WO2017070042A1 - Procédés de production de populations de lymphocytes t à l'aide d'inhibiteurs d'akt - Google Patents

Procédés de production de populations de lymphocytes t à l'aide d'inhibiteurs d'akt Download PDF

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WO2017070042A1
WO2017070042A1 PCT/US2016/057307 US2016057307W WO2017070042A1 WO 2017070042 A1 WO2017070042 A1 WO 2017070042A1 US 2016057307 W US2016057307 W US 2016057307W WO 2017070042 A1 WO2017070042 A1 WO 2017070042A1
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cells
akt inhibitor
akt
ceils
population
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PCT/US2016/057307
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Joseph Garfield CROMPTON
Nicholas P. Restifo
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The United States Of America, As Represented By The Secretary, Department Of Health 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
    • C12N5/0638—Cytotoxic T lymphocytes [CTL] or lymphokine activated killer cells [LAK]
    • 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
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • 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
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464411—Immunoglobulin superfamily
    • A61K39/464412—CD19 or B4
    • 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
    • A61K39/464466—Adhesion molecules, e.g. NRCAM, EpCAM or cadherins
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/572—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic response
    • 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
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2302—Interleukin-2 (IL-2)
    • C—CHEMISTRY; METALLURGY
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/515—CD3, T-cell receptor complex
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes
    • C12N2501/72—Transferases (EC 2.)
    • C12N2501/727—Kinases (EC 2.7.)

Definitions

  • Adoptive cell therapy (ACT) using cancer-reactive T cells can produce positive clinical responses in cancer patients. Nevertheless, several obstacles to the successful use of ACT for the treatment of cancer and other diseases remain. For example, expansion of the numbers of T cells may produce T cells with a terminally differentiated phenotype that is associated with diminished antitumor activity and poor capacity for long-term persistence in vivo. Accordingly, there is a need for improved methods of obtaining an isolated population of T ceils for ACT ' .
  • An 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 ceils having antigenic specificity for a cancer antigen in vitro in the presence of a protein kinase B (Akt) inhibitor.
  • Akt protein kinase B
  • T cells treated with an Akt inhibitor maintain their capacity to target cancer antigen-expressing ceils and have transcriptional, metabolic, and functional properties that correlate with enhanced in vivo persistence after adoptive -transfer.
  • Akt inhibitor-treated cells have features of long-lived memory T cells that are associated with prolonged antitumor immuni y.
  • an embodiment of the invention provides a method of producing an isolated population of T ceils 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 Akt 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 leukap eresis.
  • the method comprises isolating peripheral blood lymphocyte (PBL) or a peripheral blood mononuclear cell (PBMC) from a mammal.
  • PBL peripheral blood lymphocyte
  • PBMC peripheral blood mononuclear cell
  • the T cells can be obtained from a tumor 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, SupTI, etc., or a T cell obtained from a mammal. If obtained from a mammal, the T ceil 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 ceils 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, CD47CD8 "' double positive T cells, CD4 + helper T cells, e.g., ⁇ 3 ⁇ 4 and Th 2 cells, CDS " T cells (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 CD4 + T cell.
  • the term "mammal” refers to any mammal including, but not limited to, mammals of the order Logomorpha, such as rabbits; the order Carnivora, includmg Felines (cats) and Canines (dogs); the order Artiodactyla, includmg Bovines (cows) and Swines (pigs); or of the order Perssodactyia, including Equines (horses). It is preferred that the mammals are non-human primates, e.g., of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes).
  • the mammal may be a mammal of the order Rodentia, such as mice and banisters. 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. ⁇ 5
  • the method comprises cuituring tumor fragments, isolated PB.MC, or PBL (e.g., T ceils) in vitro in the presence of one or more Akt inhibitors.
  • the Akt inhibitor may be any suitable Akt inhibitor.
  • the Akt inhibitor may be an aliosteric inhibitor or a non-aliostenc inhibitor of Akt.
  • the Akt inhibitor may be Akt isoform-specific or non-Akt isoform specific.
  • the Akt inhibitor may inhibit one or both of Aktl and Akt2.
  • Examples of Akt inhibitors thai may be useful in the inventive methods include, but are not limited to. 3-[l- [[4-(7 ⁇ p.henyl-3Hrimidazo[4,3 ⁇ g]quinoxalin ⁇
  • benzimidazoi-2-one (also referred to as Akt Vlli or Akti-1/2), perifosine, 17-A G, SH-6, palonnd 529, FPA 124, SH-5, AGL 2263, mitefosine, fisetin, PIT 1 , PHT-427, API-1, MK- 2206, RX-0201 , PBI-05204, and GSK2141795.
  • the Aki inhibitor is Akt VIII.
  • the T cells may be cultured in the presence of the Akt inhibitor in any suitable manner.
  • the T cells are cultured in the presence of a cytokine such as. for example, interIeukin-2 (IL-2), interleukin- 7 (IL-7), interleukin -d 5 (IL- 15), in erleukin- 12 (IT- 12) or a combination of two or more of the foregoing.
  • a cytokine such as. for example, interIeukin-2 (IL-2), interleukin- 7 (IL-7), interleukin -d 5 (IL- 15), in erleukin- 12 (IT- 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 in the presence of an Akt inhibitor and under conditions to express the TCR by the T cells.
  • exogenous is meant that the TCR is not native to (naturally-occurring on) the T cell.
  • the exogenous TCR may be a recombinant TCR.
  • 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 recombinant TCR can be a chimeric or hybrid TCR comprised of amino acid sequences derived from I CRs 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 human TCR such that the TCR is "humanized.” Any exogenous TCR having antigenic specificity for a cancer antigen may be useful in the inventive methods.
  • the 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 cancer antigen-specific TCR can comprise any amino acid sequence, provided that the TCR can specifically 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.
  • a T cell comprising an endogenous cancer antigen-specific TCR can also be transformed, e.g., transduced or iransfected, with one or more nucleic acids encoding an exogenous (e.g., recombinant) TCR or other recombinant chimeric receptor.
  • exogenous chimeric receptors e.g., chimeric TCRs
  • the method further comprises introducing a nucleic acid encoding a chimeric antigen receptor (CAR) into the T cells in the presence of an Akt inhibitor and under conditions to express the CAR by the T cells.
  • 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.
  • 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 cancer antigen, or an epitope thereof.
  • CAE s that may be useful in the inventive methods include, but are not limited to, those disclosed in, for example, WO 2015/187528; U.S. Patents 8,465,743; 9,266,960; and 9,359,447 and U.S. Patent Application Publication Nos. 2014/0274909; and 2015/0051266, each of which is incorporated herein by reference.
  • the exogenous TCR or CAR has antigenic specificity for a cancer antigen.
  • antigen-specific and antigenic specificity mean that the TCR or CAR can specifically bind to and immunologically recognize an antigen, or an epitope thereof, such that binding of the TCR or CAR to antigen, or the epitope thereof, elicits an immune response.
  • a nucleic acid encoding the exogenous TCR or CAR is introduced into any suitable 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 DMA 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 or non ⁇ naturaliy-oceurring intemucieotide linkages, or both ty es of linkages, Preferably, the non-naturally occurring or altered nucleotides or intemucieotide linkages do not hinder the transcription or replication of the vector.
  • recombinant expression vectors examples include, but are not limited to, plasmids, viral vectors ⁇ retroviral vectors, gamma -retroviral vectors, or ientiviral vectors), and transposons.
  • the vector may then, in turn, be introduced into the isolated population of T cells by any suitable technique such as, e.g., gene editing, transfection, transformation, or transduction as described, for example. Green and Sam brook, Molecular Cloning: A Laboratory Manual (4 ,h Ed.), Cold Spring Harbor Laboratory Press (2012).
  • transfection techniques include, for example, calcium phosphate DNA co-precipitation; DEAE-dextran; eiectroporation; cationic liposome- mediated transfection; tungsten particle-facilitated micropanicle bombardment; and strontium phosphate DNA co-precipitation.
  • Phage or viral vectors can be introduced into host cells, 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 cells in the presence of an Akt inhibitor.
  • 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 cell transfer" or ''adoptive cell therapy" (ACT).
  • the T ceils are cultured under conditions to express the exogenous TCR or CAR and in the presence of an Akt inhibitor.
  • the Akt inhibitor-treated population of T ceils administered to the mammal can be allogeneic or autologous to the mammal, in "autologous" administration methods, cells are removed from a mammal, stored ⁇ and optionally modified), and returned back to the same mammal, in ' ' 'allogeneic" administration methods, a mammal receives ceils from a genetically similar, hut not identical, donor. Preferably, the cells are autologous to the mamma!. Autologous ceils may, advantageously, reduce or avoid the undesirabie immune response that may target an allogeneic cell such as, for example, graft-versus-host disease.
  • the T cells may cultured in the presence of an Akt inhibitor intermittently in vitro
  • the T cells are ciiitured in the presence of the Akt inhibitor for the entire duration of in vitro culture, including during expansion of the numbers of cells 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 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 ceils of a different state of development or maturation.
  • the cancer antigen can be additionally expressed by ceils 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 ceils are not normally found in an adult host, Examples of cancer a tigens include, but are not limited to, mesothelin, CD!
  • the cancer antigen may be a mutated antigen that is expressed or overexpressed by tumor or cancer cells and which is not expressed by norma!., non-tumor, or non-cancerous cells.
  • T cells having antigenic specificity for a cancer antigen may, advantageously, reduce or avoid cross-reactivity with normal tissues such as. for example, that which may occur using T cells having antigenic specificity for minor histocompatahiiity antigens.
  • the cancer antigen can be an antigen expressed by any cell of any cancer or tumor, including the cancel's and tumors described herein.
  • the cancer antigen may be a cancer antigen of only one type of cancer or tumor, such thai the cancer antigen is associated with or characteristic of only one type of cancer or tumor.
  • the cancer antigen may he 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 ceils in the presence of one or more non-specific ⁇ cell stimuli, one or more cytokines, and an Aki inhibitor.
  • non-specific T cell stimuli include, but are not limbed to, one or more of irradiated allogeneic feeder cells, irradiated autologous feeder cells, anti-CD3 antibodies, anii-4-lBB antibodies, and anti-CD28 antibodies.
  • the non-specific T cell stimulus may be anti-CD3 antibodies and anti ⁇ CD28 antibodies conjug ted 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, IL-21 , and IL- 15.
  • IL interleukin
  • IL-7 interleukin-7
  • IL-21 IL-21
  • IL- 15 IL- 15
  • the Akt inhibitor for expanding the numbers of ceils may be as described herein with respect to other aspects of the invention.
  • Expansion of the numbers of T ceils 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.
  • the numbers of T cells are expanded by physically contacting the T cells with one or more non-specific T cell stimuli and one or more cytokines in the presence of the Akt inhibitor.
  • expansion of the numbers of T cells may be carried out by culturing the T cells with OKT3 antibody, IL-2, and feeder PBMC (e.g., irradiated allogeneic PBMC) in the presence of the Akt inhibitor.
  • expanding the number of T ceils in the presence of the Akt inhibitor comprises cuiturisig the cells for at least about 14 days in the presence of the Akt inhibitor.
  • the invention further provides an isolated or purified population of T cells produced by any of the inventive methods.
  • the Akt inhibitor- tre ted populations of T cells produced by the inventive methods may provide many advantages.
  • the Akt inhibitor-treated populations of T cells produced by the inventive methods may be less differentiated as compared to control T cells, wherein the control T cells are identical to the T cells that were cultured in the presence of an Akt inhibitor except that the control T cells were not cultured in the presence of an Akt inhibitor.
  • the less differentiated populations of Aki inhibitor-treated T cells produced according to 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.
  • expansion of the numbers of T cells in the presence of an Akt inhibitor in accordance with the inventive methods may reduce or avoid the production of T ceils with a terminally differentiated phenotype that is associated with diminished antitumor activity and poor capacity for long-term persistence in vivo.
  • the Akt inhibitor-treated population of T cells produced by the inventive methods have a naive T cell ( ' IV).
  • the Akt inhibitor- treated population of T cells lacks an effector memory T cell (TE ) phenotype.
  • CCR7 and CD62L are expressed by T N , TSCM, and TCM ceils, but are not expressed by TEM cells.
  • the transcription factors LEF1 , FOXP1, and KLF7 are expressed by TN, TSCM, and TCM eelis, but are not expressed by TEM cells.
  • CD45RO and KLRG1 are not expressed by TN or TSCM ceils, but are expressed by TEM cells. Gattinoni et al., Nat. Rev. Cancer, 12: 671-84 (2012).
  • the Akt inhibitor-treated isolated or purified T cell of the invention may be any one or more of CD62L 1 , LRG1 " , LEF1 , ⁇ , and KLF7 + , and CD45RO " .
  • the T cells may be CD62I,' " .
  • the T ceils may be CDS ' .
  • the Akt inhibitor-treated population of T cells is both CD62L ⁇ and CD8 + .
  • T N , TSCM, and TCM ceils may be characterized by longer telomeres as compared to those of EM cells.
  • the Akt inhibitor-treated population of T cells produced according to the inventive methods has an increased expression of genes associated with a ' I ' M, TS M, or TC phenotype.
  • the Akt inhibitor-treated population of T ceils may have an increased expression of any one or more of 117r, Sell. CD28, CD27, Foxol , !f2, Tcf7, and Let! mRNA or protein as compared to control ⁇ cells, wherein the control ⁇ cells are as described herein with respect to other aspects of the invention.
  • the Akt inhibitor-treated populations of T cells produced according to the inventive methods may have a higher expression of CD27 and/or CD2S as compared to control T cells.
  • CD27 and CD28 are associated with proliferation, in vivo persistence, and a less differentiated state of T cells ⁇ the increased differentiation off cells is believed to negatively affect the capacity of T cells to function in vivo).
  • T cells expressing higher levels of CD27 are believed to have better antitumor activity than CD27-low cells.
  • the Akt inhibitor-treated population of T cells produced accordmg to the inventive methods has a decreased expression of genes associated with a TE phenotype.
  • the Akt inhibitor-treated population of T cells may have a decreased expression of any one or more of Ifng, Homes, Prfl , GzmB, and Klrgl mR A or protein as compared to control T ceils, wherein the control T cells are as described herein with respect to other aspects of the invention.
  • the Akt inhibitor-treated population of T cells produced according to the inventive methods has a decreased extracellular acidification rate as compared to control T ceils, wherein the control T cells are as described herein with respect to other aspects of the invention.
  • 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.
  • the purity can be at least about 50%, can be greater than 60%, 70% or 80%, 90% or can be 100%.
  • the Akt inhibitor-treated population of T cells produced by the inventive methods can be a heterogeneous population comprising the T cells described herein, in addition to at least one other cell, e.g. , a cell other than a T cell, e.g., a B cell, a macrophage, a neutrophil, an erythrocyte, a hepatocyte, an endothelial cell, an epithelial cell, a muscle cell, a brain cell, etc.
  • the Akt inhibitor-treated population of cells can be a substantially homogeneous population, in which the population comprises mainly T ceils described herein.
  • the Akt inhibitor-treated population also can be a clonal population of cells, in which ail celis of the population are ations of a singie T ceil.
  • the population of celis is a clonal population comprising T cells comprising a recombinant expression vector encoding the exogenous TCR or CAR as described herein.
  • inventive isolated or purified Akt inhibitor-treated population of T ceils produced according to any of the inventive methods described herein may be included in a composition, such as a pharmaceutical composition.
  • the invention provides a pharmaceutical composition comprising the Akt inhibitor --treated isolated or purified population of T ceils described herein and a pharmaceutically acceptable carrier.
  • the carrier is a pharmaceutically acceptable carrier.
  • the earner 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 of the pharmaceutical composition of the invention may include any of those for parenteral, subcutaneous, intravenous, intramuscular, intraarterial, intrathecal, intraturnoral, or interperitoneal administration. More than one route can be used to administer the Akt inhibitor-treated population of T ceils, and in certain instances, a particular route can provide a more immediate and more effective response than another route.
  • the Akt inhibitor-treated population of T cells are administered by injection, e.g., intravenously.
  • a suitable pharmaceutically acceptable carrier for the cells tor 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, DeerfiekL IL), about 5% dextrose in water, or Ringers lactate.
  • the pharmaceutically acceptable carrier is supplemented with human serum albumen.
  • the dose e.g., number of Akt inhibitor-treated T ceils administered should be sufficient to effect, e.g., a therapeutic or prophylactic response, in the mammal over a reasonable time frame.
  • the number of Akt inhibitor- treated 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 Akt inhibitor-treated T cells administered will be determined by, e.g., the efficacy of the particular population of T cells to be administered and the condition of the animal (e.g., human), as well as the body weight of the animal (e.g., human) to be treated.
  • an assay which comprises comparing the extent to which target ceils are lysed or one or more cytokines such as, e.g., IFN- ⁇ and IL-2 is secreted upon administration of a given number of such T ceils 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.
  • cytokines such as, e.g., IFN- ⁇ and IL-2
  • target cells are lysed or cytokines such as, e.g., IFN- ⁇ 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 provide an indication of the quality (e.g., phenotype and/or effectiveness) of a T cell preparation.
  • the number of Akt inhibitor-treated T ceils administered also will be determined by the existence, nature and extent of any adverse side effects thai might accompany the administration of a particular population of T cells. Typically, the attending physician will decide the number of T cells with which to treat each individual patient, taking into consideration a variety of factors, such as age, body weight, general health, diet, sex, route of administration, and the severity of the condition being treated. By way of example and not intending to limit the invention, the number of Akt inhibitor-treated T cells to be
  • administered can be about 10 x 10 6 to about 10 x 10 1 1 cells per infusion, about 10 x 1 0 cells to about 10 x ID 1 1 cells per infusion, or 10 x 10 ' ' to about 10 x 10 9 cells per infusion.
  • the Akt inhibitor-treated populations of T ceils produced according to the inventive 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 described herein in an amount effective to treat or prevent cancer in the mammal.
  • One or more additional therapeutic agents can he coadministered to the mammal
  • coadministering is meant administering one or more additional therapeutic agents and the Akt inhibitor-treated isolated population of T cells sufficiently close in time such that the Akt inhibitor- treated isolated population of T cells can enhance the effect of one or more additional therapeutic agents, or vice versa, in this regard, the Akt inhibitor- treated isolated population of T cells can be administered first and the one or more additional therapeutic agents can be administered second, or vice versa. Alternatively, the Akt inhibitor-treated isolated population of T ceils and the one or more additional therapeutic agents can he administered simultaneously.
  • Additional therapeutic agents that may enhance the function of the Akt inhibitor-treated 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 Akt inhibitor-treated isolated population of T cells is 1L-2. Without being bound to a particular theory or mechanism, it is believed that IL-2 may enhance the therapeutic effect of the Akt inhibitor- treated isolated population of T cells,
  • An embodiment of the invention further comprises b/mphodepietmg the mammal prior to administering the Akt inhibitor-treated isolated population of T cells.
  • lymphodepletion include, but may not be limited to, nonmyeloablative lymphodepieting chemotherapy, myeloabiative lymphodepieting chemotherapy, total body irradiation, etc. 10041 ]
  • the terms "treat,” and “prevent” as well as words stemming therefrom, as used herein, do not necessarily imply 100% or complete treatment or prevention. Rather, there are var ing degrees of treatment or prevention of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. n this respect, the inventive methods can provide any amount of any level of treatment or prevention of cancer in a mammal.
  • 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., Hodgkin lymphoma and non-Hodgkin lymphoma), hepatocellular carcinoma, glioma, head-neck cancer, acute lymphocytic cancer, acute myeloid leukemia, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, or anorectiim, 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,
  • leukemia e.g.
  • Akt inhibitor (Akti) (PubChem CID: 10396499; MF: C34H39N7O; MW: 551.64 g/mol) was purchased from Merck Millipore (catalog # 124018) and prepared at clinical- grade in collaboration with the Pharmaceutical Development Section at the National Institutes of Health (NIH) Clinical Center Pharmacy Department. Therapeutic concentration of Akt inhibitor used in experiments was 1 ⁇ .
  • the i.UPAC name of Akt inhibitor is 3-[ l -
  • T cell medium includes AIM V maximra. plus 5% AB semm.
  • R 10 medium includes Rosweli Park Memorial Institute (RPM1) 1640 pins 10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 pg/mL streptomycin and 2 niM L-glutamine.
  • PBMCs were suspended at a concentration of 1 x 10" cell per ml in T cell medium containing 50 rsg/ml. of the anti-CD3 monoclonal antibody OKT3 (Ortho,
  • RETRONECTIN solution was aspirated and 2 mL of a blocking solution including Hanks' balanced salt solution (HBSS) plus 2% bovine serum albumin (BSA) was added to each RETRONECTIN solution-coated well. The plates were incubated for 30 minutes at. RT. The blocking solution was aspirated, and wells were rinsed with a solution of HBSS T 2.5% HEPES. Retroviral supernatant was rapidly thawed and diluted 1 : 1 in T cell media.
  • HBSS Hanks' balanced salt solution
  • BSA bovine serum albumin
  • the plates were eentrifuged at 2000xg for 2 hours at 32 °C. The supernatant was then aspirated from the wells, and 2x 10" T cells that had been cultured with OKT3 and IL-2 for 2 days were added to each well.
  • the T cells were added to the retrovirus-coated plates, they were suspended at a concentration of 0.5x 10* cells per ml, in T cell medium plus 300 IU/ mL of IL-2. After the T cells were added to each well, the plates were eentrifuged for 10 minutes at 1000*g. The plates were incubated at 37 °C overnight.
  • T cells were removed from the plates and suspended in fresh T cell medium with 300 lU/mL of IL-2 at a concentration of 0.5x 10° cells per mL and cultured at 37 °C and 5% C ⁇ [0049]
  • FACs buffer phosphate-buffered saiine (PBS) plus 0.1% sodium azide and 0.4% BSA.
  • Fc receptors were blocked with normal goat IgG (Inviirogen).
  • Biotin-labeled polyclonal goat arrti--niouse ⁇ F(ab)2 antibodies were added to one tube to detect the FMC63 scFv.
  • the ceils were incubated at 4 °C for 25 minutes and washed once.
  • the cells were suspended in FACs buffer and blocked with normal mouse IgG (Inviirogen).
  • the ceils were then stained with phycoerythrin (PE)-labeled streptavidin (BD Phanningen, San Diego. CA) and allophycocyanin (APC)-labeled CD3 (eBiocience, San Diego, CA).
  • Flow cytometry was performed with a BD FACS JAZZ ceil sorter (BD
  • mice [00501 ttOD.Cg-Prkdd*" Il2rg' m> m fSz3 (NSG) mice were purchased from The Jackson Laboratory. Mice were housed in the NIH Clinical Research Center vivarium and maintained in compliance with the ' NIH Animal Care and Use Committee. Mice were excluded from analysis if less than 6 weeks old and not age- and gender-matched with experimental cohort. Mice were randomized to treatment group and investigators blinded when measuring outcomes of tumor burden. The following CD19-expressing immortalized cell lines were used: NALM-6 (acute lymphoid leukemia from DSMZ, Braunschweig. Germany), Toledo (B cell diffuse large cell lymphoma from ATCC), and CD!
  • NALM-6 acute lymphoid leukemia from DSMZ, Braunschweig. Germany
  • Toledo B cell diffuse large cell lymphoma from ATCC
  • NOD.Cg-iV " ⁇ U2rg mS WJI !SzS (NSG) mice were injected in tail vein with 2 ⁇ l O 6 NALM-6 cells stably-transduced with firefly iuciferase.
  • tumor-bearing mice were treated with 1 10" human CAR-expressing cells injected by tail vein that had been expanded in the presence or absence of Akti as described above.
  • mice were injected i.p. with 150 mg kg "1 body weight D- luciferin (Caliper Life Sciences) and imaged 10-12 minutes later using a XE OGEN SPECTRUM system and LIVING IMAGE v3.2 software (Caliper Life Sciences).
  • PBMCs Human PBMCs from three healthy donors were isolated, and the numbers of cells were expanded ex vivo as described above. After 10 days expansion, T lymphocytes were enriched for the CD8 'r population by MILTENY1 magnetic column separation (order no. 130- 096 -495) according to the manufacturer's instructions. RNA (100 ng) was extracted from CD8 + CAR-expressing cells using OVATION PICO WTA System V2 (NuGEN) according to the manufacturer's instructions.
  • first-strand cDNA was synthesized using the SPIA tagged random and oligo dT primer mix in 10 ⁇ ] reactions after denaturation and incubated at 65 °C for 2 minutes (min) and priming at 4 °C followed by extension at 25 °C for 30 min, 42 °C for 15 min and 77 °C for 15 min.
  • Second strand cDNA synthesis of fragmented RNA was performed using DMA polymerase at 4 °C for i min, 25 °C for 10 nun, 5 0°C for 30 min and 80 °C for 20 min.
  • 5' double stranded cDNA was used as the template for isothermal single-strand cDNA amplification following a cycle of DNA/RNA primer binding, ON A replication, strand displacement and R A cleavage at 4 °C for 1 min, 47 °C for 75 min and 95 °C for 5 rnin in total 100 ⁇ reaction.
  • Samples were fragmentated and biotinylated using the ENCORE Biotin Module (NuGEN) according to the manufacturer's instructions. Biotinylated cDNA was then hybridized to Human Gene 1.0 ST arrays (Asymetrix) overnight at 45 °C and stained on a GENECHIP Flmdics Station 450
  • CAR-expressing cells from 3 patients were enriched using MILTENYI magnetic column CDS " separation (order no. 130- 096-495) according to the manufacturer's instmctions.
  • mice per treatment group A sample size of five mice per treatment group was used to detect an effect size in all experiments unless otherwise indicated. Data assumed to have a normal distribution and differences between two groups were assessed with unpaired, 2-tailed t tests. P values less than 0.05 were considered significant. The measure of central tendency is mean and variation is SEM unless otherwise stated. All experiments were replicated at least twice in laboratory unless otherwise indicated.
  • Akti clinical -grade Akt inhibitor
  • Akt inhibition may limit ex vivo expansion of the numbers of human anti-CD 19 CAR-expressing cells because therapeutic efficacy has previously been shown to correlate with absolute number of adoptively- transferred T cells.
  • PBMCs were stimulated and transduced in the presence of Akti or absence of Akti for 10 days. The cellular number expansion was comparable between CAR-expressing cells cultured in the presence of Akti and CAR- expressing cells cultured in the absence of Akti in the three healthy donors evaluated.
  • Akt inhibition compromises a.oti-CD19 CAR transduction efficiency or capacity to recognize tumor targets.
  • the transduction efficiency was comparable between CAR-expressing cells cultured in the presence of Akti and CAR- expressing ceils cultured in the absence of Akti.
  • Akti-treated CAR-expressing cells released similar levels of interferon-gamma ( ⁇ ⁇ ) compared to CAR-expressing cells cultured in the absence of Akti upon co-culture with CD 19-expresssing tumor cells (Toledo and K562- CD1 ). No more than background ⁇ was measured when the cells were co-cuitured with CD19-negative cells CEM or K562.
  • Akti-treated CAR-expressing cells may have enhanced long-term persistence after adoptive-transfer.
  • cells expanded in the presence or absence of Akti were transferred into NOD.Cg- Prkdc d Il2rg l" " ' Wjl /Szl (NSG) mice.
  • Akti-treated CAR-expressing cells demonstrated superior engraftment and persistence 30 days after transfer as compared to CAR-expressing cells that were not treated with Akti. Therefore, Akt inhibition promotes CD62L expression and survival of CAR-expressing cells in a humanized mouse model and may represent a clinically-feasible method to prolong anti-tumor immunity of CAR-expressing cells by enhancing persistence after adoptive transfer,
  • This example demonstrates di l inhibition of Akt in human anti-CD 19 CAR- expressing cells promotes a transcriptional signature of memory T cells.
  • Expression of CD62L identifies antigen-experienced cells with a central memory phenotype (Klebanoff et al., PNAS, 1 02(27): 9571 -6 (2005); Klebanoff et a!., Immunol Rev., 21 1 : 214-24 (2006); Klebanoff et aL Clin. Cancer Res., 17(16): 5343-52 (2011)).
  • PCA principle component analysis
  • GSEA Gene Set Enrichment Analysis
  • Akti-treated CAR-expressing ceils a decrease in key glycolytic metabolites (such as, for example, 3 -phosphogiycerate, mannitol/sorbitoi, lactate, and phosphoenolpyruvate (PEP)) was noted.
  • GSEA phosphoenolpyruvate
  • Akt inhibition limits glycolysis but not cell expansion, it was sought to determine whether Akti-treated CAR-expressing cells rely on alternative metabolic programs to fuel cell proliferation and effector function. It was noted that Akt inhibition was associated with a significant disruption of amino acid metabolism.
  • tumor fragments or digests were cultured in 6,000 IU/mL TL-2 for 2 weeks, and subsequently expanded with a rapid expansion protocol using 30 ng/mL OKT3 (anti-CD3) antibody (Miltenyi Biotee) and 6,000 lU/mL IL2 in the presence of irradiated (50 Gy) allogeneic feeder cells at a 200: 1 ratio of feeder cells to TIL, TIL culture media was supplemented with 1 ⁇ /L Akt inhibitor (PubChem Compound Identification: 10196499, also called Akt inhibitor VIII or Akti-1/2) purchased from Calbiochein. TIL were harvested for myriad assays 30 days after initiation of culture, including FACS analysis, coculture with tumor targets, microarray analysis, adoptive -transfer into NSG mice, and metaboloniic analysis,
  • Thyl .1 and Ly5.1 Pmel-ITCR- transgenic (Pmel) mice have been described in Overwijk et al, J. Exp. Med., 198: 569-80 (2003).
  • KOO.Cg-Prkdc scid I!2r m /$iJ (NSG) mice and C57BL/6 N (B6) mice were purchased from The Jackson Laboratory and NCI Frederick.
  • Mice were housed in the NIK Clinical Research Center vivarium and maintained in compliance with the NIH Animal Care and Use Committee. Mice were excluded from analysis if less than 6 weeks old and not age- and gender matched with experimental cohort. Mice were randomized to treatment group and investigators blinded when measuring outcomes of tumor size, survival after adoptive transfer, and histopathologic analysis.
  • Splenocytes from Pmei mice were stimulated with peptide (1 umol/ ' L) and 100 ILT/mL recom inant human IL2 (rhIL-2;Novartis) in the presence or absence of I ⁇ L Akti-1/2 (Akti; Calbiochem) and CD8 + T cells were harvested at day 5. Secondary stimulation was performed using peptide -pulsed irradiated B6 feeder ceils.
  • the B16F10 tumor line (B16) was obtained from the National Cancer Institute tumor repository and tested for mycoplasma contamination.
  • cytokine production assays coculture of ' HI, with autologous tumor was performed and superaaiants assessed for the presence of gamma-interferon (TFNg) by enzyme-linked immunosorbent assay (ELISA) in accordance with the manufacturer's proioeol.
  • Tumor therapy was performed as described in Ovexwijk et al, J, Exp. Med., 198: 569-80 (2003). Briefly, Pmei splenocytes were stimulated with hgpl00 2 j-33 peptide and expanded in rhiL-2 (1.000 IU/ml) to model clinical protocol of expanding human T L in high-dose IL-2. Expanded splenocytes (2xl0 6 ) were subsequently transferred into irradiated (6 Gy) B6 mice with established subcutaneous B16 melanoma tumor and WhgplOO (l e7 pfu) was administered upon transfer.
  • Intraperitoneal injections of rhIL-2 were administered twice daily for 3 days after transfer.
  • 1x10° cells were adoptively transferred with coadministration of WhgplOO (I e7 pfu) into irradiated (6 Gy) B6 mice after ex vivo stimulation (with hgp 100 25 -33 peptide) and expansion in 100 IU/ml rhIL-2.
  • Transferred cells were enumerated with hemoeytometer and FACS staining with conjugated antibodies (all from BD
  • CDS catalog number 557654
  • CD27 560691
  • CD62L 55315.1
  • Thyl .1 557266
  • LyS. i 553775
  • Microarray analysis 0082 Human TIL from 3 patients were isolated and expanded ex vivo as described in "Adoptive Ceil Transfer" for Examples 6-10 above. After 30 days expansion, T lymphocytes were enriched for the CD8 + population by Miitenyi magnetic column separation (order no. 130-096-495) according to the manufacturer's instructions. RNA ( 100 ng) was extracted from CD8 + TILs using OVA TION PICO WTA System V2 (NuGEN) according to the manufacturer's instructions.
  • first-strand cDNA was synthesized using the SPIA tagged random and oligo dT primer mix in 10-mL reactions after denaturation and incubated at 65 °C for 2 minutes and priming at 4 °C followed by extension, at 25 °C for 30 minutes, 42 °C for 15 mmutes and 77 °C for 15 mmutes.
  • Second- stra d cDNA synthesis of fragmented RNA was performed using DNA polymerase at 4 °C tor 1 minute, 25 °C for 10 minutes, 50 °C for 30 minutes, and 80 °C for 20 minutes.
  • Arrays were scanned on a GENECHIP Scanner 3000 7G (Affymetrix).
  • Global gene expression profiles were rank ordered by relative fold-change values and analyzed by using GENE SET Enrichment Analysis (GSEA) software (Broad Institute, MIT). P values were calculated using the Student t test using PARTEK Genomic Suite after Robust MuUiarray Average normalization.
  • GSEA GENE SET Enrichment Analysis
  • Oxygen consumption rate was measured at 37 °C using an XF24 extracellular analyzer (Seahorse Bioscience) as described in Ferrick et al. ; Drug Discov. Today, 13: 268-74 (2008). Briefly, TIL were initially plated with XF media (nonbuffered RPMI-1640 containing 25 mmol/L glucose, 2 mraol L L-glutamme, and 1 mmol/L sodium pyruvate) and incubated in a non-COj incubator for 30 minutes at 37 °C.
  • XF media nonbuffered RPMI-1640 containing 25 mmol/L glucose, 2 mraol L L-glutamme, and 1 mmol/L sodium pyruvate
  • OCR OCR was measured under basal conditions and in response to injection port-administration of the following compounds at indicated time points: 1 ⁇ / ⁇ , oligomyem, 1.5 ⁇ L f.uorocarbonyl cyanide pheny!hydrazone (FCCP), 100 nmol/L rotenone, and 1 pmol/L antimycin A.
  • FCCP f.uorocarbonyl cyanide pheny!hydrazone
  • TIL were enriched using Miltenyi magnetic column CDS ' separation (order no. 130-096-495) according to the manufacturers instructions.
  • Splenocytes were harvested on day 10 and CD8 + T cells were enriched using a MACS Negative Selection Kit (Miltenyi Biotec order no. 130-104-075).
  • mice per treatment group A sample size of five mice per treatment group was used to detect an effect size in all experiments unless otherwise indicated. Data assumed to have a normal distribution and differences between two groups were assessed with unpaired, two-tailed t tests. Comparisons involving more than two groups were assessed using an A OVA. P values less than 0.05 were considered significant. The measure of central tendency is mean and variation is SEM unless otherwise stated. All experiments were replicated at least twice in laboratory with the exception of the histopathologic analysis of MSG mice receiving either Akti-treaied CTL or control CTL thai were not treated with Akti.
  • This example demonstrates thai inhibition of Akt promotes expression of CD62L in human tumor-specific cytotoxic lymphocytes without compromising cell expansion.
  • Akt inhibition affects expression of CD62L on the surface of human TIL
  • Akti allosterie inhibitor of all three isoforms of Akt
  • TIL When measured at acute time points after TCR stimulation, it was found that phosphorylation of Akt is inhibited at both the serine 473 and threonine 308 residues, resulting in decreased phosphorylation of its downstream substrates ribosomal protein S6 and glycogen synthase kinase 3 beta (GSK3p), TIL were then isolated from 3 patients with metastatic melanoma as described in Dudley et al., J. Immunolher., 26: 332-42 (2003), expanded the numbers of them at clinical-scale in the presence or absence of Akti, and measured surface expression of CD62L on CD4 + and CDS '1" T cells.
  • Akti-expanded TIL had significantly increased expression of CD62L. No effect of Akt inhibition on the expansion of the numbers of TIL was observed. Finally, it was sought to determine if Akt inhibition compromises tumor specificity or capacity to release IFNg when cocultured with autologous tumor cells. Akti- treated TIL released similar levels of IFNg compared with control TIL (not treated with Akti) that was restricted in an MHC class I-dependent maimer. Thus, pharmacologic inhibition of Akt enabled expansion of the numbers of TIL expressing elevated levels of CD62L without affecting their expansion or capacity to release IFNg upon recognition of tumor targets.
  • CD62L distinguishes antigen-experienced cells with a central memory phenotype (Graef et ah, immunity, 41 : 1 1 6-26 (2014)). in addition to their lymphoid homing capacity, these cells exhibit enhanced capacity for cell survival and proliferation. Given elevated expression of CD62L on the surface of Akti-expanded TIL, it was asked whether Akt inhibition causes global changes in gene transcription that are characteristic of memory ceils. To visualize the transcriptome of CDS ' TIL, principle component analysis ⁇ PC A) of microarray data was performed. Segregation of treatment groups among the 3 patients under study was observed.
  • LB.F.I, TCF ' J, KLF2, IL7R, SELL, CD28, and C.D27 were upregulated with Akt inhibition, whereas effector-associated genes such as CD500A, XCL1, XCL2, IF.NG and KLRG1 were suppressed (Kaech et al., Nat. Rev. Immunol., 12: 749--61 (2012)).
  • GSEA was performed using genes upregulated in human naive (CD62V CD45RA compared with effecior memory (CD62L-CD45RO 'r ) CD8 T cells isolated from healthy donors.
  • thai memory T cells have metabolic qualities such as reduced glycolysis (Sukumar et al.. J Clin. Invest. , 123: 4479-88 (2013)) and enhanced mitochondrial fatty acid oxidation (FAO) that support long term survival and effector function (Pearce et al., Nature, 460: 103-7 (2009)). Having shown thai pharmacologic inhibition of Akt in CD8 + TIL promotes a transcriptional profile characteristic of memory T ceils, it was asked whether Akt inhibition affects metabolism of human CDS * TiL.
  • Akti-treated CD8 'r TIL showed accumulation of both long chain and polyunsaturated fatty acids (eicosenoate, erucate, iinoleate, arachidonate, eicosapentaenoate, and oleate) that may be a result of membrane lipid turnover used to fuel FAO, This interpretation was supported by accumulation of phospholipid catabolites and elevated lysolipid levels (glycerophosphoryleholiiie, 1-eicosenoyl
  • Akt- treated TIL was merely dite to decreased cell growth and expansion, but this seems less likely because no difference in absolute cell numbers after culture was observed.
  • inhibition of Akt during expansion of TIL does not affect the abundance of glycolytic metabolites, it resulted in changes in abundance of metabolites involved in FAO,
  • Spare respiratory capacity is a measure of the bioenergetic ability of mitochondria to produce additional energy under conditions of increased stress or work and is thought to be involved in the long-term survival and function of diverse cell types.
  • Mitochondriai SRC may provide for the longe vity of memory CDS' ' T cells (van der Windt ei al,, PNAS, 1 10: 14336-41 (201 3)). Because SRC in memory CDS* T cells may be dependent on mitochondrial FAO, it was sought to determine whether Akt inhibition augments SRC in human TIL. By using an extracellular flux analyzer, the metabolism of therapeutic TIL was characterized in real time by measuring 0 2 consumption rates (OCR), an indicator of oxidative phosphorylation (OXPHOS; Ferrick et al, Drug Discov. Today 13: 268-74 (2008)). It was found that Akti-treated TIL had slightly higher basal OCR when compared with vehicle-treated TIL.
  • OCR 0 2 consumption rates
  • TIL were challenged with a well-established "mitochondrial stress test' ' in which oligomycin (to block ATP synthesis) is added after measurement of basal OCR, followed by fksorocarhonyl cyanide phenylbydrazone or FCCP (to uncouple AT? synthesis from the electron transport chain, ETC), and finally by coadministration of rotenone and antixnyem A (to block complex I and III, respectively, of ETC).
  • Akti-treated TJ.Ls demonstrated a considerably higher mitochondrial SRC compared with vehicle-treated controls as indicated by the difference between basal OCR and maximal OCR (after FCCP injection).
  • Akti-treated human TIL Superior engraftment and persistence of Akti-treated cells as compared to vehicle-treated cells was observed 30 days after transfer in both lymphoid and noniymphoid organs.
  • Akti-treated human TIL have enhanced persistence, and this correlates with phenotypic, metabolic, and transcriptional features of memory.
  • Akti-expanded tumor-specific T cells The persistence and antitumor function of Akti-expanded tumor-specific T cells was evaluated using the Pmel-1 mouse model in which transgenic T cells express a T-cell receptor specific for the melanoma-associated antigen, hgpl OO, widely expressed in B16 melanoma (Overwijk et al, J. Exp, Med., 198: 569-80 (2003)). Consistent with the findings in TIL from, patients with melanoma, it was found that Akt phosphorylation is inhibited at both the serine 473 and threonine 308 residues, resulting in decreased phosphorylation of its downstream target ribosomal protein S6. Akti-treated Pmel-1 T cells showed a gene- expression profile and surface phenotype reminiscent of long-lived memory T cells.
  • Akt inhibition On the cell-intrinsic capacity' of tumor- specific CDS T cells to engraft and persist following adoptive transfer, a 1 : 1 mixture of vehicle and Akti-expanded Pmel-l TCR-transgenic (ig) cells that can be distinguished using congenic markers were cotransferred. Their kinetics were tracked following adoptive transfer into mice. Akti-treated cells expanded to greater numbers as compared to vehicle-treated cells. Akti-treated cells persisted to form a long-lived population of memory cells that could be detected in lymphoid and rsonlymphoid organs 600 days following adoptive transfer.
  • Akti-treated CD8 T T cells were individually transferred into sublethaily ablated recipients bearing established B 16 melanoma tumors. Tumor growth and survival were measured following transfer. Consistent with human TIL, Akti-treated Pmei- T cells exhibited superior persistence and trafficking to the tumor microenvironment compared to vehicle-treated T cells. Akti-treated Pmel- l T ceils produced similar levels of IFNg compared with vehicle-treated cells that correlated with decreased tumor growth and improved survival upon transfer into the tumor-bearing mice. Thus, consistent with their transcriptional and metabolic characteristics, Akti-expanded cells exhibit enhanced persistence upon adoptive transfer that correlates with augmented tumor regression and survival following ACT.
  • This example demonstrates a method of treating or preventing cancer in a human patient, the method comprising producing an isolated population of T cells for adoptive cell therapy, the method comprising eulturmg isolated T cells in vitro in the presence of an Akt inhibitor, and administering the Aki-inhibitor-treated cells to the patient.
  • TIL tumor necrosis factor
  • the culture media will be supplemented with 1 ⁇ AKT inhibitor in half of tumor fragments (approximately 12 per ceil culture condition) from which TIL are isolated.
  • TIL tumor fragments
  • the numbers of TIL will be rapidly expanded (REF).
  • Culture media lacking Aki irsliibiior and AKTi-suppiemented media will be used tor each respective TIL group for the entirety of ex vivo culture.
  • AKTi-treated TIL will be pooled together and all control TIL will be pooled separately so that, the clonotypie diversity of each treatment group of TIL can be characterized with high-throughput deep sequencing of the TCR V-beta CDR3 region.
  • TIL from each treatment group will be enumerated and combined after normalization to a i : 1 ratio for die infusion bag.
  • AKTi-treated TIL will undergo a thorough washing to remove residual AKTi in media. Once cells exceed the potency requirement and are projected to exceed the minimum number for ACT
  • the patient will receive a lymphocyte depleting preparative regimen, followed by infusion of between 1x10' to 2x10 lymphocytes including of 1 : 1 mixture of control and AKTi-treated TIL, and high dose aldesleukin.
  • Patients will be evaluated for response approximately 4-6 weeks following the administration of the cell product.
  • Patients will receive one course of treatment.
  • the siart date of the course will be the start date of the chemotherapy; the end date will be the day of the first post-treatment evaluation.
  • Patients may undergo a second treatment.
  • Clinical response will be determined using the Response Evaluation Criteria in Solid Tumors (RECiST) guideline.

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Abstract

La présente invention concerne des procédés de production de populations isolées de lymphocytes T pour thérapie cellulaire adoptive, le procédé comprenant la culture de lymphocytes T isolés in vitro en présence d'un inhibiteur de protéine kinase B (Akt), 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.
PCT/US2016/057307 2015-10-20 2016-10-17 Procédés de production de populations de lymphocytes t à l'aide d'inhibiteurs d'akt WO2017070042A1 (fr)

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WO2020096927A1 (fr) * 2018-11-05 2020-05-14 Iovance Biotherapeutics, Inc. Expansion de til utilisant des inhibiteurs de la voie akt
WO2021026290A1 (fr) * 2019-08-07 2021-02-11 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Lymphocytes t ayant une activité antitumorale augmentée
US11111493B2 (en) 2018-03-15 2021-09-07 KSQ Therapeutics, Inc. Gene-regulating compositions and methods for improved immunotherapy
WO2021209759A1 (fr) * 2020-04-17 2021-10-21 Adaptimmune Limited Procédé amélioré de fabrication de lymphocytes t
CN113574172A (zh) * 2018-12-07 2021-10-29 亘喜生物科技（上海）有限公司 用于免疫疗法的组合物和方法
JP2022512915A (ja) * 2018-11-05 2022-02-07 アイオバンス バイオセラピューティクス，インコーポレイテッド 改良された腫瘍反応性ｔ細胞の選択
WO2023049862A1 (fr) * 2021-09-24 2023-03-30 Iovance Biotherapeutics, Inc. Processus d'expansion et agents pour lymphocytes infiltrant la tumeur

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Cited By (10)

* Cited by examiner, † Cited by third party
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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
WO2020096927A1 (fr) * 2018-11-05 2020-05-14 Iovance Biotherapeutics, Inc. Expansion de til utilisant des inhibiteurs de la voie akt
JP2022512915A (ja) * 2018-11-05 2022-02-07 アイオバンス バイオセラピューティクス，インコーポレイテッド 改良された腫瘍反応性ｔ細胞の選択
CN113574172A (zh) * 2018-12-07 2021-10-29 亘喜生物科技（上海）有限公司 用于免疫疗法的组合物和方法
EP3891272A4 (fr) * 2018-12-07 2023-03-29 Gracell Biotechnologies (Shanghai) Co., Ltd. Compositions et procédés d'immunothérapie
WO2021026290A1 (fr) * 2019-08-07 2021-02-11 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Lymphocytes t ayant une activité antitumorale augmentée
WO2021209759A1 (fr) * 2020-04-17 2021-10-21 Adaptimmune Limited Procédé amélioré de fabrication de lymphocytes t
WO2023049862A1 (fr) * 2021-09-24 2023-03-30 Iovance Biotherapeutics, Inc. Processus d'expansion et agents pour lymphocytes infiltrant la tumeur

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