of WT and PFID tKO mice following intrasplenic tumor injection,
  • T cells that have been cultured in the presence of one or both of (i) a Phd inhibitor and (ii) a low-oxygen atmosphere have an improved capacity to target cancer antigen-expressing cells.
  • CD4+ T ceils that have been cultured in the presence of one or both of (i) a Phd inhibitor and (ii) a low-oxygen atmosphere may, advantageously, adopt a Thl phenotype. Thl ceils can mediate anti-tumor immune responses at least in part through their capacity to produce the cytokine mterferon-gamma (IFN- ⁇ ), which has and- tumor properties.
  • IFN- ⁇ cytokine mterferon-gamma
  • CD8+ T DCis that have been cultured in the presence of one or both of (i) a Phd inhibitor and (ii) a low-oxygen atmosphere may, advantageously, express surface markers consistent with an effector memory phenotype. Effector memory cells may provide anti-tumor activity.
  • the prolyl hydroxylase domain containing (Phd) proteins are a family of enzymes that provide oxygen sensing machinery within T cells.
  • the Phd family of proteins includes Phd-1, Phd-2, and Phd-3,
  • the gene Eglnl encodes Phd2, the gene Egin2 encodes Phdl, and the gene EglnS encodes Phd3.
  • the main protein substrates of the Phd enzymes are the transcription factors Hypoxia Inducible Factor 1 -alpha and Hypoxia inducible Factor 2-alpha (HIFla and H!F2a). in the presence of sufficient oxygen, the Phd proteins enzymaticaily hydroxylate HIFla and HIF2a, thus targeting these proteins for proteasome-mediated degradation.
  • HIFl and HIF2a proteins are able to accumulate, translocate to the cell nucleus, and drive the expression of genes containing "hypoxia-response element" sequences in their genomic regulatory regions. Without being bound to a particular theory or mechanism, it is believed that HIFl and HIF2a may have roles in directing T cell metabolism and differentiation in both CD4 ⁇ and CDS + T cells.
  • an embodiment of the invention provides a method of producing an isolated population oi ' T cells for adoptive ceil therapy, the method comprising culturing isolated T cells having antigenic specificity for a cancer antigen in vitro in the presence of a Phd inhibitor.
  • the method may comprise isolating T cells from a mammal by any suitable method known in the art.
  • the T cells can be obtained from the mammal by a blood draw or a leukapheresis.
  • the method comprises isolating peripheral blood lymphocytes (PBL) or a peripheral blood mononuclear cells (PBMC) from a mammal.
  • PBL peripheral blood lymphocytes
  • PBMC peripheral blood mononuclear cells
  • the T cells can be obtained from a rumor sample taken from the mammal.
  • the T cells may be tumor infiltrating lymphocytes (TIL).
  • the population of T cells may include any type of T cells.
  • the T cells may be a cultured T cell, e.g., a primary T cell, or a T cell from a cultured T cell line, e.g.. Jurkat, SupTl , etc., or a T ceil obtained from a mammal, if obtained from a mammal, the T cell can be obtained from numerous sources, including but not limited to blood, bone marrow, lymph node, the thymus, tumor, or other tissues or fluids. T cells can also be enriched for or purified.
  • the T cell may be a human T cell.
  • the T cell can be any type of T cell and can be of any developmental stage, including but not limited to, CD4 ' 7CD8 + double positive T cells, CD4 'r helper T ceils, e.g., Thi and Tl3 ⁇ 4 cells, CD8 + T ceils (e.g., cytotoxic T cells), tumor infiltrating lymphocytes (TIL), memory T cells, naive T cells, and the like.
  • the T cell may be a CD8 + T cell or a CO4 ⁇ T cell.
  • the T cells are TIL.
  • mammal refers to any mammal including, but not limited to, mammals of the order Logomorpha, such as rabbits: the order Camivora, including Felines (cats) and Canines (dogs); the order Artiodactyla, including Bovines (cows) and S wines (pigs); or of the order Ferssodactyla, including Equines (horses).
  • the order Logomorpha such as rabbits: the order Camivora, including Felines (cats) and Canines (dogs); the order Artiodactyla, including Bovines (cows) and S wines (pigs); or of the order Ferssodactyla, including Equines (horses).
  • the mammals are non-human primates, e.g., of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes), in some embodiments, the mammal may be a mammal of the order Rodentia, such as mice and hamsters, in other embodiments, the mammal is not a mouse, Preferably, the mammal is a non-human primate or a human, An especially preferred mammal is the human,
  • the method comprising eulturing tumor fragments, isolated PBMC, or PBL (e.g., T cells) in vitro in the presence of one or more Phd inhibitors.
  • the Phd inhibitor may be any agent that inhibits any one or more of Eg!nl-3 R A expression, Phd protein expression, and Phd biological activity (e.g., the ability of the Phd protein(s) to enzymatically hydroxylate one or both of HIFia and HIF2a).
  • the Phd inhibitor may inhibit any one or more of the proteins in the Phd family and/or the corresponding mRNA,
  • the Phd inhibitor may inhibit any one of Phd-1, Phd-2, and Phd-3 and/or the corresponding mRNA
  • the Phd inhibitor may inhibit any two Phd proteins, such as Phd-1 and Phd-2, Phd-1 and Phd-3, or Phd-2 and Phd-3, and/or the corresponding mRNA molecules.
  • the Phd inhibitor may be a pan-Phd inhibitor thai inhibits all three members of the Phd family, i.e., Phd-1 , Phd-2, and Phd-3 and/or the corresponding mRNA molecules.
  • the Phd inhibitor is a small molecule inhibitor of Phd.
  • the small molecule Phd inhibitor may be an aliosteric inhibitor or a non-allosteric inhibitor of Phd.
  • Examples of small molecule Phd inhibitors that may be useful in the inventive methods include, but are not limited to, dirnethyloxaiylglyeine, N- (methoxyoxoacetyl) -glycine methyl ester (DMOG), IOX2, desferoxamine. 3.4,dihydroxybenzoie acid (DHB), and HIF Phd Inhibitor 4.
  • the Phd inhibitor is DMOG.
  • the Phd inhibitor is a small interfering RNA (siRNA), microRNA, antisense nucleic acid, or CRISPR-Cas9 guide RNA (gRNA).
  • the Phd inhibitor can be a nucleic acid at least about 10 nucleotides in length that specifically binds to and is complementary to a target nucleic acid encoding Phd or a complement thereof.
  • the Phd inhibitor may be introduced into the T cel ls, wherein the cells are capable of expressing Phd, in an effective amount for a time and under conditions sufficient to interfere with expression of Phd.
  • RNA interference RNA interference
  • the Phd inhibitor may comprise an RNAi agent.
  • the RNAi agent may comprise a small interfering RNA (siRNA), a microRNA (miRNA), or an antisense nucleic acid.
  • siRNA small interfering RNA
  • miRNA microRNA
  • antisense nucleic acid e.g., siRNA, miRNA, and/or antisense nucleic acid can comprise overhangs. That is, not all nucleotides need bind to the target sequence.
  • RNA interference nucleic acids employed can be at least about 19, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about I SO, at least about 200, at least about 220, at least about 240, from about 19 to about 250, from about 40 to about 240, from about 60 to about 220, from about 80 to about 200, from about 60 to about 180, from about SO to about 160, and/or from about 100 to about 140 nucleotides in length.
  • the RNAi agent e.g., siRNA or shRNA
  • a nucleotide sequence included in a cassette e.g., a larger nucleic acid construct such as an appropriate vector.
  • vectors include leniivirai and adenoviral vectors, as well as other vectors described herein with respect to other aspects of the invention.
  • An example of a suitable vector is described in Aagaard et al. Mol. Ther., 15(5): 938-45 (2007).
  • the resulting nucleic acid can be longer than the comprised RNAi nucleic acid, e.g., greater than about 70 nucleotides in length.
  • the RNAi agent employed cleaves the target mRNA. In other embodiments, the RNAi agent employed does not cleave the target mRNA.
  • the antisense nucleic acid comprises a nucleotide sequence complementary to at least about 8, at least about 15, at least about 19, or from about 19 to about 22 nucleotides of a nucleic acid encoding Phd or a complement thereof
  • the siRNA may comprise, e.g., trans-acting siRNAs (tasiRNAs) and/or repeat- associated siRNAs (rasiRNAs).
  • the miRNA may comprise, e.g., a short hairpin miRNA (shMIR).
  • the Phd inhibitor may inhibit or downregulate to some degree the expression of the protein encoded by an Egln gene, e.g., at the D. , RNA, or other level, of regulation.
  • a T cell comprising a Phd inhibitor expresses no Phd protein or lower levels of Phd protein as compared to a T cell that lacks a Phd inhibitor.
  • a T cell comprising a Phd inhibitor expresses no Egln mRNA or lower levels of. Egln mRNA as compared to a T cell that lacks a Phd inhibitor.
  • the Phd inhibitor such as an RNAi agent, such as a shMIR
  • the Egln sequence is a human sequence.
  • human Egln2 mRN A sequences include Genbank Accession Nos. NM 053046.3 (SEQ ID NO: 1) and NM 080732.3 (SEQ ID NO: 2), with corresponding human Phd-1 protein sequences NP _444274, 1 (SEQ ID NO: 3) and NP_542770.2 (SEQ ID NO: 4), respectively.
  • Human Egln I mRNA sequences include Genbank Accession Nos.
  • Human Egln-3 mRNA sequences include Genbank Accession Nos. NM ... 001308103.1 (SEQ ID NO: 11) and NM ... 022073.3 (SEQ ID NO: 12).
  • Human Phd-3 protein sequences include ⁇ ... 001295032.1 (SEQ ID NO: 13) and NP_071356.1 (SEQ ID NO: 14). Other human sequences, as well as other Phd species can be employed in accordance with the invention.
  • the Phd inhibitor such as an RNAi agent, such as a shMIR
  • a nucleotide sequence selected from the group consisting of the 5 * untranslated region (5' UTR), the V untranslated region (3" UTR), and the coding sequence oiEgln, complements thereof, and any combination thereof.
  • Egln target sequence can be employed.
  • RNAi agents can be designed against any appropriate Egln mRNA sequence,
  • the Phd inhibitor may be an artificially engineered nuclease that inhibits expression of Phd.
  • Phd expression may be inhibited in a T cell using a genome editing technique.
  • Genome editing techniques can modify gene expression in a target cell by inserting, replacing, or removing DNA in the genome using an artificially engineered nuclease.
  • nucleases may include zinc finger nucleases (ZF s) (Goniraans et a.l. J. Mo!
  • the Phd inhibitor is a CRISPR-Cas9 guide RNA (gRNA).
  • nucleic acid includes “polynucleotide,” “oligonucleotide,” and “nucleic acid molecule,” and generally means a polymer of DNA or RNA. which can be single- stranded or do ble- stranded, synthesized or obtained (e.g., isolated and/or purified) from natural sources, which can contain natural, non-natural or altered nucleotides (e.g., ribonucleic acid nucleotides as well as deoxyribonucleic acid nucleotides), and which can contain a natural, non-natural or altered internucieotide linkage, such as a phosphoroamidate linkage or a phosphorotliioatc linkage, instead of the phosphodiester found between the nucleotides of an unmodified oligonucleotide.
  • nucleotides e.g., ribonucleic acid nucleotides as well as deoxyribonucleic acid nucle
  • nucleic acid When a nucleic acid is recked, it refers genetically to nucleic acids and includes DNA and RNA unless the recitation explicitly states that the nucleic acid is a specific one, e.g., DNA or RNA. If a nucleic acid refers to a sequence that contains thymine (1). that does not necessarily indicate that the nucleic acid is DNA; in some embodiments the nucleic acid is RNA and/cr DNA. Similarly, if a nucleic acid refers to a sequence that contains uracil (u), that does not necessarily indicate that the nucleic acid is RNA; in some embodiments, the nucleic acid is DNA and/or RNA.
  • the nucleic acid does not comprise any insertions, deletions, inversions, and/or substitutions. However, it may be suitable in some instances tor the nucleic acid to comprise one or more insertions, deletions, inversions, and/or substitutions.
  • nucleic acid molecules relevant to the invention can readily be obtained in a variety of ways, including, without limitation, chemical synthesis, cDNA or genomic library screening, expression library screening, and/or PGR amplification of cDNA. These methods and others useful for isolating such DNA are set forth, for example, in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4 th Ed.), Cold Spring Harbor Laboratory Press (2012).
  • Chemical synthesis of a nucleic acid molecule can be accomplished using methods well known in the art. These methods include, inter aiia, the phosphotriester, phosphoramidite and H-phosphonate methods of nucleic acid synthesis.
  • Nucleic acids larger than about 100 nucleotides in length can be synthesized as several fragments, each fragment being np to about 100 nucleotides in length. The fragments can then be ligated together to form a full length nucleic acid encoding the polypeptide.
  • One method is polymer-supported synthesis using standard phosphoramidite chemistry.
  • the nucleic acid can be obtained by screening an appropriate cDNA library prepared from one or more tissue source(s) that express the polypeptide, or a genomic library from any subspecies.
  • the source of the genomic library may be any tissue or tissues from a mammalian or other species believed to harbor a gene encoding a protein relevant to the invention (e.g., Phd).
  • the library can be screened for the presence of a cDNA/gene using one or more nucleic acid probes (oligonucleotides.
  • cDNA or genomic D A fragments that possess an acceptable level of homology to the gene or gene homologue cDNA or gene to be cloned) that will hybridize selectively with the gene or gene homologue cDNA(s) or gene(s) that is(are) present in the library.
  • the probes preferably are complementary to or encode a small region of the DNA sequence from the same or a similar species as the species from which the library was prepared . Alternatively, the probes can be degenerate. After hybridization, the blot containing the library is washed at a suitable stringency, depending on several factors such as probe size, expected homology of probe to clone, type of library being screened, number of clones being screened, and the like. Stringent washing solutions can be low in ionic strength and are used at relatively high temperatures.
  • Another suitable method for obtaining a nucleic acid for use in accordance with the invention is the polymerase chain reac tion (PGR).
  • PGR polymerase chain reac tion
  • poiy(A) ⁇ RN A or total RKA is extracted from a tissue that expresses the gene product.
  • cDNA is then prepared from the RJ A using the enzyme reverse transcriptase.
  • Two primers typically complementary to two separate regions of the cDNA (oligonucleotides) are then added to the cD A along with a polymerase such as Taq polymerase, and the polymerase amplifies the cDNA region between the two primers.
  • a polymerase such as Taq polymerase
  • An embodiment of the invention provides for the use of isolated, purified or enriched nucleic acid sequences of, for example, about 15 to about 500 nucleotides in length, about 1 5 to about 100 nucleotides in length, about 1 5 to about 50 nucleotides in length, and about 15 to about 30 nucleotides in length, which have a sequence that corresponds to a portion of one of the nucleotides described herein.
  • the nucleic acid can be at least about 17, about 20, about 22, or about 25 nucleotides in length.
  • the nucleic acid sequence can he about 30 to about 300 nucleotides in length, or about 45 to about 200 nucleotides in length, or about 45 to about 100 nucleotides i length.
  • the nucleic acid can be at least about 5, about 6, about 7, about 8, about 9, about 10, about 12, bout 1 5, bout 17, about 20, about 22, bout 25, about 30, about 35, bout 40, about 50, about 100, about 1 0, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 1000, about 10,000, about 50,000, about 100,000 or more nucleotides in length, about 100,000, about 75,000, about 50,000, about 10,000, about 5,000, about 1000, about 750, about 500, about 250, about 200, about 100, about 50, about 40, about 30, about 25, about 22, about 20, about 17, about 15, about 12, about 10, about 9, about 8, about 7, about 6, about 5, or fewer nucleotides in length.
  • the nucleic acid can have a length in a range from any one of the above lengths to any other of the above lengths including endpoints.
  • a nucleic acid or protein relevant to the invention can be at least, e.g., about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 82%, about 84%, about 86%, about 88%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to reference sequences provided herein.
  • the nucleic acids described herein 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 vii.ro replication or in vivo replication.
  • Another embodiment of the invention provides a method of producing an isolated population of T cells for adoptive ceil therapy, the method comprising culturing isolated T cells having antigenic specificity fo a cancer antigen in vitro in a low-oxygen atmosphere, in an embodiment, the atmosphere has an oxygen content less than or equal to about 5%, less than or equal to about 4%, less than or equal to about 3%, less than or equal to about 2%, or less than or equal to about 1 %, or a r ange between any two of the foregoing values.
  • the T cells may be cultured in a !ow-oxygen atmosphere in the presence or absence of a Phd inhibitor.
  • the T cells may be cultured in ihe presence of one or both of (i) a Phd inhibitor and (ii) a low-oxygen atmosphere in any suitable manner.
  • the T cells are cultured in the presence of one or both of (i) a Phd inhibitor and (ii) a low-oxygen atmosphere with a cytokine such as, for example, interleukin-2 (IL-2), mterleukin-7 (I.L-7), mierleukin-15 (IL-15), interleukin-12 (IL-12) or a combination of two or more of the foregoing,
  • a cytokine such as, for example, interleukin-2 (IL-2), mterleukin-7 (I.L-7), mierleukin-15 (IL-15), interleukin-12 (IL-12) or a combination of two or more of the foregoing,
  • the method further comprises introducing a nucleic acid encoding an exogenous T cell receptor (TCR) into the T cells under conditions sufficient to express the 'ICR by the T cells.
  • the nucleic acid may be introduced into the T cells in ihe presence of one or both of (i) a Phd inhibitor and (ii) a low-oxygen atmosphere.
  • exogenous is meant that the TCR is not native to (naturally-occurring on) the T ceil.
  • the exogenous TCR may be a recombinant TCR.
  • a recombinant TCR is a TCR.
  • a recombinant TCR can comprise polypeptide chains derived entirely from a single mammalian species, or the recombinant TCR can be a chimeric or hybrid TCR comprised of amino acid sequences derived from TCRs from two different mammalian species.
  • the antigen-specific TCR can comprise a variable region derived from a murine TCR, and a constant region, of a huma ICR such that Ihe TCR is "humanized.
  • the TC 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 cancer antigen-specific TCR can comprise any amino acid sequence, provided that ihe TCR can specificaiiy bind to and immunologically recognize a cancer antigen or epitope thereof. Examples of exogenous TCRs that may be useful in the inventive methods include, but are not limited to, those disclosed in, for example. U.S. Patents 7,820,174;
  • a T cell comprising an endogenous cancer antigen-specific TCR can also be transformed, e.g., transduced or transfected, with one or more nucleic acids encoding an exogenous (e.g., recombinant) TCR or other recombinant chimeric receptor.
  • exogenous chimeric receptors e.g.. chimeric T CRs
  • the method further comprises introducing a nucleic acid encoding a chimeric antigen receptor (CAR) into the T cells under conditions to express the CAR by the T ceils.
  • the nucleic acid may be introduced into the T ceils in the presence of one or both of (i) a Phd inhibitor and (ii) a low-oxygen atmosphere, Typically, a CAR.
  • the antigenic specificity of a TCR of the invention can be encoded by a scFv which specifically binds to the cancer antigen, or an epitope thereof
  • Any CAR having antigenic specificity for a cancer antigen may be useful in the inventive methods.
  • CARs that may he useful in the inventive methods include, but are not limited to, those disclosed in, for example, WO 201 5/187528; U.S. Patents 8,465,743; 9,266,960: and 9359,447; and U.S. Patent Application Publication Nos.2014/0274909 and 201 5/0051266, each of which is incorporated herein by reference.
  • the exogenous TCR or CAR has antigenic specificity for a cancer antigen.
  • nucleic acids described herein can be incorporated into a recombinant expression vector.
  • 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.
  • the inventive recombinant expression vectors can comprise any type of nucleotides, including, but not limited to DNA and RNA, which can be single-stranded or double-stranded, synthesized or obtained in part from natural sources, and which can contain natural, non-natural or altered nucleotides.
  • the recombinant expression vectors can comprise naturaliy-occurring or non- naniralJy-occurring inlermicleotide linkages, or both types of linkages.
  • the non- naturally occurring or altered nucleotides or intermicleotide linkages do not hinder the transcription or replication of the vector.
  • recombinant expression vectors examples include, but are not limited to, piasmids, viral vectors (retroviral vectors, gamma-retroviral vectors, or lentiviral vectors), and transposons.
  • the vector may then, in turn, be introduced into the isolated popuiation of T cells by any suitable technique such as, e.g., gene editing, transfection, transformation, or transduction as described, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual (4 1,1 Ed,), Cold Spring Harbor Laboratory Press (2012).
  • Phage or viral vectors can be introduced into host ceils, after growth of infectious particles in suitable packaging cells, many of which are commercially available.
  • the vector is introduced into the isolated population of T ceils in the presence of one or both of (i) a Phd inhibitor and (ii) a low-oxygen atmosphere.
  • the isolated population of T cells into which a vector encoding the exogenous TCR or CAR has been introduced, can be cultured ex vivo under conditions to express the exogenous TCR or CAR, and then directly transferred into a mammal (preferably a human) affected by cancer.
  • a mammal preferably a human
  • Such a cell transfer method is referred to in the art as "adoptive ceil transfer” or “adoptive ceil therapy' 1 (ACT).
  • the T cells are cultured under conditions to express the exogenous TCR or CAR in the presence of one or both of (i) a Phd inhibitor and (ii) a low-oxygen atmosphere.
  • the Phd inhibitor is removed (e.g., washed) from the T cells prior to administering the cells to a patient, Jn another embodiment of the invention, the Phd inhibitor is not removed from the T cells prior to administering the cells to a patient.
  • the T cells administered to the mammal can be allogeneic or autologous to the mammal .
  • autologous cells are removed from a mammal, stored (and optionally modified), and returned back to the same mammal.
  • allogeneic administration methods, a mammal receives ceils from a genetically similar, but not identical, donor. Preferably, the ceils are autologous to the mammal. Autologous cells may, advantageously, reduce or avoid the undesirable immune response that may target an allogeneic ceil such as, for example, graft-versus-host disease.
  • the T cells may be cultured in the presence of one or both of (i) a Phd inhibitor and (ii) a low-oxygen atmosphere intermittently in vitro, in a preferred embodiment of the in vention, the T cells are cultured in the presence of one or both of (i) a Phd inhibitor and (ii) a low-oxygen atmosphere for the entire duration of in vitro culture, including during expansion of the numbers of ceils and during introduction of a nucleic acid encoding a CAR or an exogenous TCR into the cells.
  • the T cells may have antigenic specificity for a cancer 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.
  • the expression of the cancer antigen by normal, non-tumor, or non-cancerous cells is not as robust as the expression by tumor or cancer ceils, in this regard, the tumor or cancer cells can over-express the antigen or express the antigen at a significantly higher level, as compared to the expression of the antigen by normal, non-tumor, or non-cancerous cells.
  • the cancer antigen can additionally be expressed by ceils 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.
  • cancer antigens include, but are not limited to, mesothelin, CD 19, CD22, CD30, CD70, CD276 (B7H3), gpiOO, MART-1 , Epidermal Growth Factor Receptor Variant III (EGFRVIII), Vascular Endothelial Growth Factor Receptor 2 (VEGFR-2), TRP-1, TRP-2, tyrosinase, human papillomavirus (HPV) 16 E6, HPV 16 E7, RAS.
  • mesothelin CD 19, CD22, CD30, CD70, CD276 (B7H3), gpiOO, MART-1 , Epidermal Growth Factor Receptor Variant III (EGFRVIII), Vascular Endothelial Growth Factor Receptor 2 (VEGFR-2), TRP-1, TRP-2, tyrosinase, human papillomavirus (HPV) 16 E6, HPV 16 E7, RAS.
  • the cancer antigen may be a mutated antigen that is expressed or overexpressed by tumor or cancer cells and which is not expressed by normal, non-tumor, or non-cancerous cells.
  • 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.
  • 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.
  • the method further comprises expanding the number of T cells in the presence of one or more non-specific T cell stimuli, one or more cytokines, and. in the presence of one or both of (i) a Phd inhibitor and (ii) a low-oxygen atmosphere.
  • non-specific T cell stimuli include, but are not limited to, one or more of irradiated allogeneic feeder cells, irradiated autologous feeder cells, anti-CD3 antibodies, anti-4-lBB antibodies, and anti ⁇ CD28 antibodies.
  • the non-specific T cell stimulus may be anti-CD3 antibodies and anti-CD28 antibodies conjugated to beads. Any one or more cytokines may be used in the inventive methods.
  • Exemplary cytokines that may be useful for expanding the numbers of cells include interleukin (iL)-2, IL-7, LL-21 , and 1.1.-1.5,
  • the Fhd inhibitor and low-oxygen atmosphere for expanding the numbers of cells may be as described herein with respect to other aspects of the invention.
  • the numbers of T cells can be accomplished by any of a number of methods as are known in the art as described in, for example, U.S. Patent 8,034,334; U.S. Patent 8,383,099; and U.S. Patent Application Publication No, 2012/024 133.
  • the numbers of T cells are expanded by physically contacting the T ceils with one or more non-specific T cell stimuli and one or more cytokines in the presence of one or both of (i) a Phd inhibitor and (ii) a low-oxygen atmosphere.
  • expansion of the numbers of T cells may be carried out by culturing the T cells with O T3 antibody, 1L-2, and feeder PBMC (e.g., irradiated allogeneic PBMC) in the presence of one or both of (i) a Phd inhibitor and (ii) a low-oxygen atmosphere
  • expanding the number of T cells in the presence of one or both of (i) a Phd inhibitor and (ii) a low-oxygen atmosphere comprises culturing the cells for at least about 14 days in the presence of one or both of (i) a Phd inhibitor and (ii) a low-oxygen, atmosphere.
  • the invention further provides an isolated or purified population of T cells produced by any of the inventive methods.
  • the populations of T cells produced by the J 6 inventive methods may provide many advantages.
  • the populations of T cells produced by the inventive methods may, advantageously, demonstrate any one or more of greater persistence, proliferation, trafficking to tumor site(s), and antitumor activity upon in vivo transfer as compared to control T cells.
  • the population of T cells cultured in the presence of the Phd inhibitor has an increased expression of one or both of interferon-gamma (IFN- ⁇ ) and tumor necrosis factor alpha (TNF-a) as compared to control T cells, wherein the control T celis are identical to the T cells that are cultured in the presence of a Phd inhibitor except that the control ceils have not been cultured in the presence of a Phd inhibitor.
  • IFN- ⁇ interferon-gamma
  • TNF-a tumor necrosis factor alpha
  • lFN- ⁇ may, advantageously, inhibit tumor ceil proliferation
  • the population of T cells cultured in the presence of a low-oxygen atmosphere has an increased expression of one or both of in erferon-gamma (IFN- ⁇ ) and tumor necrosis factor alpha (TNF-a) as compared to control T cells, wherein the control T cells are identical to the T cells that are cultured in the presence of a low-oxygen atmosphere except that the control cells have not. been cultured in the presence of a low- oxygen atmosphere,
  • IFN- ⁇ in erferon-gamma
  • TNF-a tumor necrosis factor alpha
  • the population of T cells cultured in the presence of the Phd inhibitor are CD4H- and have an increased expression of Tbet as compared to control CD4+ T cells, wherein the control CD4+ T cells are identical to the T cells thai are cultured in the presence of a Phd inhibitor except that the control cells have not been cultured in the presence of a Phd inhibitor.
  • the population of T cells cultured in the presence of a low oxygen atmosphere are CD4+ and have an increased expression of Tbet as compared to control CD4+ T cells, wherein the control CD4+ T cells are identical to the T ceils that are cultured in the presence of a low oxygen atmosphere except that the control cells have not been cultured in the presence of a low oxygen atmosphere.
  • the population of T cells cultured in the presence of the Phd inhibitor are CD4+ and have a decreased expression of Foxp3 as compared to control CD4+ T cells, wherein the control CD4+ T cells are identical to the T ceils that are cultured in the presence of a Phd inhibitor except that the control cells have not been cultured in the presence of a Phd inhibitor.
  • the population of T cells cultured in the presence of a low oxygen atmosphere are CD4+ and have a decreased expression of Foxp3 as compared to control CD4+ T cells, wherein the control CD4+ T cells are identical to the T cells that are cultured in the presence of a low oxygen atmosphere except that the control ceils have not been cultured in the presence of a low oxygen atmosphere,
  • the population of T cells cultured in the presence of the Phd inhibitor are CD8+ and have a decreased expression of CD62L as compared to control CD8+ T ceils, wherein the control CD8+ T cells are identical to the T cells that are cultured in the presence of a Phd inhibitor except that the control cells have not been cultured in the presence of a Phd inhibitor.
  • the population of T cells cultured in the presence of a low oxygen atmosphere are CD8+ and have a decreased expression of CD62L as compared to control CD8+ T cells, wherein the control CD8+ T cells are identical to the T ceils that are cultured in the presence of a low oxygen atmosphere except that the control cells have not been cultured in the presence of a low oxygen atmosphere.
  • the population of T ceils cultured in the presence of a Phd inhibitor provides greater antitumor acti vity upon in vivo transfer as compared to control T cells, wherei the control T cells are identical to the T cells that are cultured in the presence of a Phd inhibitor except that the control cells have not been cultured in the presence of a Phd inhibitor.
  • the population of T ceils cultured in the presence of a low-oxygen atmosphere provide greater antitumor activity upon in vivo transfer as compared to control T cells, wherein the control T cells are identical to the T cells that are cultured in the presence of a low-oxygen atmosphere except that the control cells have not been cultured in tire presence of a low-oxygen atmosphere.
  • the term "isolated,” as used herein, means having been removed from, its natural environment.
  • the term “purified,” as used herein, means having been increased in punty, wherein “purity” is a relative term, and not to be necessarily construed as absolute purity.
  • the punty can be at least about 50%. can be greater than about 60%, about 70% or about 80%, about 90% or can be about 100%.
  • the population off cells produced by the inventive methods can be a
  • the population of T cells produced by the inventive methods can be a substantially homogeneous population, in which the population comprises mainly T cells described herein.
  • the population also can be a clonal population of ceils, in which all cells of the population are clones of a single T cell.
  • the population of cells is a clonal population comprising T cells comprising a recombinant, expression vector encoding the exogenous ICR or CAR as described herein.
  • inventive isolated or purified population of T cells produced according to any of the inventive methods described herein may be included in a composition, such as a pharmaceutical composition, in this regard, the invention provides a pharmaceutical composition comprising die isolated or purified population of T cells described herein and a pharmaceutically acceptable carrier.
  • the earner is a phamiaeeutically acceptable carrier.
  • the carrier can be any of those conventionally used for the administration of cells.
  • Such pharmaceutically acceptable carriers are well-known to those skilled in the art and are readily available to the public. It is preferred that the
  • pharmaceutically acceptable carrier be one which has no detrimental side effects or toxicity under the conditions of use.
  • Suitable formulations may include any of those for parenteral, subcutaneous, intravenous, intramuscular, intraarterial, intrathecal, intra tumoral, or imerperitoneai administration. More than one route can be used to administer the population of T ceils, and in certain instances, a particular route can provide a more immediate and more effective response than another route.
  • the population of T cells is administered by injection, e.g., intravenously.
  • a suitable pharmaceutically acceptable carrier for the cells for injection may include any isotonic carrier such as, for example, normal saline (about 0.90% w/v of NaCl in water, about 300 mOsm/L NaCl in water, or about 9.0 g NaCl per liter of water),
  • NORMOSOL R electrolyte solution (Abbott, Chicago, IL), PLA SMA-LYTF.
  • A Boxter, Deeriield, IL
  • about 5% dextrose in water or Ringer's lactate
  • the pharmaceutically acceptable carrier is supplemented with human serum albumen.
  • the dose e.g., number of T cells administered should be sufficient to effect, e.g., a therapeutic or prophylactic response, in the mammal over a reasonable time frame.
  • the number of T cells administered should be sufficient to bind to a cancer antigen or treat or prevent cancer in a period of from about 2 hours or longer, e.g., 12 to 24 or more hours, from the time of administration. In certain embodiments, the time period could be even longer.
  • the number of T cells administered will be determined by, e.g., the efficacy of the particular population of T ceils to be administered and the condition of the animal (e.g.. human), as well as the body weight of ihe animal (e.g., human) to be treated.
  • an assay which comprises comparing the extent to which target, cells are iysed or one or more cytokines such as, e.g., IFN-v and 1L-2 is secreted upon administration of a given number of such T cells to a mammal among a set of mammals of which is each given a different number of the T cells, could be used to determine a starting number to be administered to a mammal.
  • the extent to which target cells are lysed or cytokines such as, e.g., IFN-y and IL-2 are secreted upon administration of a certain number can be assayed by methods known in the art. Secretion of cytokines such as, e.g., IL-2, may also pro vide an indication of the quality (e.g., phenotype and/or effectiveness) of a T cell preparation.
  • the number of T ceils administered also will be determined by the existence, nature and extent of any adverse side effects that might accompany the administration of a particular population of T cells. Typically, the attending physician will decide the number of T cells with which to treat each individual patient, taking into consideration a variety of factors, such as age, body weight, general health, diet, sex, route of administration, and the severity of the condition being treated.
  • the number of T cells to be administered can be about 10 x 10 ⁇ to about 10 x 1 CT 1 cells per infusion, about 10 x 10 v cells to about 10 x 10 : : cells per infusion, or 10 x 10' ' to about 10 x 10'' ceils per infusion. While it may be possible to administer the cells together with the Phd inhibitor, in a preferred embodiment, the Fhd inhibitor is washed from the ceils prior to administering to a mammal.
  • the populations of T cells produced according to the mventive methods can be used in methods of treating or preventing cancer in a mammal
  • the invention provides a method of treating or preventing cancer in a mammal, comprising administering to the mammal any of the pharmaceutical compositions or populations of T cells produced by any of the methods described herein in an amount effective to treat or prevent cancer in tire mammal.
  • One or more additional therapeutic agents can be coadministered to the mammal.
  • administering i.s meant administering one or more additional therapeutic agents and the isolated, population of T cells sufficiently close in time such that the isolated population of T ceils can enhance the effect of one or more additional therapeutic agents, or vice versa.
  • the isolated population of T cells can be administered first and the one or more additional therapeutic agents can be administered second, or vice versa.
  • the isolated population of T cells and the one or more additional therapeutic agents can be administered simultaneously.
  • Additional therapeutic agents that may enhance the function of the isolated population of T cells may include, for example, one or more cytokines or one or more antibodies (e.g., antibodies that inhibit PD-1 function).
  • An exemplary therapeutic agent that can be co-administered with the isolated population of T cells is lL-2. Without being bound to a particular theory or mechanism, it is believed that IL-2 may enhance the therapeutic effect of the isolated population of T cells.
  • An embodiment of the invention further comprises lymphodepieting tire mammal prior to administering the isolated population of T cells.
  • lymphodepieting tire mammal prior to administering the isolated population of T cells.
  • iymphodepletion include, but may not be limited to, nonmyeloablaiive lymphodepieting chemotherapy, myeloablative lymphodepleiing chemotherapy, total body irradiation, etc.
  • the treatment or prevention provided by the inventive method can include treatment or prevention of one or more conditions or symptoms of the disease, e.g., cancer, being treated or prevented.
  • prevention can encompass delaying the onset or recurrence of the disease, or a symptom or condition thereof.
  • the T cells can be cells that are allogeneic or autologous to the host.
  • the cells are autologous to the host.
  • the cancer can be any cancer, including any of leukemia (e.g.. B cell leukemia), sarcomas (e.g., synovial sarcoma, osteogenic sarcoma, leiomyosarcoma uteri, and alveolar rhabdomyosarcoma), lymphomas (e.g..
  • leukemia e.g.. B cell leukemia
  • sarcomas e.g., synovial sarcoma, osteogenic sarcoma, leiomyosarcoma uteri, and alveolar rhabdomyosarcoma
  • lymphomas e.g..
  • mice (B6J29S7-Ragl lm i Moi3l/J ), Ly5.1 +/+ (B6.SJL-Ptprc a .Pepc b BoyJ) ! TRP-1 (B6.Cg- Rag 1 !m, Mom Tyrp 1 Bw Tg(Tcra,Tcrb)9Rest/J), and Cd4-Cre (Tg(CD4-Cre) 1 Cwi/Bflu J) were purchased from The Jackson Laborator . The following mice have been described: Eglnl "'", Egln2 n/n , and Egm3 n a mice (Takeda et ai., Mol. Cell. Biol,.
  • mice Hifl a i f mice (Ryan et a!., Cancer Res., 60: 4010-4015 (2000)), Epasl ' ⁇ mice (Gruber et a! ., Proc. Natl. Acad. Sci. USA, 104: 2301 -2306 (2007) ⁇ .
  • Deletion of /oxP-flanked genes in T cells was achieved by crossing to Cd4-Cre mice to obtain animals with homozygous loxP-flanked alleles without Cre or hemizygous for Cre. All mice were previously backcrossed over ten generations to the C57BL/6 background.
  • age and sex matched WT and PHD-tKO mice were used with at least 5 mice per genotype.
  • CD4 ! T cells from spleens and lymph nodes of 6-12-week-old mice were purified by negative magnetic selection (Miltenyi) followed by sorting of naive CD4 " CD62L 1 CD44 " CD25 " ceils using a FACSARiA II sorter (BD).
  • Naive CD4 + T cells were activated with platebound anti-CD3 and soluble anti-CD28 (5 ⁇ ml '1 each; eBioscience) in media for 72h either under: ThO conditions (media alone); Thl conditions (IL-12 (10 ng ml '1 , R&D
  • anti -11,-4 neutralizing antibodies (10 pg ml " '): Thl 7 conditions (I.L-6 (20 ng ml “1 , R&D Systems), human TGF-pl (0.2 ng ml “ ', R&D Systems), anti-IFN- ⁇ neutralizing antibodies (10 ⁇ ml " 1 ) and anti-IL-4 neutralizing antibodies (10 ug ml "'1 ); or iTreg conditions (human TGF- ⁇ (0-2 ng ml. "1 , as indicated)).
  • naive CD43 ⁇ 4D45RA ⁇ CD45RQ " CD62L ⁇ CCR7 ⁇ cells were FACS purified from three biologically independent healthy donor buffy coats and activated with plate-bound human anti-CD3 and soluble anti-CD28 (5 pg nil "1 each; eBioscience) with huma TGF- ⁇ (5 ng mL "1 , R&D Systems) and recombinant human IL-2 (20 CU).
  • Mouse and human cells were cultured under standard incubator conditions or 5% or 2% oxygen as indicated (Thermo Seientific-HERA CELL incubator equipped to replace oxygen with nitrogen) through the entire duration of culture.
  • the FHD protein inhibitor DMQG (0.5 - 1.0 mM, Sigma-Aldrich), Rapamycin (50 nM. Sigma- Aldrich), or 2-deoxyglucose (1 ⁇ , Sigma- Aldrich) was added for the duration of in vitro culture.
  • J Lungs were isolated from. WT and FHD-tKO mice and fixed m 10% formalin, embedded in methylacrylate, sectioned, and H&E stained. Sections were blindly evaluated by a veterinary pathologist. The presence of chronic, active inflammation, vasculitis, and hemorrhage in the alveoli was quantified on a standardized severity score system (0 least severe, 3 most severe).
  • WT and PHD-tKO mice were sensitized with 25 fig of house dust mite ( ⁇ , D. Pteronyssinus: !ow-endotoxin, Greer) delivered to the retropharyngeal space in sterile PBS on days 0, 1 and 2, Animals were challenged with 5 .ug of HDM on days 15, 16, 17 and 18, and euthanized on day 19.
  • Antibodies and Flow Cytometry 25 fig of house dust mite ( ⁇ , D. Pteronyssinus: !ow-endotoxin, Greer) delivered to the retropharyngeal space in sterile PBS on days 0, 1 and 2, Animals were challenged with 5 .ug of HDM on days 15, 16, 17 and 18, and euthanized on day 19.
  • NKT cells For the identification of NKT cells, a-ga actosyl ceramide loaded, PE-conjugated recombinant CD id tetramers were used (Proimmune). Cells were incubated with specific antibodies for 30 min on ice in the presence of 2.4G2 monoclonal antibody to block FcyR binding. All samples were acquired with a Fortessa or LSR flow cytometer (Becton
  • RNA-Seq analyses were performed using R 2 biological replicates. Raw data from replicate measurements are pubiically available from the GEO repository. Total RNA was prepared from cells using the RNEASY PLUS Mini kit (QIAGEN). 200 ng of total RNA was subsequently used to prepare RNA-Seq library by using TRUSEQ RNA sample prep kit (Iliumina) according to manufacturer's instructions. Paired-end RNA sequencing was performed on a HiSEQ 2000 sequencing system (Iliumina). Sequenced reads were aligned to the mouse genome (NCBI37/mm9) with TOPHAT 2.0.1 1 software (Kim and Salzberg, Genome. Biol., 12 ⁇ 201 1 )) and uniquely mapped reads were used to calculate gene expression.
  • REFSEQ gene database (mm9) was downloaded from the UCSC genome browser for RNA-Seq analysis.
  • RNA-Seq reads were mapped to mni9 (UCSC) using BOWTTE aligner.
  • Gene expression, of annotated transcripts was calculated from mapped RNA-Seq reads using the CUFFDIFF program to obtain RPKM-normalized gene expression values (Trapnell et al., Nat. Protoc, 7: 562-578 (2012)). Two-tailed t tests were performed to identify different! l y expressed genes after applying the Benjamini-Hochberg correction for multiple testing.
  • Gene set enrichment analysis was performed as previously described (Subrarnanian et al., Froc. Natl. Acad. ScL, 102: 15545-15550 (2005)).
  • ELISA assays were performed on mouse serum according to manufacturer s instructions (Alpha Diagnostic international). ELISA quantification of IFN- ⁇ in cell supematants collected after 72h or stimulation was performed according to manufacturer's instructions (Ebioscience),
  • RNA from pelleted cells was isolated using the RNEASY Plus mini kit (QIAGEN).
  • First-strand cDNA synthesis was performed using random priming with the high-capacity cD A synthesis kit (Applied Biosystems) in the presence of SLiPERASEIN RNase inhibitor (Ambion).
  • cDNA was used as a template for quantitative PGR reactions using TAQMAN primer-probes against specified mRNA transcripts (Applied Biosystems). Reactions were performed using Universal PGR Masiermix (Applied Biosystems). FAM channel intensity were normalized to ROX intensity, and C, values were calculated using automatically determined threshold values using SDS software (Applied Biosystems).
  • Nuclear extracts were isolated (NE-PER kit; Pierce) and ⁇ 15 , ug protein was loaded in each lane of a mini-protean precast TGX gel (Biorad). Protein was iransten'ed onto activated PVDF membrane (Biorad), blocked with 5% BSA in TBST at RT for Ih, and incubated in 1° antibody at 4 °C overnight. HiFl (NB 100-449, Novus) and loading control HDAC1 ( 10E2, Cell Signaling Technology) antibodies were used at 1 : 1000 dilution in TBST.
  • RagP' ' mice were injected intravenously with 4 X 10 ' ' CD4 ' CD25* cells from wild-type or PHD-tKO mice. On day 7, transferred cells were isolated and analyzed for FoxP3 expression by flow cytometry.
  • CD4 *' CD25 ' Treg cells were FACS purified from wild-type or PHD-tKO mice and stimulated in vitro with anti-CD 3 (1 pg mL '! ) in the presence of IL-2 (100 IU). Foxp3 expression was analyzed by flow cytometry 72 h post-stimulation.
  • Naive CD4 ⁇ T cells were FACS purified from Foxp3-GFP mice and stimulated i vitro in the presence of TGF- ⁇ ⁇ DMOG for 72h. Foxp3-GFP+ iTreg cells were then FACS purified from VEH and DMOG treated cultures.
  • VEH and DMOG iTreg cells were cultured in 96-well round-bottom plates with 5x10 4 CFSE-labeled naive CD45.1 CD4+ CD25- (Tresp) cells along with I xt O 4 CD! l e t dendritic cells used as antigen-presenting cells, isolated by immunomagnetic selection (Miltenyi). Cells were stimulated with anti-CD. ' :? antibody (1 ⁇ ig niL-i, BD Biosciences) for 72h at 37 and 5% CO ?. . Tresp cell proliferation was measured by CFSE dilution by flow cytometry.
  • Extracellular acidification rates (ECAR) and oxygen consumption rates (OCR.) were measured ai 37 °C using an XF24 extracelkilar analyzer (Seahorse Bioscience). ECAR was measured in XF media (nonhuffered RPMI 1640 containing 25 mM glucose, 2 rnM L- glutariiine, and 1 mM sodium pyruvate) under basal conditions. To determine glucose uptake, T cells were incubated with 100 ⁇ 2-NBDG (Invitrogen for 2 hr before measuring fluorescence by flow cytometry.
  • 2-NBDG Invitrogen for 2 hr before measuring fluorescence by flow cytometry.
  • PYROSEQUENC1NG primer was annealed to the purified single-stranded PCR product and sequencing was performed using the PYROSEQUE C1NG PSQ96 HS System according to manufacturer's instructions (QIAGEN). The methylation status of each locus was analyzed individually as a T/C S P using QCpG software (QIAGEN),
  • B16F1G (B 16) melanoma cell line was obtained from the NCI tumor repository and passaged in Dulbecco's Modified Eagle Medium (Invitrogen) supplemented with 10% fetal calf scrum. 2.5 ⁇ 0 5 B 16 cells were injected subcutaneously in flanks and intravenously through the tail vein. Tumor implantation experiments were performed in littermate mice of 8-32 weeks of age. Subcutaneous tumors were measured at serial time points following implantation using digital calipers and the tumor area was calculated as the product of perpendicular diameter. Lung tumor nodules were enumerated by gross count of visible sites of disease.
  • Micrometasiatic lesions were quantified following H&E stain of lung sections, and sections consistent with melanoma histology were circumscribed by a certified veterinarian. All subcutaneous and pulmonary tumor measurements were performed in a blinded manner. IFN- ⁇ depletion was performed using intraperitoneal injection of 250 mg anti-IF -y (XM.G1.2, BioXcell) at indicated time points.
  • Tr l transgenic splenocytes were stimulated for 72 hours with Trp peptide and irradiated feeder cells under ThO or iTreg (0.2 ng/mL TGF- ⁇ ) conditions in the absence (vehicle) or presence of the Ph.d inhibitor DMOG ( i rriM). Flow cytometric analysis of Foxp3 and Tbet were earned out. The results are shown in Table A (ThO cells) and Table B (iTreg cells). Tables A and B show the percentage of cells with the indicated phenotypes.
  • Trp " CD4 ⁇ T cells adopted a Thl phenotype when stimulated in the presence of the small molecule pan-Phd inhibitor DMOG, even under conditions that would normally promote the development of immunosuppressive regulatory T cells ⁇ Tables A and B and Figures 1 A and IB).
  • mice bearing 10-day established subcutaneous B 16 melanoma tumors were treated with no cells or 1 x10 b Trp+ CD4 ⁇ T cells that were treated with vehicle or DMOG. Trp + T cells recognize the tyrosinase melanocyte differentiation antigen expressed on many melanomas. Eight mice were in each treatment group. Tumor growth and overall survival of die mice were measured. The results are shown in Figiire I C (tumor growth) and Figure ID (survival). These data are representative of three independently performed tumor treatment experiments. All mice treated in Figures IC and ID received exogenous inierleiikin (IL)-2,
  • Thl cells are more effective than ThO cells at inducing tumor rejection (Muranski et ah, Blood, 1 12: 362-373 (2008).
  • Augmented antitumor function in mice administered the DMOG-treated Trp + T ceils is consistent with the induction of a Thl phenotype in these cells.
  • DMOG-treated Trp* T cells were as effective in mediating tumor regression as cells polarized under conventional Thl cell specifying conditions.
  • inhibition of PHD proteins provides a viable strategy to enforce Thl cell differentiation under conditions used for ACT, thereby providing a potential means to improve the efficacy of ACT.
  • CD8 'r PmeF transgenic splenocytes were stimulated for 5 days with gplOO peptide and IL-2 (100 KJ) in the presence or absence of the Phd inhibitor DMOG (1 niM).
  • the cells were analyzed for the expression of CD44 and CD62L by flow cytometry. Representative results are shown in Table C.
  • Table C shows the percentage of cells with the indicated phenoiypes.
  • Figure 2A shows the population distribution of nai e, central memory, and effector memory cells in the CD8* PmeF transgenic splenocyies ihat were stimulated in the absence or presence of the Phd inhibitor DMOG.
  • D and Figure 2B show the pei -eeniage of cells with the indicated cytokine pro files.
  • mice bearing 10-day established subcutaneous B16 melanoma tumors transduced with humanized gplOO antigen were treated with no cells or 1x106 PmeH- CD8+ T cells thai were treated with vehicle or DMOG. Eight mice were in each treatment group. Tumor growth and overall survival of the mice were measured. The results are shown in Figure 2C (tumor growth) and Figure 2D (.survival). All mice treated in Figures 2C and 2D received exogenous interleuk (JL)-2. and total body irradiation (600 Gy) prior to ceil transfer.
  • JL interleuk
  • 600 Gy total body irradiation
  • CDS + T cells can mediate dramatic tumor regression in adoptive cell transfer therapies.
  • Pmef CD8 + T cells expanded ex vivo in the presence of DMOG were able to mediate stronger tumor regression leading to improved overall survival of the tumor bearing hosts.
  • CD4 T cells were stimulated for 72 hours with anti-CD3 antibody and anti-CD28 antibody (5 mg/tr.L each) under ThO or iTreg (0.2 ng/mL TGF-b) conditions in an ambient air incubator (20% i3 ⁇ 4) or a hypoxic chamber incubator (1 % 0 2 ).
  • ThO ThO
  • iTreg 0.2 ng/mL TGF-b
  • mice w th a T ceil specific deletion of all three Phd proteins are protected from hmg tumor colonization.
  • mice with a T ceil specific deletion of all three Phd proteins ⁇ Phd-tKO) and wild- type (WT) littermate matched mice were injected intravenously with 2 I0 5 B16 melanoma cells.
  • the total number of Sung tumors formed was enumerated 21 days after injection. The results are shown in Figures 4A--4C.
  • mice with a T cell specific deletion of all three Phd proteins were protected from lung tumor colonization. It was also observed that lung resident P d-tKO CD4+ and CD8+ T ceils had an increased capacity to produce IF - ⁇ as compared to WT T cells. Lung resident CD4+ T cells from Phd-tKO mice also demonstrated a reduced percentage of Nrpi-Lo iTreg as compared to hmg resident CD4+ T cells from WT mice.
  • the tumor burden in the livers of WT and PHD-tKO mice was also measured by liver weight (Fig. 5C), visible tumor nodules (Fig. 5D), and histologic enumeration (Fig. 5E), As shown in Figs, 5C-5.E, the tumor burden in the livers of PHD-tKO mice, as measured by liver weight, visible tumor nodules, and histologic enumeration, was significantly reduced as compared to WT livers.
  • PHD-tKO mice Upon gross evaluation, a patchy hemorrhagic appearance was observed within the lungs of PHD-tKO mice that was not present among wild-type (WT) littermales. Histologic examination revealed the presence of diffuse alveolar hemorrhage (DAH) of variable severity in PHD-tKO mice. PHD-tKO mice had increased serum autoantibodies, which can be elevated upon immune-mediated tissue damage. Pathology in PHD-tKO mice was only observed in the lung, and abnormal iiver and pancreas enzymes were not detected in the blood.
  • DASH diffuse alveolar hemorrhage
  • PHD-tKO animals showed no defects in thymocyte number or phenotypic distribution. Similar numbers of CD4+and CD8 ⁇ T lymphocytes were detected in peripheral blood and secondary lymphoid organs. In the bone marrow of PHD-tKO mice, there was a slight reduction in CD8+ T cells but similar numbers of CD + T cells. As the bone marrow is a reservoir of memory T cells, the differentiation state of T cells in WT and PHD-tKO mice was evaiulated. There was a similar distribution of naive and effector CD4+ T cells in WT and PHD-tKO mice.
  • T cells can drive immunopat ology in the lung. Increased numbers of pulmonary CD4+ and CD8+ T celis were observed in PHD-tKO mice. Additionally, pulmonary T cells in PHD-tKO mice had increased expression of the activation markers CD44, CD25, CTLA-4, and GiTR. In PHD-tKO mice, increased expression of IFN-g by CD4+ and CD8+ T c eils isolated from multiple lymphoid and non-lymphoid organs was measured, yet this was most pronounced in the lung. Elevated IFN-g production was more significant in the lung, including among pulmonary tissue resident memory (TRM) T cells, than in T ceils residing in mediastinal lymph nodes (meLN).
  • TRM pulmonary tissue resident memory
  • Treg cell stability is proportional to the level of demetbylaiion within the Treg-specific-demethylated region (TSDR) of the Foxp3 locus (Floess et al., PLoS Bio!. , 5: e.38 (2007)).
  • TSDR CpG methylation was similar in WT and PHD-tKO Treg celis.
  • the stability of Foxp3 expression was similar in WT and PHD-tKO Treg cells following restimulation in vitro or upon in vivo transfer into Ragl " ' " hosts.
  • a reduction in the frequency and number of Nrp-lLo Treg cells was detected in the lungs of PHD-tKO mice.
  • Nrp-i expression can be induced on " freg cells in inflammatory environments (Weiss et al., J. Exp. Med., 209: 1723-42 (2012)). This raised the possibility that the observed reduction in pulmonary Nrp-lLo Treg cells in PHD-tKO mice is the result of induced rp-1 expression in the inflamed lung environment. To exclude this possibility, it was tested whether differences in Nrp-1 expression between WT and PHDtKO Treg cells exist when cells are exposed to an identical pulmonary environment. Lethally ablated Ragl- /- mice were reconstituted with bone marrow isolated from congemcally distinguishable WT or PHD-tKO mice in a 1 : 1 mixture.
  • Pulmonary Treg cells were evaluated following reconsiitution, A reduced frequency of Nrp- lLo cells was observed within PHD-tKO pulmonary Treg cells.
  • the reduced frequency of Nrp-lLo Treg cells in lungs of PHD- tKO mice is not secondary to the inflamed environment but instead is a cell-intrinsic phenomenon.
  • HDM challenge promoted similar expansion of pulmonary CD4+ T cells in WT and PHD-tKO mice.
  • HDM challenge induced a robust expansion of pulmonary Th2 cells in WT mice but failed to do so in PHD-tKO mice, instead, a dramatic increase in IFNg + Thl cells was observed in lungs of PHD-tKO mice upon HDM challenge.
  • HDM challenge induced accumulation of pulmonary Treg cells that was reduced in PHD-tKO mice compared to WT controls.
  • PHD-tKO mice also exhibited greater expansion of CD8+ T cells and induction of IFN-g expression among these ceils upon HDM challenge.
  • Ti ls example demonstrates that FHD proteins regulate reciprocal iTreg and Thl differentiation programs.
  • naive CD4+ T ceils isolated from WT and PHD-tKO mice were stimulated in the presence of titrated concenirations of TGF-b.
  • PHD proteins were required for TGF-b-driven induction of Foxp3 and repression of T-bet. Consistently, higher amounts of IFN-g were detected in snpematants collected from PHD-tKO cell cultures.
  • Naive PHD-tKO CD4+ T cells also demonstrated impaired iTreg conversion in vivo following transfer into Ragl -/- recipient animals.
  • T-bet expression can be induced in Treg cells in response to IFN-g (Koch et al., Immunity, 37: 501 -510 (2012)). Indeed, blockade of IFN-g limited T-bet expression in PHD-tKO Foxp3+ iTreg ceils. Foxp3+ T-bet+ Treg cells exert suppressive function, especially in inflammatory contexts (Koch et al,. Nat. Immunol.., 10: 595-602 (2009)).
  • FACS fluorescence-activated cell sorting
  • T is example demonstrates that extracellular oxygen promotes iTreg cell differentiation in a PHD-dependent manner.
  • GSEA Gene set enrichment analysis
  • HIFl a protein expression was similar in naive cells but significantly elevated in PHD- ⁇ T cells upon activation. Consistent with their functional redundancy, each PHD protein limited HIFl a accumulation in T cells, Furthermore, HIFla accumulation was suppressed in CD4+ T cells stimulated in high oxygen environments in a PHD-dependent manner.
  • HIFl a HIFl a
  • Foxp3. and T- bet protein expression was analyzed in CD4-J- T cells isolated from each PHD KO genotype.
  • HIFla accumulation inversely correlated with iTreg cell differentiation and positively correlated with Thl effector cell differentiation.
  • loss of HIFla and HIF2a partially rescued iTreg cell differentiation and reversed excessive Thl differentiation when the enzymatic activity of PHD proteins was inhibited using DMOG.
  • HIF transcriptional activity drives expression of genes involved in multiple cellular programs including glycolysis.
  • Expression of components of the glycolytic machinery was elevated in PHDtKO and DMOG-treated VVT T cells in a HIFa-dependent manner.
  • T ceils stimulated in the presence of TGF-b demonstrated a PflD-dependent reduction in extracellular acidification rate (ECAR), a measure of glycolytic activity.
  • ECAR extracellular acidification rate
  • PHDtKO CD4 ⁇ T ceils demonstrated increased glucose uptake and adopted an anaerobic metabolic signature.
  • PHD proteins also restrained glycolysis in CD8+ T cells,
  • WT and PHD-tKO mice were injected with B 16 melanoma tumors subcuianeously in the flank and intravenously (i.v.) through the tail vein, to introduce rumor cells at each site within the same animal. While subcutaneous tumor growth was similar in WT and PHDiKO mice, PHD-tKO mice were significantly protected from tumor colonization in the lung. PHD-tKO animals had fewer detectable lung tumors upon gross and microscopic evaluation, resulting in a significant reduction in total pulmonary tumor burden.
  • PHD proteins support pulmonary umor colonization through suppression of IFN-g-mediaied anti-tumor immunity.
  • PHD-tKO mice were injected intravenously with B1.6 melanoma and received IFN-g neutralizing or control antibodies at serial time points following tumor implantation. The protection from lung tumor colonization observed in PHD-tKO mice was abrogated by IFN-g n utralization. Thus, PHD proteins limit IFN-g-mediated tumor clearance in the lung.

Abstract

La présente invention concerne des procédés de production de populations isolées de lymphocytes T pour thérapie cellulaire adoptive, le procédé consistant à cultiver des lymphocytes T isolés in vitro en présence soit (i) d'un inhibiteur de protéines contenant un domaine prolyl hydroxylase (Phd), soit (ii) d'une atmosphère à faible teneur en oxygène, ou des deux, les lymphocytes T présentant une spécificité antigénique pour un antigène cancéreux. La présente invention concerne également des populations isolées de lymphocytes T apparentées, des compositions pharmaceutiques et des méthodes de traitement ou de prévention du cancer chez un mammifère.
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019075055A1 (fr) * 2017-10-11 2019-04-18 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Procédés de production de populations de lymphocytes t au moyen d'inhibiteurs de mapk p38
WO2020081987A1 (fr) * 2018-10-18 2020-04-23 Board Of Regents The University Of Texas System Méthodes de production de lymphocytes t de type à mémoire résidant dans un tissu et leur utilisation
US11111493B2 (en) 2018-03-15 2021-09-07 KSQ Therapeutics, Inc. Gene-regulating compositions and methods for improved immunotherapy

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8034334B2 (en) 2002-09-06 2011-10-11 The United States Of America As Represented By The Secretary, Department Of Health And Human Services Immunotherapy with in vitro-selected antigen-specific lymphocytes after non-myeloablative lymphodepleting chemotherapy
US20120244133A1 (en) 2011-03-22 2012-09-27 The United States of America, as represented by the Secretary, Department of Health and Methods of growing tumor infiltrating lymphocytes in gas-permeable containers
US8383099B2 (en) 2009-08-28 2013-02-26 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Adoptive cell therapy with young T cells

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8034334B2 (en) 2002-09-06 2011-10-11 The United States Of America As Represented By The Secretary, Department Of Health And Human Services Immunotherapy with in vitro-selected antigen-specific lymphocytes after non-myeloablative lymphodepleting chemotherapy
US8383099B2 (en) 2009-08-28 2013-02-26 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Adoptive cell therapy with young T cells
US20120244133A1 (en) 2011-03-22 2012-09-27 The United States of America, as represented by the Secretary, Department of Health and Methods of growing tumor infiltrating lymphocytes in gas-permeable containers

Non-Patent Citations (27)

* Cited by examiner, † Cited by third party
Title
AAGAARD ET AL., A1OL. THER., vol. 15, no. 5, 2007, pages 938 - 945
ANDREW L DOEDENS ET AL: "Hypoxia-inducible factors enhance the effector responses of CD8+ T cells to persistent antigen", NATURE IMMUNOLOGY, vol. 14, no. 11, 29 September 2013 (2013-09-29), GB, pages 1173 - 1182, XP055333988, ISSN: 1529-2908, DOI: 10.1038/ni.2714 *
CHENG ET AL., CELL RES., vol. 23, 2013, pages 1163 - 1171
CHIARA BONINI ET AL: "Adoptive T-cell therapy for cancer: The era of engineered T cells", EUROPEAN JOURNAL OF IMMUNOLOGY, vol. 45, no. 9, 21 August 2015 (2015-08-21), DE, pages 2457 - 2469, XP055333980, ISSN: 0014-2980, DOI: 10.1002/eji.201545552 *
CLEVER DAVID ET AL: "Oxygen Sensing by T Cells Establishes an Immunologically Tolerant Metastatic Niche", CELL, vol. 166, no. 5, 25 August 2016 (2016-08-25), pages 1117, XP029706252, ISSN: 0092-8674, DOI: 10.1016/J.CELL.2016.07.032 *
DANG ET AL., CELL, vol. 146, 2011, pages 772 - 784
DOEDENS ET AL., NAT. IMMUNOL., vol. 14, 2013, pages 1173 - 1182
FLOESS ET AL., PLOS BIOL.,, vol. 5, 2007, pages E38
GOMMANS ET AL., J. MO! BIOL., vol. 354, no. 3, 2005, pages 507 - 519
GREEN; SAMBROOK: "Molecular Cloning: A Laboratory Manual", 2012, COLD SPRING HARBOR LABORATORY PRESS
GRUBER ET AL., PROC. NATL. ACAD. SCI. USA, vol. 104, 2007, pages 2301 - 2306
KAELIN; RATCLIFFE, MOL. CELL, vol. 30, 2008, pages 393 - 402
KIM; SALZBERG, GENOME BIOL., 2011, pages 12
KOCH ET AL., IMMUNITY, vol. 37, 2012, pages 501 - 510
KOCH ET AL., NAT. IMMUNOL., vol. 10, 2009, pages 595 - 602
MURANSKI ET AL., BLOOD, vol. 112, 2008, pages 362 - 373
MURANSKI ET AL., BLOOD, vol. 112, no. 2, 2008, pages 362 - 373
OVERWIJK ET AL., J. EXP. MED, vol. 188, no. 2, 1998, pages 277 - 286
RIVIERE ET AL., GENE THER., vol. 21, no. 5, 2014, pages 529 - 532
RYAN ET AL., CANCER RES., vol. 60, 2000, pages 4010 - 4015
SUBRAMANIAN ET AL., PROC. NATL. ACAD. SCI., vol. 102, 2005, pages 15545 - 15550
T. BOLLINGER ET AL: "HIF-1 - and hypoxia-dependent immune responses in human CD4+CD25high T cells and T helper 17 cells", JOURNAL OF LEUKOCYTE BIOLOGY, vol. 96, no. 2, 1 August 2014 (2014-08-01), US, pages 305 - 312, XP055333992, ISSN: 0741-5400, DOI: 10.1189/jlb.3A0813-426RR *
TAKEDA ET AL., MOL. CELL. BIOL,, vol. 26, 2006, pages 8336 - 8346
TANAKA ET AL: "Regulation of early T cell development by the PHD finger of histone lysine methyltransferase ASH1", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, ELSEVIER, AMSTERDAM, NL, vol. 365, no. 3, 5 November 2007 (2007-11-05), pages 589 - 594, XP022372623, ISSN: 0006-291X, DOI: 10.1016/J.BBRC.2007.10.159 *
TRAPNELL ET AL., NAT. PROTOC., vol. 7, 2012, pages 562 - 578
WEISS ET AL., J. EXP. MED., vol. 209, 2012, pages 1723 - 1742
ZHANG ET AL., NATURE BIOTECHNOL., vol. 29, 2011, pages 149 - 153

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019075055A1 (fr) * 2017-10-11 2019-04-18 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Procédés de production de populations de lymphocytes t au moyen d'inhibiteurs de mapk p38
US11111493B2 (en) 2018-03-15 2021-09-07 KSQ Therapeutics, Inc. Gene-regulating compositions and methods for improved immunotherapy
US11421228B2 (en) 2018-03-15 2022-08-23 KSQ Therapeutics, Inc. Gene-regulating compositions and methods for improved immunotherapy
US11608500B2 (en) 2018-03-15 2023-03-21 KSQ Therapeutics, Inc. Gene-regulating compositions and methods for improved immunotherapy
WO2020081987A1 (fr) * 2018-10-18 2020-04-23 Board Of Regents The University Of Texas System Méthodes de production de lymphocytes t de type à mémoire résidant dans un tissu et leur utilisation
CN113056276A (zh) * 2018-10-18 2021-06-29 得克萨斯大学体系董事会 用于产生组织驻留记忆样t细胞的方法及其用途

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