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WO2020102726A1 - Procédé de fabrication de lymphocytes t/th2 régulateurs hybrides humains (lymphocytes treg/th2 hybrides) à partir de lymphocytes t dédifférenciés - Google Patents

Procédé de fabrication de lymphocytes t/th2 régulateurs hybrides humains (lymphocytes treg/th2 hybrides) à partir de lymphocytes t dédifférenciés Download PDF

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WO2020102726A1
WO2020102726A1 PCT/US2019/061811 US2019061811W WO2020102726A1 WO 2020102726 A1 WO2020102726 A1 WO 2020102726A1 US 2019061811 W US2019061811 W US 2019061811W WO 2020102726 A1 WO2020102726 A1 WO 2020102726A1
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cells
cell
treg
culture
population
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Daniel Harding FOWLER
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Rapa Therapeutics LLC
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Rapa Therapeutics LLC
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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
    • C12N5/0637—Immunosuppressive T lymphocytes, e.g. regulatory T cells or Treg
    • 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
    • C12N2501/10—Growth factors
    • C12N2501/15—Transforming growth factor beta (TGF-β)
    • 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
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2302—Interleukin-2 (IL-2)
    • 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
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2304—Interleukin-4 (IL-4)
    • 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
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/515—CD3, T-cell receptor complex

Definitions

  • Hybrid T REG /Th2 Method for Manufacturing of Human Hybrid Regulatory T/Th2 Cells
  • Adoptive T cell therapy is an emerging intervention for the effective treatment of cancer and infectious disease, auto-immunity, and neuro-degenerative disease. It is increasingly clear that the transfer of T cells with a more primitive differentiation state, which translates into a higher proliferative potential and other key attributes, is associated with improved in vivo effects after adoptive transfer.
  • most forms of adoptive T cell therapy require an ex vivo manufacturing step, which typically results in further cellular differentiation; this is particularly problematic, as T cells from the adult human are already primarily in an advanced state of differentiation (termed effector memory cells) and often exist in a senescent state that is under the control of checkpoint inhibitory molecules.
  • differentiated cells possess an inherent capacity for de- differentiation towards a more primitive state. Indeed, in the most extreme examples, differentiated cells can be manipulated to attain an induced pluripotent stem cell (iPSC) state, whereby such iPS cells share key characteristics with embryonic stem cells and can then be further modulated towards re-differentiation to divergent tissue fates; cellular therapy using such iPSC methodologies has numerous potential clinical applications.
  • iPSC induced pluripotent stem cell
  • Generation of iPS cells from differentiated somatic cells was initially demonstrated by the transfer of key transcription factors via viral or non-viral mediated approaches, including Sox2, Oct3/4, KLF4, and c-myc or Sox2, Oct3/4, Nanog, and Lin28.
  • the ability to convert somatic cells to iPS cells is inefficient and dependent in part upon the degree of somatic cell differentiation.
  • the ability to convert mature murine immune T cells into iPS cells is 300-fold less efficient relative to conversion of murine hematopoietic stem cells into iPS cells. Nonetheless, using gene transfer methods, it was demonstrated that mature human peripheral blood T cells maintain a capacity for conversion to an iPS cell state.
  • investigators have also characterized transcription factors associated with the earliest stages of T cell differentiation.
  • the re-differentiation of T cells from various types of stem cell precursors is a relatively inefficient process that typically takes one-to-two months.
  • rapamycin which is an immune suppression drug that inhibits the mammalian target of rapamycin (mTOR)
  • mTOR mammalian target of rapamycin
  • KLF2 transcription factor 2
  • rapamycin and resultant inhibition of mTOR signaling is critical for the maintenance of cellular quiescence in naive T cells having a reduced state of differentiation.
  • the mTOR pathway is comprised of both an mTORCl complex (which contains the Raptor sub unit) and the mTORC2 complex (which contains the Rictor sub-unit).
  • rapamycin can only directly inhibit mTORCl; however, with prolonged rapamycin-mediated inhibition of mTORCl, down-stream inhibition of mTORC2 can occur.
  • Reduction in T cell growth factor signaling via mTOR inhibition or other pathway inhibition is also known to up-regulate another key molecule associated with T cells of more primitive differentiation status, namely, IL-7 receptor alpha (CD127).
  • T cell mTOR pathway through the pharmacologic agent rapamycin or the Wnt-P-catenin signaling activator TWS11 promoted the de-differentiation of human naive T cells towards a less- differentiated, T stem cell memory population that was previously identified and characterized in murine and human T cells.
  • pharmacologic inhibition of the ART signaling pathway or combined inhibition of the PI3 kinase and vasoactive intestinal peptide signaling pathways resulted in the generation of T cells with a reduced differentiation status and increased T cell function upon adoptive transfer.
  • cytokine secretion molecules such as cytokine secretion molecules and cytolytic effector molecules.
  • Vitamin D can inhibit human T cell effector function.
  • the inhibitory effect of Vitamin D on human T cell proliferation can be synergistic with immune suppressive drug exposure using agents such as cyclosporine A or rapamycin.
  • agents such as cyclosporine A or rapamycin.
  • Vitamin D was shown to promote the immune suppressive regulatory T (TREG) cell population.
  • Vitamin D was critical for macrophage elimination of intracellular pathogens through a mechanism that involved IFN-g production and autophagy.
  • Vitamin D signaling can promote a cytotoxic form of autophagy that contributes to an anti-tumor effect when combined with radiation.
  • Vitamin D receptor signaling promotes autophagy in normal human mammary tissue; loss of such Vitamin D receptor signaling was associated with an increased risk of developing breast cancer.
  • TREG Regulatory T cells are essential for the maintenance of immune tolerance.
  • a reduction in the quantity or quality of TREG cells is a fundamental cause of a multitude of primary auto-immune diseases, including type I diabetes mellitus (T1DM), multiple sclerosis, rheumatoid arthritis, and systemic lupus erythematosus, to name just a few.
  • T1DM type I diabetes mellitus
  • TREG-deficiency has been associated with acceleration in the natural history of primary neurodegenerative diseases.
  • TREG-deficiency is associated with severe complications in the solid organ and hematopoietic cell transplantation setting, most notably, an increased rate of graft rejection and graft-versus-host disease (GVHD).
  • GVHD graft-versus-host disease
  • TREG cells which exist in two main sub-types: (1) natural (n) TREG cells, which are derived from the thymus, which involutes with age, thereby reducing the number of nTREG cells available for adoptive transfer; and (2) inducible (i) TREG cells, which are converted in the periphery from the more plentiful pool of effector T cells. Because nTREG cells are limiting in number, attempts to use nTREG cells for adoptive T cell therapy have relied upon ex vivo manufacturing methods for isolation and subsequent expansion of nTREG cells.
  • nTREG cells for adoptive cell therapy are in the early stages of implementation, primarily in phase I/phase II clinical trials for the prevention of GVHD and for the treatment of T1DM.
  • ITREG cells ex vivo produced TREG cells
  • peripheral effector T cells are relatively plentiful, they primarily exist in a state of effector memory maturation with limited replicative and therapeutic potential; and (2) such peripheral effector T cells have a high degree of pre-existing effector differentiation towards T cell subsets that contribute to disease pathogenesis, namely, the Thl- and Thl7-type subsets.
  • ITREG cell therapy is to become highly feasible, it will be necessary to develop an ex vivo manufacturing method that both: (1) causes a de-differentiation of effector T cells towards a less-differentiated memory phenotype that has an increased proliferative potential and demonstrable improvement in TREG cell therapeutic potential; and (2) extinguishes pathogenic Thl- and Thl7-type pathways while promoting T cell differentiation towards the TREG phenotype.
  • ITREG cells The manufacture of ITREG cells is initiated by collection of lymphocyte-containing peripheral blood mononuclear cells from the subject to be treated (in the case of autologous therapy) or from a normal donor (in the case of allogeneic therapy). Typically, this collection is performed in the steady-state, that is, without any growth factor administration; however, in the allogeneic context, collection is sometimes performed in the context of administration of molecules such as granulocyte colony stimulating factor (G-CSF) or plerixifor, as described in DiPersio JF, Stadtmauer EA, Nademanee A, et al.
  • G-CSF granulocyte colony stimulating factor
  • plerixifor as described in DiPersio JF, Stadtmauer EA, Nademanee A, et al.
  • the anti-TNF-a agent etanercept which is a recombinant receptor that preferentially inhibits serum, cell-free TNF-a with relative preservation of the cell surface, membrane-bound form of TNF-a, induces a global change in the T cell receptor (TCR) repertoire when measured by RNA sequencing.
  • TCR T cell receptor
  • membrane-bound TNF-a provides a positive signal to TREG cells through the TNFR2 receptor
  • our method offers a robust intervention to enrich for TREG cells prior to ITREG cell manufacturing.
  • Other therapeutics that preferentially inhibit serum, cell-free TNF-a can also be used for this intervention, including but not limited to the anti-TNF-a monoclonal antibody, adalimumab.
  • the present disclosure is directed to methods for de-differentiation of T cells and re- differentiation of such cells to TREG or TREG/T1I2 cells.
  • the initial de-differentiation method is can include of initiating the culture with an input cell populations harvested in the steady-state (without drug
  • the method comprises initiating the de-differentiation culture with an input cell populations harvested from a subject (in the autologous context) or a normal donor (in the allogeneic context) who has been or is being treated with an anti-TNF-a therapeutic agent that is preferentially selective for inhibition of the serum, cell-free form of TNF-a with relative preservation of membrane-bound TNF-a.
  • therapeutic agents include but are not limited to the recombinant receptor etanercept, which can be administered at the conventional dose of 25 or 50 mg per week by subcutaneous injection, or the monoclonal antibody adalimumab, which can be administered at the conventional dose of 40 mg per week or 40 mg every other week by intravenous injection.
  • the dosing of the anti- TNF-a therapeutic can be adjusted according to the desired biomarker change, which can include but is not limited to alteration of the TCR repertoire by RNA sequencing analysis and a shift towards type 2 TNF receptors (TNFR2) and a shift away from type 1 TNF receptors (TNFR1), as measured by flow cytometry.
  • the desired biomarker change can include but is not limited to alteration of the TCR repertoire by RNA sequencing analysis and a shift towards type 2 TNF receptors (TNFR2) and a shift away from type 1 TNF receptors (TNFR1), as measured by flow cytometry.
  • the method comprises inoculating a culture input population of cells comprising T cells from a subject at a cell density in a culture medium comprising vitamin D, temsirolimus and an IL-2 signaling inhibitor; adding anti-CD3/anti-CD28 coated magnetic beads to said T cells and culture medium at a relatively low bead:T cell ratio of 1 : 1 or less to stimulate said T cells, or, in the most extreme example, no addition of anti-CD3/anti-CD28 co stimulation; incubating said culture input population of cells and culture medium for a period of time to yield de-differentiated T cells. It is also possible to perform this de-differentiation procedure in the absence of any bead co-stimulation. [17] In any of the foregoing embodiments, the method may further comprise harvesting said de-differentiated T cells.
  • the method may further comprise, after harvesting said de-differentiated T cells: packaging at least a portion of said de-differentiated T cells in a package; and freezing said package containing said portion of said de-differentiated T cells.
  • the method may further comprise before inoculating said culture input population of cells into said culture medium: harvesting said culture input population of cells from said subject.
  • the method may further comprise measuring an expression level of RAPTOR or RICTOR in said culture input population of cells wherein said period of time lasts until the expression level of RAPTOR or RICTOR, respectively, in said culture input population of cells is reduced by at least 50% and more preferably 90% relative to a control population of T cells, and wherein said control population of T cells are manufactured under the same conditions as the culture input population of cells without temsirolimus, IL-2 signaling inhibitor and Vitamin D.
  • the method may further comprise measuring an expression level of RAPTOR or RICTOR and a housekeeping protein in said culture input population of cells, wherein said period of time lasts until the expression level of RAPTOR or RICTOR, normalized by the housekeeping protein, in the manufactured T cells is at least 50% and more preferably 90% lower than the expression level of RAPTOR or RICTOR, respectively, normalized by the housekeeping protein, in the control population of T cells manufactured under the same conditions as the culture input population of cells without temsirolimus, IL-2 signaling inhibitor and Vitamin D.
  • the present disclosure is also directed to a de-differentiated T cell produced by the methods of any of the foregoing embodiments.
  • the present disclosure is also directed to a composition
  • a composition comprising a population of de differentiated cells, wherein at least a portion of said population of said de-differentiated cells express at least 50% and more preferably 90% less of RAPTOR or RICTOR as compared to a control population of T cells manufactured under the same conditions as the culture input population of cells without temsirolimus, IL-2 signaling inhibitor and Vitamin D.
  • the method may further comprise measuring at least a portion of said population of said de-differentiated cells whereby they express at least a 10% and more preferably a 50% change in RNA expression of the following molecules relative to a control population of T cells, namely: reduction in T cell effector molecules including but not limited to granzyme B, IL-10, and IFN-g; increase in transcription factors associated with cells of reduced differentiation status, including but not limited to Nanog, KLF4, and KLF10; increase in expression of molecules preferentially expressed on naive T cell subsets, including but not limited to CD 127, the IL-7 receptor alpha chain; reduction in transcription factors associated with Thl-type differentiation, including but not limited to T-BET and STAT1; and relative preservation of transcription factors that promote cell survival, including but not limited to HIF-1 alpha.
  • T cell effector molecules including but not limited to granzyme B, IL-10, and IFN-g
  • increase in transcription factors associated with cells of reduced differentiation status including but not limited to Nanog, KLF4, and
  • the method may further comprise measuring at least a portion of said population of said de-differentiated cells whereby they express at least a 10% and more preferably a 50% change in expression of molecules indicative of cells that have undergone autophagy.
  • the said de-differentated cells have increased expression of p62 by western blot analysis relative to control T cells.
  • Other standard methods that measure autophagy may also be used, such as those described in Yoshii SR, Mizushima N. Monitoring and Measuring Autophagy. International Journal of Molecular Sciences. 2017;18(9): 1865.
  • the present disclosure is also directed to a de-differentiated T cell produced by the methods of any of the foregoing embodiments.
  • the present disclosure is also directed to a composition
  • a composition comprising a population of de differentiated cells, wherein at least a portion of said population of said de-differentiated cells express at least a 10% and more preferably a 50% change in RNA expression of the following molecules relative to a control population of T cells, namely: reduction in T cell effector molecules including but not limited to granzyme B, IL-10, and IFN-g; increase in transcription factors associated with cells of reduced differentiation status, including but not limited to Nanog, KLF4, and KLF10; increase in expression of molecules preferentially expressed on naive T cell subsets, including but not limited to CD 127, the IL-7 receptor alpha chain; reduction in transcription factors associated with Thl-type differentiation, including but not limited to T-BET and STAT1; and relative preservation of transcription factors that promote cell survival, including but not limited to HIF-1 alpha.
  • the present disclosure is also directed to a composition
  • a composition comprising a population of de differentiated cells, as defined by said de-differentiated cells expressing at least a 10% and more preferably a 50% change in expression of molecules indicative of cells that have undergone autophagy.
  • the said de-differentated cells have increased expression of p62 by western blot analysis relative to control T cells.
  • Other methods that measure autophagy can also be applied, such as those described in Yoshii SR, Mizushima N. Monitoring and Measuring Autophagy. International Journal of Molecular Sciences. 2017;18(9): 1865.
  • the present disclosure is also directed to a composition
  • a composition comprising a population of de differentiated cells, wherein at least a portion of said population of said de-differentiated cells express less than 50% of both RAPTOR and RICTOR as compared to a control population of T cells.
  • the present disclosure is directed to methods for differentiating de-differentiated T cells to TREG or TREG/T1I2 cells.
  • the method comprises culturing de-differentiated T cells of the present disclosure or that are otherwise de-differentiated, in a culture medium comprising IL-2, IL-4 and TGF-b; adding anti-CD3/anti-CD28 coated magnetic beads at a ratio of 3: 1 (bead:T cell ratio); and incubating said de-differentiated T cells for a period of time to yield TREG/T1I2 cells.
  • the method comprises culturing de-differentiated T cells having reduced expression of at least 50% less of RAPTOR or RICTOR relative to a control population of T cells manufactured under the same conditions as the culture input population of cells without temsirolimus, IL-2 signaling inhibitor and Vitamin D, in a culture medium comprising IL-2, IL-4 and TGF-b; adding anti-CD3/anti-CD28 coated magnetic beads at a ratio of 3: 1 (bead:T cell ratio); and incubating said de-differentiated T cells for a period of time to yield TRE G /Th2 cells.
  • the method comprises culturing de-differentiated T cells having reduced expression of at least 90% less of RAPTOR or RICTOR relative to a control population of T cells manufactured under the same conditions as the culture input population of cells without temsirolimus, IL-2 signaling inhibitor and Vitamin D, in a culture medium comprising IL-2 and TGF-b; adding anti-CD3/anti-CD28 coated magnetic beads at a ratio of 3: 1 (bead:T cell ratio); and incubating said de-differentiated T cells for a period of time to yield TREG cells.
  • the culture medium can further comprise pemetrexed.
  • the present disclosure is also directed to a TREG/T1I2 cell produced by any of the foregoing methods.
  • FIGURE 1 A depicts normalized GAPDH mRNA expression for the control cells and cells treated under various conditions.
  • FIGURE IB depicts normalized granzyme B mRNA expression for the control cells and cells treated under various conditions.
  • FIGURE 1C depicts normalized IL-10 mRNA expression for the control cells and cells treated under various conditions.
  • FIGURE ID depicts normalized IFN-g mRNA expression for the control cells and cells treated under various conditions.
  • FIGURES 1 A-1D illustrate that the combination of Vitamin D and temsirolimus reduces effector molecule expression in human CD4+ and CD8+ T cells.
  • FIGURE 2A depicts normalized NANOG mRNA expression for the control cells and cells treated under various conditions.
  • FIGURE 2B depicts normalized KLF4 mRNA expression for the control cells and cells treated under various conditions.
  • FIGURE 2C depicts normalized KLF10 mRNA expression for the control cells and cells treated under various conditions.
  • FIGURE 2D depicts normalized IL-7 receptor mRNA expression for the control cells and cells treated under various conditions.
  • FIGURES 2A-2D illustrate that the combination of Vitamin D and temsirolimus increases expression of stem cell-associated transcription factors and the primitive T cell molecule IL-7 receptor-alpha in human CD4+ and CD8+ T cells.
  • FIGURE 3 A depicts normalized T-BET mRNA expression for the control cells and cells treated under various conditions.
  • FIGURE 3B depicts normalized STAT1 mRNA expression for the control cells and cells treated under various conditions.
  • FIGURE 3C depicts normalized HIF-1-a mRNA expression for the control cells and cells treated under various conditions.
  • FIGURES 3A-3C illustrate that the combination of Vitamin D and temsirolimus reduces expression of transcription factors associated with effector Thl/Tcl cells without reducing expression of a transcription factor associated with T cell survival, HIF-1-a.
  • FIGURE 4 depicts p62 expression normalized by actin expression for cells treated under various conditions and illustrates that the combination of Vitamin D, temsirolimus, and anti-IL-2 receptor blockade induces expression of the autophagy-related molecule, p62.
  • FIGURE 5 depicts Raptor expression normalized by actin expression for cells treated under various conditions and illustrates that the combination of Vitamin D, temsirolimus, and anti-IL-2 receptor blockade reduces expression of the mTORCl -related molecule, Raptor.
  • FIGURE 6 depicts a Western blot of GAPDH, p70S6K, SGK1, Raptor and Rictor expression for cells treated under various conditions and illustrates that the combination of Vitamin D, temsirolimus, and anti-IL-2 receptor blockade reduces expression of the mTORCl- related molecule, Raptor, and the mTORC2-related molecule, Rictor.
  • FIGURE 7 depicts BIM expression normalized by actin expression for cells treated under various conditions and illustrates that the combination of Vitamin D, temsirolimus, and anti-IL-2 receptor blockade reduces expression of the pro-apoptosis molecule, BIM.
  • FIGURE 8 illustrates the effect of culture components during the de-differentiation interval on subsequent T cell yield (at day 13 of culture).
  • FIGURE 8 illustrates the effect of culture components during the de-differentiation interval on subsequent T cell yield (at day 13 of culture).
  • FIGURE 9A depicts the percent of CD4 cells that are CD45RA+ for cells treated under various conditions.
  • FIGURE 9B depicts the percent of CD4 cells that are CD62L+ and CCR7+ for cells treated under various conditions.
  • FIGURE 9C depicts the percent of CD4 cells that are CD62L+, CCR7+, and CD127+ for cells treated under various conditions.
  • FIGURES 9A-9C illustrate the effect of culture components during the de-differentiation interval on CD4+ T cell expression of memory markers (at day 13 of culture).
  • FIGURE 10A depicts the percent of CD8 cells that are CD62L+ and CCR7+ for cells treated under various conditions.
  • FIGURE 10B depicts the percent of CD8 cells that are CD62L+, CCR7+, and CD127+ for cells treated under various conditions.
  • FIGURES 10A-10B illustrate the effect of culture components during the de- differentiation interval on CD8+ T cell expression of memory markers.
  • FIGURES 11 A-l ID depict the inflammatory Thl/Thl7 cytokine analysis of cultured de differentiated T cells in polarization-neutral media.
  • FIGURE 11 A depicts the IFN-g secretion for cells treated under various conditions.
  • FIGURE 1 IB depicts the GM-CSF secretion for cells treated under various conditions.
  • FIGURE 11C depicts the TNF-a secretion for cells treated under various conditions.
  • FIGURE 1 ID depicts the IL-17 secretion for cells treated under various conditions.
  • FIGURES 12A-12D depict the IL-2 and Th2-type cytokine analysis of cultured de differentiated T cells in polarization-neutral media.
  • FIGURE 12A depicts the IL-2 secretion for cells treated under various conditions.
  • FIGURE 12B depicts the IL-4 secretion for cells treated under various conditions.
  • FIGURE 12C depicts the IL-5 secretion for cells treated under various conditions.
  • FIGURE 12D depicts the IL-13 secretion for cells treated under various conditions.
  • FIGURE 13 depicts favorable expansion of de-differentiated T cells in hybrid Th2/TREG polarization condition relative to Thl polarization condition.
  • FIGURE 14A depicts the percentage of CD4 + CD45RA + cells out of total CD4 + cells for cells treated under various conditions.
  • FIGURE 14B depicts the percentage of CD4 + CD62L + CCR7 + cells out of total CD4 + cells for cells treated under various conditions.
  • FIGURE 14C depicts the percentage of CD4 + CD62L + CCR7 + CD127 + cells out of total CD4 + cells for cells treated under various conditions.
  • FIGURES 14-A14C illustrate that the culture of de-differentiated T cells in hybrid Th2/TRe g polarization condition results in the generation of naive and triple-positive T central memory CD4 + T cells.
  • FIGURE 15A depicts the percentage of CD8 + CD62L + CCR7 + cells out of total CD8 cells for cells treated under various conditions.
  • FIGURE 15A depicts the percentage of CD8 + CD62L + CCR7 + CD127 + cells out of total CD8 cells for cells treated under various conditions.
  • FIGURES 15A-15B illustrates that culture of de-differentiated T cells in hybrid Th2/TR eg polarization condition results in the generation of triple-positive T central memory CD8 + T cells.
  • FIGURE 16A depicts IL-2 secretion for cells treated under various conditions.
  • FIGURE 16B depicts IL-4 secretion for cells treated under various conditions.
  • FIGURE 16C depicts IL-5 secretion for cells treated under various conditions.
  • FIGURES 16A-16C illustrate that the culture of de-differentiated T cells in hybrid Th2/TRe g polarization condition results in the generation of T cells with a primitive Th2 cell cytokine phenotype: IL-2, IL-4, and IL-5 secretion.
  • FIGURE 17A depicts IL-10 secretion for cells treated under various conditions.
  • FIGURE 17B depicts IL-13 secretion for cells treated under various conditions.
  • FIGURE 17C depicts IL-17 secretion for cells treated under various conditions.
  • FIGURES 17A-17C illustrate that the culture of de-differentiated T cells in hybrid Th2/TRe g polarization condition results in the generation of T cells with a primitive Th2 cell cytokine phenotype: IL-10, IL-13, and IL-17 secretion.
  • FIGURE 18A depicts IFN-g secretion for cells treated under various conditions.
  • FIGURE 18B depicts TNF-a secretion for cells treated under various conditions.
  • FIGURE 18C depicts GM-CSF secretion for cells treated under various conditions.
  • FIGURES 18A-18C illustrates that the culture of de-differentiated T cells in hybrid T1I2/TREG polarization condition results in the generation of T cells with a primitive Th2 cell cytokine phenotype: IFN-gamma, TNF-alpha, and GM-CSF secretion.
  • FIGURE 19A depicts the percent of CD4 + T cells in culture by day and culture inhibitor.
  • FIGURE 19B depicts the percent of CD4 + FOXP3 + T cells in culture by day and culture inhibitor.
  • FIGURE 19C depicts the percent of CD4 + Tbet + T cells in culture by day and culture inhibitor.
  • FIGURE 19D depicts the percent of CD4 + GATA3 + T cells in culture by day and culture inhibitor.
  • FIGURES 19A-19D illustrate that extended culture of de-differentiated T cells in the hybrid Th2/TREG polarization condition containing pemetrexed results in the generation of CD4 + T cells expressing FOXP3 and GATA3 transcription factors.
  • FIGURE 20A depicts the percent of CD8 + T cells in culture by day and culture inhibitor.
  • FIGURE 20B depicts the percent of CD8 + FOXP3 + T cells in culture by day and culture inhibitor.
  • FIGURE 20C depicts the percent of CD8 + Tbet + T cells in culture by day and culture inhibitor.
  • FIGURE 20D depicts the percent of CD8 + GATA3 + T cells in culture by day and culture inhibitor.
  • FIGURES 20A-20D illustrate that extended culture of de-differentiated T cells in the hybrid Th2/TREG polarization condition containing pemetrexed results in the generation of CD8 + T cells expressing FOXP3 and GATA3 transcription factors.
  • FIGURE 21A depicts IL-4 secretion for cells in culture by day and culture inhibitor.
  • FIGURE 21B depicts IL-5 secretion for cells in culture by day and culture inhibitor.
  • FIGURE 21C depicts IL-13 secretion for cells in culture by day and culture inhibitor.
  • FIGURES 21 A-21C illustrate that extended culture of de-differentiated T cells in the hybrid T1I2/TREG polarization condition results in the generation of T cells expressing with a predominant Th2 cytokine phenotype: IL-4, IL-5, and IL-13 secretion.
  • FIGURE 22A depicts IL-2 secretion for cells in culture by day and culture inhibitor.
  • FIGURE 22B depicts IFN-g secretion for cells in culture by day and culture inhibitor.
  • FIGURE 22C depicts GM-CSF secretion for cells in culture by day and culture inhibitor.
  • FIGURES 22A-22C illustrate that extended culture of de-differentiated T cells in the hybrid T1I2/TREG polarization condition results in the generation of T cells expressing with a predominant Th2 cytokine phenotype: IL-2, IFN-gamma, and GM-CSF secretion.
  • FIGURES 23A and 23B illustrate that the anti-TNF-a therapy etanercept therapy results in marked alteration of the TCR repertoire when measured by RNA sequencing, thereby representing a new approach for subject treatment prior to lymphocyte collection by apheresis.
  • FIGURE 24 illustrates that extended culture of de-differentiated T cells in the hybrid T1I2/TREG polarization condition results in the generation of T cells expressing increased levels of the following molecules relative to control Thl/Tcl cells: CD25, CD27, 2B4, BTLA, and CTLA.
  • FIGURE 25 illustrates that extended culture of de-differentiated T cells in the hybrid T1I2/TREG polarization condition results in the generation of T cells expressing increased levels of the following molecules relative to control Thl/Tcl cells: TIGIT, TIM3, ICOS, LAIR1, and 0X40.
  • FIGURE 26A depicts FOXP3 expression in CD4 + and CD8 + T cells at culture initiation and after culture as measured by flow cytometry.
  • FIGURE 26B depicts GATA3 expression in CD4 + and CD8 + T cells at culture initiation and after culture as measured by flow cytometry.
  • FIGURE 27A depicts CD73 expression in CD4 + and CD8 + T cells at culture initiation and after culture as measured by flow cytometry.
  • FIGURE 27B depicts CD103 expression in CD4 + and CD8 + T cells at culture initiation and after culture as measured by flow cytometry.
  • FIGURE 28A depicts CD150 frequency as measured by flow cytometry in CD4 + and CD8 + T cells at culture initiation, after culture and for control T cells not exposed the mTOR inhibitors, as measured by flow cytometry.
  • FIGURE 28B depicts CD27 versus CD95 expression for CD4 + T cells at culture initiation and after culture as measured by flow cytometry.
  • FIGURE 29 depicts the IL-4, IL-2, IFN-g, TNF-a, IL-17 and GM-CSF for differently cultured cells and control cells.
  • FIGURE 30A depicts the cytokine content for a transwell assay of Thl/Tcl cells with or without RAPA-501 cells.
  • FIGURE 3 OB depicts the flow cytometry results for assays of CD4 and PD1 in Example 24.
  • FIGURE 31 A depicts RAPA-501 GATA3 and FOXP3 as measured by flow cytometry for CD4 + cells.
  • FIGURE 3 IB depicts RAPA-501 GATA3 and FOXP3 as measured by flow cytometry for CD8 + cells.
  • FIGURE 32 depicts an exemplary workflow of a de-differentation method of the present disclosure.
  • the present disclosure provides a method for T cell de-differentiation and resulting cells and a method for manufacturing of human hybrid regulatory T/Th2 cells (hybrid TREG/T1I2 cells) from de-differentiated T cells.
  • the term“de-differentiated T cell” refers to a T cell that has been de differentiated by any of the methods of the present disclosure.
  • the de differentiated T cell has reduced expression of RAPTOR or RICTOR relative to a control population of T cells manufactured under the same conditions without temsirolimus, IL-2 signaling inhibitor and Vitamin D.
  • The“de-differentiated T cell” does not include T cells as collected from a patient, i.e. naturally occurring T cells.
  • anti-CD3/anti-CD28 should be understood to refer to anti- CD3/anti-CD28 antibodies.
  • “anti-CD3/anti-CD28 magnetic beads” should be understood to refer to magnetic beads having anti-CD3/anti-CD28 antibody moieties associated therewith. In instances where it is disclosed that no anti-CD3/anti-CD28 co-stimulation is provided, even by a specific form such as anti-CD3/anti-CD28 magnetic beads, it should be understood that this can also exclude co-stimulation with other forms of anti-CD3/antiCD28.
  • this co-stimulation can be provided in any form of anti- CD3/anti-CD28 antibodies.
  • anti-CD3/anti-CD28 nanoparticles or microparticles can be used in an amount sufficient to achieve the equivalent effect.
  • The“human hybrid TREG/T1I2 cells,”“ITREG” and“TREG/T1I2 cells” of the present disclosure do not include T cells as collected from a patient, i.e. naturally occurring T cells.
  • control Thl/Tcl cells refers to cells that have not been treated with vitamin D, temsirolimus or the IL-2 signaling inhibitor and, rather, have been co-stimulated with anti-CD3/anti-CD28 magnetic coated beads at a ratio of 3: 1 (beads:T cell) in media supplemented with 20 IU/mL IL-2 and 20,000 IU/mL of IFN-a and otherwise cultured the same as the cells to which they are being compared.
  • control T cell a control population of cells (or control T cell) is referred to as having been treated without culture additives, including temsirolimus, vitamin D and the IL-2 signaling inhibitor, or in the context of de-differentiated cells
  • this population has been further co-stimulated with anti-CD3/anti-CD28 magnetic coated beads at a ratio of 3: 1 (beads:T cell) in media supplemented with 20 IU/mL IL-2 and 20,000 IU/ml of IFN-a and otherwise cultured the same as the cells to which they are being compared, i.e. they are“control Thl/Tcl cells.”
  • the present disclosure provides new methodology for the ex vivo generation of T cells of a reduced differentiation state that is based upon the conversion of differentiated effector memory T cells into less differentiated central-memory type T cells using a novel pharmacologic combination and defined T cell co-stimulatory conditions.
  • a de-differentiated T cell of the present disclosure can have a quiescent phenotype with low or no expression of checkpoint inhibitor receptors (such as PD1, CTLA4, TIM3, and LAG3), memory markers (such as CD45RO) and fate molecules (such as TBET, RORy, FOXP3 and GATA3).
  • the re-differentiated T cell can have a hybrid fate characterized GAT A3 and FOXP3 expression, as well as stem cell memory characterized by CD45RA and CD 150 expression and no checkpoint protein expression.
  • the method comprises inoculating a culture input population of cells comprising T cells from a subject at a cell density in a culture medium comprising vitamin D, temsirolimus and an IL-2 signaling inhibitor; adding anti-CD3/anti-CD28 coated magnetic beads to said T cells and culture medium at a bead:T cell ratio of 1 : 1 or less to stimulate said T cells or without adding any co-stimulation beads; incubating said culture input population of cells and culture medium for a period of time to yield de-differentiated T cells.
  • the subject has been treated with an anti-TNF-a therapy prior to collection of the culture input population of cells.
  • the anti-TNF-a therapy is etanercept or adalimumab.
  • no co-stimulation with anti-CD3/anti-CD28 is performed.
  • said culture medium can not contain IL-2 and no IL-2 can be added to said culture medium.
  • said cell density can be about 1.5 x 10 6 T cells per mL to 18 x 10 6 T cells per mL.
  • 6 x 10 6 T cells per mL to 18 x 10 6 T cells per mL 12 x 10 6 T cells per mL to 18 x 10 6 T cells per mL, 1.5 x 10 6 T cells per mL to 12 x 10 6 T cells per mL, 1.5 x 10 6 T cells per mL to 6 x 10 6 T cells per mL, 6 x 10 6 T cells per mL to 12 x 10 6 T cells per mL, or 1.5 x 10 6 T cells per mL, 3 x 10 6 T cells per mL, 6 x 10 6 T cells per mL, 9 x 10 6 T cells per mL, 12 x 10 6 T cells per mL, 15 x 10 6 T cells per mL, or 18 x 10 6 T cells per mL.
  • said temsirolimus can be present at a concentration of about 0.3 mM to about 10 pM.
  • said temsirolimus can be present in said culture medium at a concentration of about 0.3 pM to about 1 pM, 0.3 pM to about 0.75 mM, 0.3 mM to about 0.5 mM, 0.5 mM to about 1 mM, 0.75 mM to about 1 mM, 0.5 mM to about 0.75 mM, 0.3 mM to about 10 mM, 0.3 mM to about 5 mM, 0.3 mM to about 3.3 mM, 1 mM to about 3.3 mM, 5 mM to about 10 mM, 3.3 mM to about 10 mM, 3.3 mM to about 5 mM, or, by way of example but not limitation, at a concentration of about 0.3 mM, 0.4
  • said IL-2 signaling inhibitor can be an anti-IL-2 receptor antibody or fragment thereof.
  • said IL-2 signaling inhibitor can be basiliximab or daclizumab.
  • said IL-2 signaling inhibitor is present in said culture medium at a concentration of 5 to 50 pg/mL, 5 to 40 pg/mL, 5 to 30 pg/mL, 5 to 20 pg/mL, 5 to 10 pg/mL, 10 to 50 pg/mL, 20 to 50 pg/mL, 30 to 50 pg/mL, 40 to 50 pg/mL, 30 to 40 pg/mL, 20 to 40 pg/mL, 10 to 40 pg/mL, 5 to 40 pg/mL, 5 to 30 pg/mL, 5 to 20 pg/mL, 5 to 10 pg/mL, 10 to 20 pg/mL, 10 to 20 pg/mL, 5 to 30 pg/mL, 5
  • said period of time can be about 1.5 days to about 5 days, 1.5 days to about 3.5 days, 1.5 days to about 2.5 days, 2.5 days to about 3.5 days, 2.5 days to about 5 days, 3.5 days to about 5 days, or, about 1.5 days, 2 days, 2.5 days, 3 days, 3.5 days, 4 days, 4.5 days, or 5 days.
  • the level of mTORCl and mTORC2 reduction may be used as a guide to determine optimal culture interval.
  • RNA expression of T cell effector molecules i.e., decreased IFN-g
  • RNA expression of transcription factors i.e., increased KLF4
  • evidence of an autophagy signature i.e., increased p62
  • up-regulation of markers present on naive T cell subsets i.e., increased CD 127.
  • said bead:T cell ratio can be 1 :3 or no co-stimulation can be performed.
  • said bead:T cell ratio can be between 1 : 1 and 1 : 12, 1 : 1 and 1 :3, 1 :3 to 1 : 12.
  • said bead:T cell ratio can be 1 : 1, 1 :2, 1 :3, 1 :4, 1 :5, 1 :6, 1 :7, 1 :8, 1 :9, 1 : 10, 1 : 11 or 1 :12.
  • no anti-CD3/anti-CD28 co stimulation can be utilized, i.e. in some embodiments, no anti-CD3/anti-CD28 co-stimulation is performed during the initial de-differentiation process.
  • co-stimulation of the culture input population of cells can be achieved using anti-CD3/anti-CD28 containing nanoparticles which can be used at a reduced concentration than recommended.
  • such nanoparticles can be used at about 0.01X to about 0. IX, about 0.025X to about 0. IX, about 0.05X to about 0.1X, about 0.075X to about 0.
  • a reagent such as Miltenyi ® T Cell TransActTM could be used at a reduced dose compared to the recommended dose of 10 pL per 1 x 10 6 T cells such as, by way of example but not limitation,
  • no anti- CD3/anti-CD28 co-stimulation can be utilized, i.e. in some embodiments, no anti-CD3/anti- CD28 co-stimulation is performed during the initial de-differentiation process.
  • the source of co-stimulation can be provided by dissolvable anti-CD3/anti- CD28 microparticles.
  • the dissolvable anti-CD3/anti- CD28 microparticles can be used at 20% of the strength recommended by the manufacturer (e.g. Cloudz®; Bio-Techne).
  • the dissolvable anti-CD3-anti-CD28 microparticles can be used at 5%, 10%, 15%, 20%, 25% or 30% of the manufacturer’s recommended strength.
  • the anti-CD3/anti-CD28 stimulation if performed, can be performed using anti-CD3/anti-CD28 in an amount sufficient to achieve the desired de differentiated cell properties.
  • said culture medium can further comprise 5% human serum.
  • said culture medium can comprise at least 1%, 2% 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%,
  • said culture medium can comprise X-Vivo 20 medium. Any appropriate culture medium for culturing T cells can be used.
  • said vitamin D can be present in said culture medium at about 0.03 nM to about 1 nM, 0.03 nM to about 0.5 nM, 0.03 nM to about 0.1 nM, 0.03 nM to about 0.05 nM, 0.05 nM to about 0.1 nM, 0.05 nM to about 0.5 nM, 0.05 nM to about 1 nM, 0.1 nM to about 1 nM, 0.1 nM to about 0.5 nM, or 0.5 nM to about 1 nM, or by way of example but not limitation, said vitamin D is present at a concentration of about 0.03 nM, 0.05 nM, 0.1 nM, 0.5 nM, or 1 nM.
  • the method can further comprise measuring an expression level of RAPTOR or RICTOR and a housekeeping protein in said culture input population of cells, wherein said period of time lasts until the expression level of RAPTOR or RICTOR, respectively, in the manufactured T cells is at least 50% reduced relative to a control population of T cells, wherein said control population of T cells are manufactured under the same conditions as the culture input population of cells without temsirolimus, IL-2 signaling inhibitor and Vitamin D.
  • the period of time lasts until the expression level of RAPTOR or RICTOR, respectively, in the manufactured T cells is reduced by 50% or more relative to the control population of T cells manufactured under the same conditions as the culture input population of cells without temsirolimus, IL-2 signaling inhibitor and Vitamin D.
  • the period of time can last until the expression level of RAPTOR or RICTOR, respectively, is reduced by at least 50%, 60%, 70%, 80%, 90%, 95%,
  • said housekeeping protein can be actin. In some embodiments, the housekeeping protein can be GAPDH. In any of the foregoing embodiments, the step of measuring the expression level can be performed by Western blot analysis. [152] In any of the foregoing embodiments, the period of time can last until the expression level of RAPTOR or RICTOR in said culture input population of cells is reduced by at least 50% relative to a control population of T cells manufactured under the same conditions as the culture input population of cells without temsirolimus, IL-2 signaling inhibitor and Vitamin D. In some embodiments, the reduction in the expression level of RAPTOR or RICTOR can be at least 50%, 60%, 70%, 80%, 90%, 95%, 99% or more relative to the control population of T cells.
  • the period of time of the initial de-differentiation culture can last until the RNA expression pattern is at least 10% and more optimally 50% different relative to control T cells cultured under the same conditions without temsirolimus, vitamin D and the IL-2 signaling inhibitor, namely: reduction in T cell effector molecules including but not limited to granzyme B, IL-10, and IFN-g; increase in transcription factors associated with cells of reduced differentiation status, including but not limited to Nanog, KLF4, and KLF10; increase in expression of molecules preferentially expressed on naive T cell subsets, including but not limited to CD 127, the IL-7 receptor alpha chain; reduction in transcription factors associated with Thl-type differentiation, including but not limited to T-BET and STAT1; and relative preservation of transcription factors that promote cell survival, including but not limited to HIF-1 alpha.
  • the period of time of the initial de-differentiation culture can last until the RNA expression pattern is at least 10% and more optimally 50% different relative to control T cells cultured under the same conditions without temsirolimus, vitamin D and the IL-2 signaling inhibitor, namely: whereby there is least a 10% and more preferably a 50% change in expression of molecules indicative of cells that have undergone autophagy.
  • the said de-differentated cells have increased expression of p62 by western blot analysis relative to control T cells; other methods of measuring autophagy can also be utilized, by way of example but not limitation, those described in Yoshii SR, Mizushima N. Monitoring and Measuring Autophagy. International Journal of Molecular Sciences.
  • the culture medium may not contain human serum, temsirolimus, Vitamin D, the IL-2 signaling inhibitor or any combination thereof can be absent from the culture medium at the time of culture initiation.
  • human serum, temsirolimus, Vitamin D or the IL-2 signaling inhibitor can be added to the culture medium at about the same time as inoculation of the culture input population of cells or at a subsequent time.
  • an intravenous formulation of 1,25-vitamin D (“Calcitriol”) can be used.
  • This formulation is preferable because it is fully soluble in culture media and has the 1, 25 hydroxylation that is naturally produced in the kidneys and therefore must be present when adding vitamin D to culture.
  • Trade name for calcitriol includes Rocaltrol, Calcijex, and Decostriol).
  • VDR vitamin D receptor
  • other vitamin D receptor (VDR) ligands may be substituted for calcitriol, including but not limited to lithocholic acid, as described in Maestro et al; Vitamin D receptor 2016: novel ligands and structural insights; Expert Opinion on Therapeutic Patents; Volume 26, 2016, issue 11.
  • a de-differentiated T cell that can be obtained by any of the methods of the present disclosure is provided.
  • a composition comprising a population of de-differentiated T cells is provided.
  • at least a portion of the de-differentiated T cells express less than 50% of RAPTOR or RICTOR relative to a control population of T cells manufactured under the same conditions as the culture input population of cells without temsirolimus, IL-2 signaling inhibitor and Vitamin D.
  • a de differentiated T cell expresses less than 50% RAPTOR or RICTOR relative to a control population of T cells manufactured under the same conditions as the culture input population of cells without temsirolimus, IL-2 signaling inhibitor and Vitamin D.
  • said de-differentiated T cell or population of de-differentiated T cells can express 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1% or less RAPTOR or RICTOR relative to a control T cell or population of T cells, respectively, manufactured under the same conditions as the culture input population of cells without temsirolimus, IL-2 signaling inhibitor and Vitamin D.
  • a de-differentiated T cell population or de-differentiated T cell can be characterized by a reduction in RNA expression for cytolytic molecules relative to a control T cell population incubated under the same conditions without temsirolimus, vitamin D and the IL-2 signaling inhibitor, including, but not limited granzyme B and/or for cytokine molecules including, but not limited to IFN-g.
  • a reduction can be, by way of example but not limitation, at least 10%, at least 20%, at least 30%, at least 40%, at least 50% or more.
  • a de-differentiated T cell population or de-differentiated T cell can be characterized by an increase in RNA expression for transcription factors associated with iPSCs relative to a control T cell population incubated under the same conditions without temsirolimus, vitamin D and the IL-2 signaling inhibitor, including, but not limited to Nanog, KLF4, and KLF10 and/or for molecules associated with naive T cells including, but not limited to the IL-7 receptor, CD127.
  • Such an increase can be, by way of example but not limitation, at least 10%, at least 20%, at least 30%, at least 40%, at least 50% or more.
  • a de-differentiated T cell population or de-differentiated T cell can be characterized by a reduction in RNA expression for transcription factors associated with Thl effector T cells relative to a control T cell population incubated under the same conditions without temsirolimus, vitamin D and the IL-2 signaling inhibitor, including, but not limited T- Bet and STAT1 with a concomitant maintenance about equivalent HIF-1-a expression.
  • a reduction can be, by way of example but not limitation, at least 10%, at least 20%, at least 30%, at least 40%, at least 50% or more.
  • the HIF-1-a expression can be within about 20%, 15%, 10% or 5% or the control T cell population.
  • a de-differentiated T cell population or de-differentiated T cell can be characterized by an increase in protein expression of p62 relative to a control population of T cells incubated under the same conditions without temsirolimus, vitamin D and the IL-2 signaling inhibitor.
  • Such an increase can be, by way of example but not limitation, at least 10%, at least 20%, at least 30%, at least 40%, at least 50% or more.
  • the present disclosure provides an ex vivo manufacturing process that results in the generation of ITREG cells enhanced for an early state of differentiation combined with depletion of Thl- and Thl7-type polarization.
  • This method requires a two-step process, the first step consisting of T cell de-differentiation, the second step ITREG cell manufacturing.
  • Manufacture of human ITREG cells from this de-differentiated T cell substrate can be performed using a novel combination of cytokines (standard ITREG use of IL-2 and TGF-b cytokines plus additional use of the cytokine classically-associated with Th2 differentiation, IL-4) and, optionally, a novel pharmaceutical agent, pemetrexed as described herein.
  • the ITREG cells can be generated without pemetrexed. Because such cells have expression of both TREG and Th2 molecules, cells generated by this method are termed‘human hybrid TREG/Th2 cells’.
  • the method comprises culturing de-differentiated T cells of the present disclosure in a culture medium comprising IL-2, IL-4 and TGF-b; adding anti-CD3/anti- CD28 coated magnetic beads, such as at a ratio of 3: 1 (bead:T cell ratio); and incubating said de differentiated T cells for a period of time to yield TREG/Th2 cells.
  • the method comprises culturing a population of de-differentiated T cells of the present disclosure.
  • the ratio of anti-CD3/anti-CD28 beads can be varied so long as the co-stimulation is sufficient to differentiate the cells.
  • the method comprises culturing de-differentiated T cells having reduced expression of RAPTOR or RICTOR relative to a control population of T cells manufactured under the same conditions without temsirolimus, IL-2 signaling inhibitor and Vitamin D, in a culture medium comprising IL-2, IL-4 and TGF-b; adding anti-CD3/anti-CD28 coated magnetic beads, such as at a ratio of 3: 1 (bead:T cell ratio); and incubating said de differentiated T cells for a period of time to yield TREG/Th2 cells.
  • the method comprises culturing a population of de-differentiated T cells of the present disclosure.
  • the ratio of anti-CD3/anti-CD28 beads can be varied so long as the co-stimulation is sufficient to differentiate the cells.
  • the expression of RAPTOR or RICTOR is normalized by a housekeeping protein, such as, by way of example, but not limitation, actin or GAPDH.
  • IL-2 can be present in said culture medium at a concentration of about 100 IU/ml to 10,000 IU/ml, 100 IU/ml to 1,000 IU/ml, 1,000 IU/ml to 10,000 IU/ml, or, about 100 IU/ml, 1,000 IU/ml, or 10,000 IU/ml.
  • the culture medium can further comprise IL-4.
  • IL-4 can be present in said culture medium at a concentration of about 100 IU/mL to 1000 IU/M1, 100 IU/mL to 1000 IU/mL, 100 IU/mL to 250 IU/mL, 100 IU/mL to 500 IU/mL, 250 IU/mL to 1000 IU/mL, 500 IU/mL to 1000 IU/mL, 250 IU/mL to 500 IU/mL, or, 100 IU/mL, 200 IU/mL, 300 IU/mL, 400 IU/mL, 500 IU/mL, 600 IU/mL, 700 IU/mL, 800 IU/mL, 900 IU/mL, or 1000 IU/mL.
  • lower concentrations such as 100 IU/mL can be used if there is a desire to achieve reduced Th2 polarization.
  • TGF-b can be present in said culture medium at a concentration of about 10 ng/mL.
  • concentration of TGF-b can be about 5 ng/mL, 6 ng/mL, 7 ng/mL, 8 ng/mL, 9 ng/mL or 10 ng/mL.
  • said bead:T cell ratio can be 3 : 1.
  • an equivalent amount, having the same effect, of alternative forms of anti-CD3/anti-CD28 can be used.
  • the amount of co-stimulation is sufficient to saturate the cells.
  • the amount of co-stimulation can be sufficient to increase the expression of GATA3 and FOXP3 in the human hybrid TREG/Th2 cells.
  • the culture medium can further comprise pemetrexed.
  • pemetrexed can be present in said culture medium at a concentration from about 1 nM to 100 nM, 5 nM to 100 nM, 10 nM to 100 nM, 25 nM to 100 nM, 50 nM to 100 nM, 75 nM to 100 nM, 50 nM to 75 nM, 25 nM to 75 nM, 10 nM to 50 nM, 10 nM to 25 nM, or at such values as 5 nM, 10 nM, 25 nM, 50 nM, 75 nM, or 100 nM.
  • the culture medium does not comprise pemetrexed and pemetrexed is not added to the culture medium.
  • differentiated T cells can be, by way of example but not limitation, between 3 days to 40 days, 2 days to 20 days, 3 days to 10 days, 3 days to 6 days, 6 days to 10 days, 10 days to 40 days, 10 days to 20 days, 10 days to 15 days, 15 days to 40 days, 20 days to 40 days, 30 days to 40 days, 20 days to 30 days, or 15 days to 30 days, or 15 days to 20 days.
  • shorter intervals of culture such as 3 days to 10 days can be considered if hybrid T1I2/TREG cells of very limited differentiation status.
  • the present disclosure is also directed to methods and a TREG cell produced by any of the foregoing methods without the use of IL-4.
  • a TREG or TREG/T1I2 cell produced by the methods of the present disclosure can have increased expression by flow cytometry of at least one of CD25, CD27, 2B4, BTLA, CTLA4, TIGIT, TIM3, ICOS, LAIR1, and 0X40 relative to control Thl/Tcl cells.
  • this increase can be, by way of example but not limitation, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more.
  • a TREG or TREG/T1I2 cell produced by the methods of the present disclosure can have decreased secretion of inflammatory cytokines relative to control Thl/Tcl cells.
  • cyotkines can include IFN-g and TNF-a.
  • this decrease can be, by way of example but not limitation, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more.
  • a TREG or TREG/T1I2 cell produced by the methods of the present disclosure can have reduced TBET and increase FOXP3 expression relative to control Thl/Tcl cells and/or increased IL-4 secretion and increased expression of GATA3 relative to control Thl/Tcl cells.
  • this decrease orincrease can be, by way of example but not limitation, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more.
  • a population of TREG or TREG/T1I2 cells can have at least 5% of CD4 + or CD8 + cells that express GAT A3.
  • a population of TREG or TREG/T1I2 cells can have at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 45%, at least 50%, or at least 60% of CD4 + or CD8 + T cells that express GAT A3.
  • whether the cells express GAT A3 is determined by flow cytometry.
  • population of TREG or TREG/T1I2 cells can exhibit an increased frequency of CD4 + or CD8 + T cells expressing GATA3 relative to a control T cell population characteristic of the T cells from which the population of TREG or TREG/T1I2 cells was produced .
  • the increased frequency can be an increase of 50% or more.
  • the increase can be by 50%, 100%, 200%, 300%, 500%, 1000%, 2000%, 3000% or more.
  • a population of TREG or TREG/T1I2 cells can have at least 5% of CD4 + or CD8 + cells that express FoxP3.
  • a population of TREG or TREG/T1I2 cells can have at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, or at least 45% of CD4 + or CD8 + T cells that express FoxP3.
  • whether the cells express FoxP3 is determined by flow cytometry.
  • population of TREG or TREG/T1I2 cells can exhibit an increased frequency of CD4 + or CD8 + T cells expressing FOXP3 relative to a control T cell population characteristic of the T cells from which the population of TREG or TREG/T1I2 cells was produced .
  • the increased frequency can be an increase of 50% or more.
  • the increase can be by 50%, 100%, 200%, 300%, 500%, 1000%, 2000%, 3000% or more.
  • a population of TREG or TREG/T1I2 cells can have at least 10% of CD4 + or CD8 + cells that express CD73.
  • a population of TREG or TREG/T1I2 cells can have at least 10%, at least 15%, at least 20%, or at least 25% of CD4 + T cells that express CD73.
  • a population of TREG or TREG/T1I2 cells can have at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, or at least 80% of CD8 + T cells that express CD73.
  • whether the cells express CD73 is determined by flow cytometry.
  • population of TREG or TREG/T1I2 cells can exhibit an increased frequency of CD4 + or CD8 + T cells expressing CD73 relative to a control T cell population characteristic of the T cells from which the population of TREG or TREG/T1I2 cells was produced .
  • the increased frequency can be an increase of 50% or more.
  • the increase can be by 50%, 100%, 200%, 300%, 500%, 1000%, 2000%, 3000% or more.
  • a population of TREG or TREG/Th2 cells can have at least 10% of CD4 + or CD8 + cells that express CD 103.
  • a population of TREG or TREG/T1I2 cells can have at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of CD4 + or CD8 + T cells that express CD 103.
  • whether the cells express CD 103 is determined by flow cytometry.
  • population of TREG or TREG/T1I2 cells can exhibit an increased frequency of CD4 + or CD8 + T cells expressing CD 103 relative to a control T cell population characteristic of the T cells from which the population of TREG or TREG/T1I2 cells was produced .
  • the increased frequency can be an increase of 50% or more.
  • the increase can be by 50%, 100%, 200%, 300%, 500%, 1000%, 2000%, 3000% or more.
  • a population of TREG or TREG/T1I2 cells can have at least 5% of CD4 + or CD8 + cells that express both FOXP3 and GATA3 as measured by flow cytometry.
  • the population of TREG or TREG/T1I2 cells can have at least 5%, 10%, 20%, 30%, 40%, or 50% of CD4 + or CD8 + cells that express both FOXP3 and GAT A3.
  • a population of TREG or TREG/T1I2 cells can have at least 20% of CD4 + or CD8 + T cells that express CD 150.
  • a population of TREG or TREG/T1I2 cells can have at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% or at least 50% of CD4 + or CD8 + T cells that express CD 150.
  • a population of TREG or TREG/T1I2 cells can an increased frequency of cells that express CD 150 relative to a control population of T cells incubated without exposure to mTOR inhibitors.
  • whether the cells express CD 150 is determined by flow cytometry.
  • population of TREG or TREG/T1I2 cells can exhibit an increased frequency of CD4 + or CD8 + T cells expressing CD 150 relative to a control T cell population characteristic of the T cells from which the population of TREG or TREG/T1I2 cells was produced .
  • the increased frequency can be an increase of 50% or more.
  • the increase can be by 50%, 100%, 200%, 300%, 500%, 1000%, 2000%, 3000% or more.
  • a population of TREG or TREG/T1I2 cells can express at least 5 pg/mL/1 x 10 6 cells/day of IL-4 after co-stimulation with anti-CD3/anti-CD28 beads at a bead:T cell ratio of 3 : 1.
  • a population of TREG or TREG/T1I2 cells can express at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 pg/mL/1 x 10 6 cells/day IL-4.
  • a population of TREG or TREG/T1I2 cells can express at least 100 pg/mL/1 x 10 6 cells/day of IL-2 after co-stimulation with anti-CD3/anti-CD28 beads at a bead:T cell ratio of 3 : 1.
  • a population of TREG or TREG/T1I2 cells can express less than 100 pg/mL/1 x 10 6 cells/day of IFN-g or GM-CSF after co-stimulation with anti-CD3/anti-CD28 beads at a bead:T cell ratio of 3 : 1.
  • a population of TREG or TREG/T1I2 cells can express less than 10 pg/mL/1 x 10 6 cells/day of TNF-a or IL-17 after co-stimulation with anti-CD3/anti-CD28 beads at a bead:T cell ratio of 3 : 1.
  • a TREG or TREG/T1I2 cell can express both GATA3 and FOXP3.
  • a TREG or TREG/T1I2 cell can express GAT A3, FOXP3, CD 103 and CD73.
  • a population of TREG or TREG/T1I2 cells can be characterized by at least 5% of the T cells expressing GATA3, at least 5% of the T cells expressing FOXP3, at least 5% of the T cells expressing CD103 and at least 5% of the T cells expressing CD73, as measured by flow cytometry.
  • the TREG or TREG/T1I2 cell or population thereof can have at least one or any combination of the foregoing recited properties to the extent the properties are not incompatible.
  • FIGURES 1 A-1D illustrate that the combination of Vitamin D and temsirolimus reduces effector molecule expression in human CD4 + and CD8 + T cells.
  • T cells were subjected to a 3-day de-differentiation interval that included a low-level of anti- CD3/anti-CD28 co-stimulation (bead-to-T cell ratio; 1 :3); a high-dose of temsirolimus (1 pM); vitamin D (0.1 or 1.0 nM); and culture in X-Vivo 20 media.
  • the first column represents a control culture (no temsirolimus, no Vitamin D, use of a bead-to-T cell ratio of 3 : 1 ; and inclusion of the type I polarizing cytokine IFN-a (20,000 IU/mL, unless otherwise stated, this amount is used in the following examples 1-11 for the control culture)).
  • the second column represents the culture that had the low bead-to-T cell ratio and temsirolimus but did not contain Vitamin D; in contrast, the third column represents the culture that had Vitamin D (0.1 nM) but no temsirolimus.
  • the fourth column represents the culture with high-dose (“HD”) vitamin D (1.0 nM) but no temsirolimus.
  • the fifth column represents the culture that had both a high-dose of vitamin D (1.0 nM) combined with temsirolimus.
  • vitamins D 1.0 nM
  • RNA was harvested and the level of effector molecule expression was compared to the control culture.
  • FIGURE 1 A shows, the various cultures had similar RNA expression of
  • Temsirolimus at a dose of 1 mM acts alone beneficially as an agent of de-differentiation (column 2, reduction in granzyme B and IFN-gamma) and does not abrogate the effect of Vitamin D when used in combination (column 5).
  • Example 2 Combination Vitamin D and Temsirolimus Alters Key Transcription Factors Associated With De-Differentiation
  • FIGURES 2A-2D illustrate that the combination of Vitamin D and temsirolimus increases expression of stem cell-associated transcription factors and the primitive T cell molecule IL-7 receptor-alpha in human CD4 + and CD8 + T cells.
  • the combination of Vitamin D and temsirolimus reduces effector molecule expression in human CD4 + and CD8 + T cells.
  • T cells were subjected to a 3-day de-differentiation interval that included a low-level of anti-CD3/anti-CD28 co-stimulation (bead-to-T cell ratio; 1 :3); a high-dose of temsirolimus (1 mM); vitamin D (0.1 or 1.0 nM); and culture in X-Vivo 20 media.
  • the first column represents a control culture (no temsirolimus, no Vitamin D, use of a bead-to-T cell ratio of 3: 1; and inclusion of the type I polarizing cytokine IFN-a).
  • the second column represents the culture that had the low bead-to-T cell ratio and temsirolimus but did not contain Vitamin D; in contrast, the third column represents the culture that had Vitamin D (0.1 nM) but no
  • the fourth column represents the culture with high-dose (“HD”) vitamin D (1.0 nM) but no temsirolimus.
  • the fifth column represents the culture that had both a high-dose of vitamin D (1.0 nM) combined with temsirolimus.
  • HD high-dose
  • the fifth column represents the culture that had both a high-dose of vitamin D (1.0 nM) combined with temsirolimus.
  • FIGURE 2A shows, temsirolimus or the combination of temsirolimus plus Vitamin D resulted in up-regulation of the Nanog transcription factor, which is recognized as one of the few key factors required for somatic cell de-differentiation towards an iPSC state.
  • mTOR inhibition using rapamycin was found to increase Nanog expression; in contrast, Vitamin D receptor signaling was found to reduce the expression of transcription factors associated with the iPSC state.
  • temsirolimus increases the iPSC transcription factor Nanog; this promoting effect of temsirolimus is not abrogated by Vitamin D at concentrations ranging from 0.1 to 1.0 nM.
  • KLF4 molecule which is also one of the classical transcription factors associated with the iPSC state.
  • Vitamin D 1.0 nM
  • KLF4 RNA expression it is preferable to include both temsirolimus and Vitamin D in T cell de-differentiation attempts.
  • FIGURE 2B shows, although neither temsirolimus or Vitamin D acts alone to beneficially up-regulate the de-differentiation molecule KLF4, the combination of temsirolimus (1 mM) and Vitamin D (1.0 nM) synergistically up- regulate KLF4.
  • KLF10 A related transcription factor, KLF10 was also up-regulated when the combination of temsirolimus plus Vitamin D (1.0 nM) was utilized.
  • Vitamin D 1.0 nM
  • FIGURE 2C shows, temsirolimus at a dose of 1 mM acts alone to beneficially up-regulate the de-differentiation molecules KLF10, Nanog, and IL-7 receptor alpha; although Vitamin D does not act alone to up-regulate these molecules, it does not abrogate the effect of temsirolimus when used in combination (column 5).
  • Example 3 Combination Vitamin D and Temsirolimus Reduces Key Transcription Factors Associated With Thl Differentiation While Maintaining HIF-1 -a expression
  • FIGURES 3A-3C illustrate that the combination of Vitamin D and temsirolimus reduces expression of transcription factors associated with effector Thl/Tcl cells without reducing expression of a transcription factor associated with T cell survival, HIF-1-a.
  • the combination of Vitamin D and temsirolimus reduces effector molecule expression in human CD4 + and CD8 + T cells.
  • T cells were subjected to a 3-day de-differentiation interval that included a low-level of anti-CD3/anti-CD28 co-stimulation (bead-to-T cell ratio; 1 :3); a high-dose of temsirolimus (1 pM); vitamin D (0.1 or 1.0 nM); and culture in X-Vivo 20 media.
  • the first column represents a control culture (no temsirolimus, no Vitamin D, use of a bead-to-T cell ratio of 3: 1; and inclusion of the type I polarizing cytokine IFN-a).
  • the second column represents the culture that had the low bead-to-T cell ratio and temsirolimus but did not contain Vitamin D; in contrast, the third column represents the culture that had Vitamin D (0.1 nM) but no temsirolimus.
  • the fourth column represents the culture with high-dose (“HD”) vitamin D (1.0 nM) but no temsirolimus.
  • the fifth column represents the culture that had both a high-dose of vitamin D (1.0 nM) combined with temsirolimus.
  • cells were harvested, RNA was isolated, and RNA expression analysis was performed by Luminex Quantigene method. All results shown represent relative RNA expression, with results normalized to a value of 1.0 for the Thl/Tcl control culture.
  • each agent or the combination of agents down-regulated both T-BET RNA (FIGURE 3 A) and STAT1 RNA (FIGURE 3B).
  • Vitamin D at a dose of 0.1 to 1.0 nM acts alone to beneficially down-regulate the differentiation molecules T- BET and STATE Temsirolimus at a dose of 1 mM does not detrimentally down-regulate the pro survival transcription factor, HIF-1 alpha, even when combined with 1.0 nM Vitamin D.
  • temsirolimus at a dose of 1 pM acts alone to beneficially down-regulate the differentiation molecules T-BET and STAT1; combination with Vitamin D yields a similar result (these two agents are not antagonistic).
  • Example 4 The Combination Vitamin D, Temsirolimus, and an Anti-IL-2 Receptor
  • FIGURE 4 illustrates that the combination of Vitamin D, temsirolimus, and anti-IL-2 receptor blockade induces expression of the autophagy-related molecule, p62.
  • Human CD4 + and CD8 + T cells were subjected to the de-differentiation protocol, which involved a 3-day culture using low level co-stimulation (1 :3 bead-to-T cell ratio), temsirolimus (“TEM”, as indicated in FIGURE 4; concentration of 1.0 or 0.3 mM), Vitamin D (“D”, as indicated; concentration of 0.01, 0.03, 0.1, 0.3, or 1.0 nM), and an anti-IL-2 receptor monoclonal antibody (Daclizumab, 50 pg/ml;“DAC”, as indicated).
  • the T cells were harvested, and protein was isolated and subjected to western blot analysis for the autophagy-related gene, p62, and the housekeeping gene, Actin.
  • Vitamin D included in the T cell culture was critical for increasing autophagy, as measured by up-regulated p62.
  • Vitamin D at a dose of 0.01 to 0.1 nM works in concert with temsirolimus at a concentration of 0.3 to 1.0 pM to beneficially up- regulate the autophagy marker p62 during de-differentiation.
  • FIGURE 4 also demonstrates that Vitamin D without anti-IL-2 receptor monoclonal antibody addition and Vitamin D without temsirolimus addition was sufficient for induction of T cell autophagy.
  • Vitamin D with low-level co-stimulation is an efficient method for the induction of T cell autophagy either alone or in combination with other T cell inhibitors, namely the anti-IL-2 receptor reagents or the mTOR inhibitor
  • Example 5 The Combination Vitamin D, Temsirolimus, and an Anti-IL-2 Receptor Monoclonal Antibody Results In Optimal Disruption of the mTORCl Complex
  • FIGURE 5 illustrates that the combination of Vitamin D, temsirolimus, and anti-IL-2 receptor blockade reduces expression of the mTORCl -related molecule, Raptor.
  • Human CD4 + and CD8 + T cells were subjected to the de-differentiation protocol, which involved a 3-day culture using low level co-stimulation (1 :3 bead-to-T cell ratio), temsirolimus (“TEM”, as indicated in FIGURE 5; concentration of 1.0 or 0.3 mM), Vitamin D (“D”, as indicated;
  • T cells were harvested, and protein was isolated and subjected to western blot analysis for the mTORCl complex protein, Raptor, and the housekeeping gene, Actin.
  • FIGURE 5 demonstrates optimal inhibition of the mTORCl complex, as indicated by reduction in Raptor expression, occurred when T cells were co-stimulated at a low bead-to-T cell ratio (1 :3) in combination with temsirolimus (1.0 mM), Vitamin D (0.1 nM), and the anti-IL- 2 receptor monoclonal antibody daclizumab (50 pg/nl) (first column shown; culture 1).
  • FIGURE 5 demonstrates that the omission of daclizumab resulted in a modest increase in Raptor expression, thereby indicating a role for an anti-IL-2 receptor reagent for optimal mTORCl inhibition.
  • anti-IL-2 receptor monoclonal antibody Daclizumab dose, 50 pg/ml
  • Vitamin D is a Vitamin D dose between 0.03 to 0.1 nM; concentrations lower than or higher than this range led to less optimal suppression of Raptor.
  • levels of Vitamin D as low as 0.03 nM are sufficient for optimal inhibition of Raptor; reducing the Vitamin D level to 0.01 nM, however, results in a sub-optimal Raptor inhibition.
  • increasing the Vitamin D level beyond the 0.1 nM concentration can be detrimental, as indicated by culture 7 (0.3 nM concentration of Vitamin D), which had a higher level of Raptor expression.
  • the optimal down-regulation of Raptor requires the combination of Vitamin D plus temsirolimus, with the temsirolimus dose optimally being 1.0 mM, as culture 9 that was supplemented with temsirolimus at the concentration of 0.3 mM had higher levels of Raptor expression.
  • Example 6 The Combination of Vitamin D, Temsirolimus, and an Anti-IL-2 Receptor Monoclonal Antibody Disrupts Both the mTORCl Complex and the mTORC2 Complex
  • FIGURE 6 illustrates that the combination of Vitamin D, temsirolimus, and anti-IL-2 receptor blockade reduces expression of the mTORCl -related molecule, Raptor, and the mTORC2-related molecule, Rictor.
  • Human CD4 + and CD8 + T cells were subjected to the de- differentiation protocol, which involved a 3-day culture using low level co-stimulation (1 :3 bead- to-T cell ratio), temsirolimus (“TEM”, as indicated in FIGURE 6; concentration of 1.0 pM), Vitamin D (“D”, as indicated; concentration of 0.03, 0.1, 0.3, or 1.0 nM), and an anti-IL-2 receptor monoclonal antibody (Daclizumab, 50 ng/ml;“DAC”, as indicated).
  • the T cells were harvested, and protein was isolated and subjected to western blot analysis for the mTORCl complex protein, Raptor; the mTORC2 complex protein, Rictor; the post-mTORCl protein p70S6K; the post-mTORC2 protein, SGK1; and the housekeeping gene, GAPDH.
  • FIGURE 6 illustrates, relative to the control culture that did not contain any of the three inhibitors, T cell culture in media containing temsirolimus, Vitamin D, and the anti-IL-2 receptor antibody daclizumab had a reduction in both the mTORCl molecule Raptor and the mTORC2 molecule Rictor. Levels of the post-mTORCl molecule p70S6K and the post- mTORC2 molecule SGK1 were relatively preserved. Thus, Vitamin D (concentration between 0.03 and 1.0 nM) was effective during combination agent de-differentiation for down-regulation of the mTORC2 sub-unit, Rictor.
  • Vitamin D concentration between 0.03 and 1.0 nM
  • temsirolimus at a concentration of 1 mM was effective during combination agent de-differentiation for down-regulation of the mTORC2 sub-unit, Rictor.
  • anti-IL-2 receptor monoclonal antibody Daclizumab dose, 50 pg/ml did not abrogate the ability of temsirolimus and Vitamin D to down-regulate the mTORC2 sub-unit Rictor.
  • T cell culture using a low level of co-stimulation and a three-part inhibitory regimen of temsirolimus, Vitamin D, and anti-IL-2 receptor monoclonal antibody represents a novel method to reduce both Raptor and Rictor subunits.
  • Example 7 The Combination of Vitamin D, Temsirolimus, and an Anti-IL-2 Receptor Monoclonal Antibody Reduces Expression of the Pro-Apoptotic Bcl2-family Member Gene, BIM
  • FIGURE 7 illustrates that the combination of Vitamin D, temsirolimus, and anti-IL-2 receptor blockade reduces expression of the pro-apoptosis molecule, BIM.
  • Human CD4 + and CD8 + T cells were subjected to the de-differentiation protocol, which involved a 3-day culture using low level co-stimulation (1 :3 bead-to-T cell ratio), temsirolimus (“TEM”, as indicated in FIGURE 7; concentration of 1.0 or 0.3 mM), Vitamin D (“D”, as indicated; concentration of 0.01, 0.03, 0.1, 0.3, or 1.0 nM), and an anti-IL-2 receptor monoclonal antibody (Daclizumab, 50 ng/ml;“DAC”, as indicated).
  • the T cells were harvested, and protein was isolated and subjected to western blot analysis for the pro-apoptosis-related gene, BIM, and the housekeeping gene, Actin.
  • FIGURE 7 illustrates, the T cell culture that contained the combination of temsirolimus, Vitamin D (0.1 nM), and the anti-IL-2 receptor monoclonal antibody daclizumab had the lowest level of BIM expression. But still, FIGURE 7 shows that anti-IL-2 receptor monoclonal antibody Daclizumab (dose, 50 pg/ml) plays a beneficial role in suppressing the pro-apoptotic molecule, BIM (column 2). Each of the three inhibitors appeared to play a role in BIM inhibition because absence of any single inhibitor increased the BIM level.
  • the combination inhibitor regimen represents a method for inducing a favorable shift in the mitochondrial control of apoptotic tendency.
  • Example 8 The Three Inhibitor De-differentiation Regimen Results in T Cells With
  • FIGURE 8 illustrates the effect of culture components during the de-differentiation interval on subsequent T cell yield (at day 13 of culture).
  • Human CD4 + and CD8 + T cells were subjected to a 3-day de-differentiation interval that included a low-level of anti-CD3/anti-CD28 co-stimulation (bead-to-T cell ratio; 1 :3 or 1 : 1, as indicated); a temsirolimus (1 mM; or, low-dose [“Lo”], 0.1 pM); vitamin D (0.1 nM; or, high-dose [‘‘HD’] of 1.0 nM; or, low-dose of 0.01 nM); an anti-IL-2 receptor monoclonal antibody (daclizumab, 50 pg/ml); and culture in X-Vivo 20 media supplemented with 5% human AB serum.
  • the first column represents a control culture (no temsirolimus, Vitamin D, or anti-IL-2R antibody).
  • the ninth column represents results using the higher ratio of beads.
  • media was exchanged to fresh X-Vivo 20 without inhibitors, high-level co-stimulation was provided (3: 1 bead-to-T cell ratio), and the T cell growth cytokines IL-2 (100 IU/ml) and IL-7 (10 ng/ml) were added.
  • viable T cells were enumerated and the overall yield is shown relative to day 0 input number.
  • FIGURE 8 shows the T cell counts after the re-differentiation stage. As these data show (column #3), T cells that were initially maintained for the first 3-day de-differentiation interval using a low-level of co-stimulation, temsirolimus, Vitamin D, and anti-IL-2 receptor monoclonal antibody had a satisfactory T cell yield (more than 250% of culture input).
  • Vitamin D concentration of Vitamin D
  • the preferable concentration of Vitamin D is 0.1 nM.
  • the preferable concentration of temsirolimus is 1.0 mM.
  • FIGURES 9A9C illustrate the effect of culture components during the de-differentiation interval on CD4 + T cell expression of memory markers (at day 13 of culture).
  • Human CD4 + and CD8 + T cells were subjected to a 3-day de-differentiation interval that included (as indicated in above FIGURES 9A-9C) a low-level of anti-CD3/anti-CD28 co-stimulation (bead-to-T cell ratio; 1 :3); temsirolimus (1 mM or 0.1 mM [low-dose;“Lo”]); Vitamin D (0.1 nM; or, 0.01 nM [low- dose;“Lo”]); an anti-IL-2 receptor monoclonal antibody (daclizumab, 50 pg/ml); and culture in X-Vivo 20 media supplemented with 5% human AB serum.
  • a low-level of anti-CD3/anti-CD28 co-stimulation bead-to-T cell ratio; 1 :3
  • T cells were subjected to flow cytometry for evaluation of co-expression of CD4 + and CD45RA + markers (results shown in top panel; evaluated at day 13 of culture); co-expression of CD4 + , CD62L + , and CCR7 + markers (bottom left panel; evaluated at day 3 of culture); and co expression of CD4 + , CD62L + , CCR7 + , and CD127 + markers (bottom right panel; evaluated at day 10 of culture). All results are shown relative to the value of CD4 + T cells at culture initiation (FIGURES 9A-9C;“Day 0 Input Value”).
  • T cells initially propagated in the combination of temsirolimus, Vitamin D, and anti-IL-2 receptor monoclonal antibody had relatively preserved expression of the CD45RA marker that is expressed on naive T cells (column #3).
  • absence of these three molecules during the initial culture interval resulted in depletion of the naive T cell population (culture #1).
  • elimination of temsirolimus during the initial culture interval resulted in depletion of the naive T cell population (culture #6).
  • each of the T cell cultures that were initially propagated in the 3 -day interval that incorporated a low-level of co-stimulation had an increase in T cell expression of the central memory molecules CD62L and CCR7.
  • temsirolimus from the initial culture interval also greatly reduced the frequency of triple-positive T cells (columns #5 and 6).
  • Example 10 The Initial Three-Component Culture Interval Results in the Generation of CD8 + T Cells Expressing Cell Surface Molecules Consistent With Reduced Differentiation
  • FIGURES 10A-10B illustrate the effect of culture components during the de- differentiation interval on CD8 + T cell expression of memory markers.
  • T cells were subjected to a 3-day de-differentiation interval that included (as indicated in above FIGURES 10A-10B) a low-level of anti-CD3/anti-CD28 co-stimulation (bead-to-T cell ratio; 1 :3); temsirolimus (1 mM or 0.1 mM [low-dose;“Lo”]); Vitamin D (0.1 nM; or, 0.01 nM [low- dose;“Lo”]); an anti-IL-2 receptor monoclonal antibody (daclizumab, 50 pg/ml); and culture in X-Vivo 20 media supplemented with 5% human AB serum.
  • a low-level of anti-CD3/anti-CD28 co-stimulation bead-to-T cell ratio; 1 :3
  • temsirolimus (1 mM or 0.1 mM [low-dose;“Lo”]
  • Vitamin D 0.1 nM; or, 0.01 nM [low-
  • T cells were subjected to flow cytometry for evaluation of co-expression of CD8 + , CD62L + , and CCR7 + markers (left panel; evaluated at day 10 of culture); and co-expression of CD8 + , CD62L + , CCR7 + , and CD127 + markers (right panel; evaluated at day 10 of culture). All results are shown relative to the value of CD8 + T cells at culture initiation (last column in FIGURES 10A-10B;“Day 0 Input Value”).
  • each of the T cell cultures that were initially propagated in the 3 -day interval that incorporated a low-level of co-stimulation had an increase in CD8 + T cell expression of the central memory molecules CD62L and CCR7.
  • T cells initially propagated in the combination of temsirolimus, Vitamin D, and anti-IL-2 receptor monoclonal antibody had greatly increased expression (relative to the Day 0 input cells) of CD8 + T cells that were triple-positive for CD62L, CCR7, and IL-7 receptor alpha (CD 127).
  • Elimination of the three inhibitors during the initial 3 -day culture (column #1) abrogated the ability of the initial culture interval to promote the expansion of this triple-positive population.
  • reducing or eliminating only temsirolimus from the initial culture interval also greatly reduced the frequency of triple-positive T cells (columns #5 and 6).
  • Example 11 De-differentiated T Cells Have an Inherent Bias Towards Low Cytokine Potential
  • FIGURES 11 A-l ID highlight the components of the de-differentiation process, including use of: a low-level of co-stimulation (an anti-CD3/anti-CD28 bead to T cell ratio of 1 :3, which is reduced relative to conventional methods as described in Kalamasz D, Long SA, Taniguchi R, Buckner JH, Berenson RJ, Bonyhadi M. Optimization of human T-cell expansion ex vivo using magnetic beads conjugated with anti-CD3 and Anti-CD28 antibodies) Journal of immunotherapy (Hagerstown, Md: 1997). 2004;27(5):405-418.; the mTOR inhibitor temsirolimus; vitamin D; and an anti-IL-2 receptor monoclonal antibody.
  • a low-level of co-stimulation an anti-CD3/anti-CD28 bead to T cell ratio of 1 :3, which is reduced relative to conventional methods as described in Kalamasz D, Long SA, Taniguchi R, Buckner JH, Berenson RJ, Bony
  • FIGURES 11A-11D depict the inflammatory Thl/Thl7 cytokine analysis of cultured de differentiated T cells in polarization-neutral media.
  • Human CD4 + and CD8 + T cells were subjected to a 3 -day de-differentiation procedure that included the following culture components, as indicated: temsirolimus (Y, indicates concentration of 1 mM; Y, Lo, indicates concentration of 0.1 mM); Vitamin D (Y, indicates concentration of 0.1 nM; Y, Lo, indicates concentration of 0.01 nM); an anti-IL-2 receptor monoclonal antibody (Daclizumab, 50 pg/ml); co-stimulation with anti-CD3/anti-CD28 (3/28) coated magnetic beads at a low ratio (bead-to-T cell ratio, 1 :3), and supplementation with 5% human serum.
  • temsirolimus indicates concentration of 1 mM
  • Y, Lo indicates concentration of 0.1 mM
  • Vitamin D indicates concentration of
  • the de-differentiated T cells were co stimulated (typical bead-to-T cell ratio of 3: 1) in media supplemented with the T cell growth cytokines rhu IL-2 (100 IU/ml) and rhu IL-7 (10 ng/ml), which are not potent in terms of inducing T cell polarization.
  • the T cells were harvested, washed, and re-stimulated with 3/28 beads (3: 1 ratio) for 24 hr; the resultant supernatant was harvested and tested for cytokine content by Luminex multi-analyte method. All results shown are expressed as cytokine level in pg per ml per 1 x 10 6 cells/ml/24 hr.
  • the resultant T cells that were re-differentiated from each of the de-differentiated precursor states had very low levels of secretion of inflammatory cytokines, including IFN-g (most values below 1000 pg/ml), TNF-a (most values below 100 pg/ml), and IL-17 (all values below 10 pg/ml).
  • IFN-g most values below 1000 pg/ml
  • TNF-a most values below 100 pg/ml
  • IL-17 all values below 10 pg/ml
  • GM-CSF was secreted in some conditions at much higher levels, in some cases, greater than 10,000 pg/ml.
  • the GM-CSF value was moderated in the de-differentiated condition that was comprised of higher dose temsirolimus (1.0 mM) and higher dose vitamin D (0.1 nM); as such, for resultant moderation of T cell cytokine secretion of GM-CSF, it is desirable to expand T cells from a de-differentiation method that incorporates these higher concentrations of temsirolimus and vitamin D.
  • the resultant T cells that were re- differentiated from each of the de-differentiated precursor state T cells had very low level secretion of IL-2, although again, the level was lower in the condition that incorporated the higher concentrations of temsirolimus and vitamin D relative to the conditions that used a lower concentration of these agents.
  • FIGURES 12A-12D depict the IL-2 and Th2-type cytokine analysis of cultured de differentiated T cells in polarization-neutral media.
  • Human CD4 + and CD8 + T cells were subjected to a 3 -day de-differentiation procedure that included the following culture components, as indicated: temsirolimus (Y, indicates concentration of 1 mM; Y, Lo, indicates concentration of 0.1 mM); Vitamin D (Y, indicates concentration of 0.1 nM; Y, Lo, indicates concentration of 0.01 nM); an anti-IL-2 receptor monoclonal antibody (Daclizumab, 50 pg/ml); co-stimulation with anti-CD3/anti-CD28 (3/28) coated magnetic beads at a low ratio (bead-to-T cell ratio, 1 :3), and supplementation with 5% human serum.
  • temsirolimus indicates concentration of 1 mM
  • Y, Lo indicates concentration of 0.1 mM
  • Vitamin D indicates concentration of 0.1
  • the de-differentiated T cells were co stimulated (typical bead-to-T cell ratio of 3: 1) in media supplemented with the T cell growth cytokines rhu IL-2 (100 IU/ml) and rhu IL-7 (10 ng/ml), which are not potent in terms of inducing T cell polarization.
  • the T cells were harvested, washed, and re-stimulated with 3/28 beads (3: 1 ratio) for 24 hr; the resultant supernatant was harvested and tested for cytokine content by Luminex multi-analyte method.
  • results shown are expressed as cytokine level in pg per ml per 1 x 10 6 cells/ml/24 hr.
  • the resultant T cells also had very low level secretion of the Th2-type cytokine IL-4 (values less than 20 pg/ml) and the Th2-type cytokine IL-5 (values less than 60 pg/ml).
  • the levels of IL-13 were elevated in several of the T cell culture conditions, with lower cytokine secretion detected in the condition that incorporated the higher concentrations of temsirolimus and vitamin D relative to the conditions that used a lower concentration of these agents.
  • the de-differentiation step incorporates low-level co-stimulation and propagation in media that contains temsirolimus at a concentration of 1.0 mM, vitamin D at a concentration of 0.1 nM, and inclusion of an anti-IL-2 receptor monoclonal antibody.
  • Example 12 Favorable Expansion of De-differentiated T Cells in a Hybrid TREG/TII2 Polarization Condition and in the Presence of the Novel Pharmaceutical Agent Pemetrexed
  • T cell re-differentiation incorporated TREG polarizing cytokines IL-2 and TGF-b, or a Thl polarizing cytokine, IFN-a.
  • Human ITREG cells with a hybrid Th2 component may be favorable for adoptive T cell therapy because ITREG cells have been characterized as having a propensity to in vivo differentiation plasticity whereby an ITREG cells can convert to a pathogenic Thl-type or Thl7- type subset.
  • the Th2 bias will predictably limit plasticity towards the Thl/Thl7 phenotypes.
  • FIGURE 13 depicts favorable expansion of de-differentiated T cells in hybrid T1I2/TREG polarization condition relative to Thl polarization condition.
  • Human CD4 + and CD8 + T cells were subjected to a 3-day de-differentiation procedure (“STEP 1”).
  • this STEP 1 de-differentiation intervention variably included: no inhibitor (“no”); temsirolimus alone (“T”; 1.0 mIU ⁇ ); Vitamin D alone (“D”; 0.1 nM); the anti-IL-R monoclonal antibody Basiliximab alone (“B”; 10 pg/ml); or various combinations of the inhibitors (T, D; or T, D, B)
  • the de-differentiated T cells were co-stimulated (typical bead-to-T cell ratio of 3: 1) in media variably supplemented with: Thl polarization condition (rhu IFN-a; 10,000 IU/ml); TREG polarization (rhu IL-2, 100 IU/ml; rhu TGF-b, 10 ng/ml); or a hybrid T1I2-TREG polarization condition (IL-2, TGF-b, plus addition of rhu IL-4 [1000 IU/ml]).
  • Thl polarization condition rhu I
  • the T cell culture in the presence of the variable polarization conditions was performed without the novel inhibitory molecule, pemetrexed (“0”) or in the presence of variable concentrations of pemetrexed, as indicated (10 nM [“10”]; 33 nM [“33”]; or 100 nM [“100”].
  • FIGURE 13 shows, an ability to re-differentiate T cells after step 1 de-differentiation depended on: the specific components added during de-differentiation; the specific cytokines added during re-differentiation; and the presence of pemetrexed during re-differentiation.
  • step 1 de-differentiation process followed by step 2 re-differentiation in various cytokine polarizing conditions/various pemetrexed conditions on T cell memory status. That is, studies show that T cells of limited differentiation status have improved therapeutic utility for adoptive cell therapy; thus, limited T cell differentiation would be a favorable feature of the step 1/step 2 T cell manufacturing method.
  • FIGURES 14A-14C illustrate that the culture of de-differentiated T cells in hybrid Th2/TREG polarization condition results in the generation of naive and triple-positive T central memory CD4 + T cells.
  • the TREG conditions above all contained IL-2, TGF-b, and IL-4 (“TReg”) unless indicated (“TReg, No IL4”).
  • naive CD4 + T cells expressed as % of total CD4 + T cells that co expressed CD45RA; FIGURE 14A
  • central-memory CD4 + T cells expressed % of total CD4 + T cells that co-expressed both CD62L and CCR7; FIGURE14B
  • triple-positive central- memory CD4 + T cells expressed % of total CD4 + T cells that co-expressed CD62L, CCR7, and CD127; FIGURE 14C).
  • step 2 re-differentiation in the hybrid TREG-T1I2 condition resulted in a high frequency of the CD4 + CD45RA + naive T cell subset that was favorable in experimental murine models of adoptive T cell therapy.
  • step 2 re-differentiation in the hybrid TREG-T1I2 condition resulted in a high frequency of CD4 + T cells that had triple positive co-expression of the memory markers CD62L, CCR7, and CD127.
  • This triple-positive memory phenotype is a marker of T cells having a very primitive differentiation status.
  • step 1 de-differentiation condition that included not only temsirolimus and vitamin D but also an anti-IL-2 receptor monoclonal antibody yielded the highest frequency of CD4 cells that were triple positive for CD62L, CCR7, and CD 127 using the hybrid TREG-T1I2 polarizing condition.
  • FIGURES 15A-15B illustrates that culture of de-differentiated T cells in hybrid T1I2/TREG polarization condition results in the generation of triple-positive T central memory CD8 + T cells.
  • the TREG conditions above all contained IL-2, TGF-b, and IL-4 (“TReg”) unless indicated (“TReg, No IL4”).
  • the frequency of CD8 cells that were triple positive for CD62L, CCR7, and CD 127 was higher in the hybrid TREG-T1I2 polarizing condition relative to the pure TREG polarizing condition.
  • use of the more stringent step 1 de-differentiation condition that included not only temsirolimus and vitamin D but also an anti-IL-2 receptor monoclonal antibody yielded the highest frequency of CD8 + T cells that were triple positive for CD62L, CCR7, and CD127 using the hybrid TREG-Th2 polarizing condition.
  • Example 14 Culture of De-differentiated T Cells in the Hybrid TKEO/PI2 Polarizing Condition Results in T Cells With a Primitive Th2 Cell Cytokine Phenotype
  • T cells re-differentiated in the step 2 culture conditions after step 1 de-differentiation were also evaluated for cytokine secretion pattern.
  • Cytokine secretion is an indicator of T cell effector function, and as such, it is generally desirable that TREG cells have reduced cytokine secretion potential, particularly with respect to key inflammatory cytokines such as IL-17, IFN-g and TNF-a.
  • cytokines such as IL-17, IFN-g and TNF-a.
  • the proposed hybrid TREG-T1I2 cell population it would be expected that such cells would also secrete some distribution of Th2 cytokines.
  • FIGURES 16A-16C illustrate that the culture of de-differentiated T cells in hybrid Th2/TReg polarization condition results in the generation of T cells with a primitive Th2 cell cytokine phenotype, as indicated by high levels of IL-2 and IL-4 secretion and a low level of IL- 5 secretion.
  • TReg IL-2, TGF-b, and IL-4
  • TReg No IL4
  • T cells re-differentiated in the hybrid TREG-Th2 cytokine polarizing condition (IL-2, TGF-b, and IL-4) with or without pemetrexed added to culture had the highest values for IL-2 secretion.
  • IL-2 secretion in T cells is a characteristic of T cells in an early state of differentiation, which T cells re-differentiated in the hybrid culture conditions possess.
  • FIGURES 17A-17C illustrate that the culture of de-differentiated T cells in hybrid Th2/TREG polarization condition results in the generation of T cells with a primitive Th2 cell cytokine phenotype, as indicated by low levels of IL-10, IL-13, and IL-17 secretion.
  • TReg IL-2, TGF-b, and IL-4
  • TReg No IL4
  • FIGURES 18A-18C illustrate that the culture of de-differentiated T cells in hybrid Th2/TREG polarization condition results in the generation of T cells with a primitive Th2 cell cytokine phenotype, as indicated by low levels of IFN-gamma, TNF-alpha, and GM-CSF secretion.
  • TReg IL-2, TGF-b, and IL-4
  • TReg No IL4
  • T cells re-differentiated in the hybrid TREG-T1I2 condition (IL-2, TGF-b, and IL-4) with or without pemetrexed added to culture had the highest values for IL-4 secretion.
  • IL-4 is the key cytokine that dictates Th2 polarization
  • T cells manufactured in the hybrid condition are indeed Th2 polarized.
  • Example 15 Culture of De-differentiated T Cells in the Hybrid TKEO/TH2 Condition Results in T Cells With an Enhanced Hybrid TREc/Th2 Transcription Factor Profile
  • T cell cytokine phenotype is determined by key transcription factors.
  • the association of transcription factors with T cell subsets is as follows: FOXP3 dictates TREG cell development; TBET dictates Thl-type cell development; and GAT A3 dictates Th2-type development.
  • T cells were first subjected to the step 1 de-differentiation procedure and then re-differentiated in the hybrid TREG- Th2 culture condition (IL-2, TGF-b, and IL-4).
  • IL-2 hybrid TREG- Th2 culture condition
  • IL-4 hybrid TREG- Th2 culture condition
  • pemetrexed we compared the effects of pemetrexed with a classical mTOR inhibitor.
  • rapamycin rapolimus
  • the ITREG phenotype is considered to be unstable; as such, we evaluated the stability of T cells re-differentiated using the hybrid TREG-T1I2 culture condition at delayed time points, including days 20 and 32 of culture. In addition, to test phenotype stability in a rigorous manner, between day 24 and day 32 of culture, T cells received a high level of co-stimulation (3 : 1 bead- to-T cell ratio) and were propagated in media without cytokines or pharmacologic agents.
  • FIGURES 19A-19D illustrate that extended culture of de-differentiated T cells in the hybrid T1I2/TREG polarization condition containing pemetrexed results in the generation of CD4 + T cells expressing FOXP3 and GATA3 transcription factors.
  • Human CD4 + and CD8 + T cells were subjected to a 3-day de-differentiation procedure and subsequently were co-stimulated (3: 1 bead-to-T cell ratio) and propagated in media containing the hybrid T1I2/TREG polarizing condition (IL-2; TGF-b; IL-4) either without or with the pharmacologic inhibitors temsirolimus (1.0 mM) or pemetrexed (10 nM).
  • T cells were harvested and subjected to surface flow cytometry (CD4 marker) and intra-cellular staining for the following transcription factors, FOXP3, Tbet, and GAT A3.
  • FIGURE 19A The data above show the percent CD4 cells out of the total cultured population (FIGURE 19A); the percent of CD4 cells that expressed the TREG transcription factor FOXP3 (FIGURE 19B); the percent of CD4 cells that expressed the Thl transcription factor Tbet (FIGURE 19C); and the percent of CD4 cells that expressed the Th2 transcription factor GAT A3 (FIGURE 19D).
  • FIGURES 19A-19D T cells re-differentiated in the TREG-Th2 condition had a gradual shift towards CD4 cell predominance over time in culture (FIGURE 19A).
  • FIGURE 19B CD4 cells expressed FOXP3 at a high frequency and in a stable manner from day 12 through day 32 of culture independent of temsirolimus or pemetrexed presence in culture.
  • FIGURE 19C indicates, there was a very low frequency of contamination with the Thl transcription factor TBET even without pharmacologic inhibitor presence. However, the most consistently reduced TBET values were observed in the hybrid polarization conditions that also included pemetrexed. Finally, as shown in FIGURE 19D, the highest end-of-culture Th2- associated GATA3 expression was observed in the T cells manufactured in the hybrid TREG-T1I2 condition that was supplemented with pemetrexed.
  • FIGURES 20A-20D illustrate that extended culture of de-differentiated T cells in the hybrid T1I2/TREG polarization condition containing pemetrexed results in the generation of CD8 + T cells expressing FOXP3 and GATA3 transcription factors.
  • Human CD4 + and CD8 + T cells were subjected to a 3-day de-differentiation procedure and subsequently were co-stimulated (3: 1 bead-to-T cell ratio) and propagated in media containing the hybrid T1I2/TREG polarizing condition (IL-2; TGF-b; IL-4) either without or with the pharmacologic inhibitors temsirolimus (1.0 mM) or pemetrexed (10 nM).
  • T cells were harvested and subjected to surface flow cytometry (CD8 marker) and intra-cellular staining for the following transcription factors, FOXP3, Tbet, and GAT A3.
  • FIGURE 20A The data above show the percent CD8 cells out of the total cultured population (FIGURE 20A); the percent of CD8 cells that expressed the TREG transcription factor FOXP3 (FIGURE 20B); the percent of CD8 cells that expressed the Thl transcription factor Tbet (FIGURE 20C); and the percent of CD8 cells that expressed the Th2 transcription factor GATA3 (FIGURE 20D).
  • CD8 cell content gradually and modestly diminished over time in culture.
  • TREG cell function is generally attributed to the CD4 cell subset
  • CD8 + TREG cells have also been well described; it is possible that use of a TREG population that contains both CD4 + and CD8 + T cell subsets may be advantageous due to diversification of antigen-specificity. As such, the method we describe is potentially advantageous in-part because it generates both CD4- and CD8-type TREGS.
  • FIGURE 20B shows the CD8 + T cells manufactured using this method were indeed enriched for FOXP3 expression, which was stable over time in culture and stable independent of pharmacologic inhibitor presence.
  • FIGURE 20C shows, re-differentiation in the TREG-T1I2 polarization condition in general led to CD8 + T cell expression of low levels of the Thl transcription factor TBET; however, the lowest levels were observed most consistently in the presence of pemetrexed.
  • FIGURE 20D shows, re-differentiation in the TREG-T1I2 condition indeed resulted in CD8 + T cells that were also shifted towards Th2-type differentiation, as indicated by increased expression of the GATA3 transcription factor.
  • FIGURES 31A-31B also depict flow cytometry of GATA3 and FOXP3 for the re- differentiated TREG-Th2 cells in both the CD4 + and CD8 + subsets.
  • Example 16 Culture of De-differentiated T Cells in the Hybrid TREG/HI2 Condition Results in T Cells With an Enhanced Th2 Cytokine Secretion Profile
  • FIGURES 21A-21D illustrate that extended culture of de-differentiated T cells in the hybrid T1I2/TREG polarization condition results in the generation of T cells expressing with a predominant Th2 cytokine phenotype: IL-4, IL-5, and IL-13 secretion.
  • Human CD4 + and CD8 + T cells were subjected to a 3-day de-differentiation procedure and subsequently were co-stimulated (3: 1 bead-to-T cell ratio) and propagated in media containing the hybrid T1I2/TREG polarizing condition (IL-2; TGF-b; IL-4) either without or with the pharmacologic inhibitors temsirolimus (1.0 mM) or pemetrexed (10 nM).
  • Cultures were restimulated with 3/28 beads at both day 14 and day 24 of culture; at day 24 of culture, to evaluate stability of the transcription factor expression, the culture media did not contain exogenous cytokines or pharmacologic inhibitors.
  • the T cells were harvested, washed, and re-stimulated with 3/28 beads (3: 1 ratio) for 24 hr; the resultant supernatant was harvested and tested for cytokine content by Luminex multi-analyte method. All results shown are expressed as cytokine level in pg per ml per 1 x 10 6 cells/ml/24 hr.
  • the Th2 cytokine IL-10 was also evaluated: all values were less than 20 pg/ml per 1 x 10 6 cells/ml/24 hr.
  • T cells re-differentiated in the TREG-T1I2 polarizing condition had relatively low level expression of IL-2 (FIGURE 22A), IFN-g, (FIGURE 22B), IL-17 (all values less than 20 pg/ml), and TNF-a (all values less than 20 pg/ml).
  • FIGURES 22A-22D illustrate that extended culture of de-differentiated T cells in the hybrid T1I2/TREG polarization condition results in the generation of T cells expressing with a predominant Th2 cytokine phenotype: IL-2, IFN-g, and GM-CSF secretion.
  • Human CD4 + and CD8 + T cells were subjected to a 3-day de-differentiation procedure and subsequently were co stimulated (3: 1 bead-to-T cell ratio) and propagated in media containing the hybrid T1I2/TREG polarizing condition (IL-2; TGF-b; IL-4) either without or with the pharmacologic inhibitors temsirolimus (1.0 mM) or pemetrexed (10 nM).
  • Cultures were restimulated with 3/28 beads at both day 14 and day 24 of culture; at day 24 of culture, to evaluate stability of the transcription factor expression, the culture media did not contain exogenous cytokines or pharmacologic inhibitors.
  • the T cells were harvested, washed, and re stimulated with 3/28 beads (3: 1 ratio) for 24 hr; the resultant supernatant was harvested and tested for cytokine content by Luminex multi-analyte method. All results shown are expressed as cytokine level in pg per ml per 1 x 10 6 cells/ml/24 hr.
  • the inflammatory cytokines IL-17 and TNF-a were also evaluated: all values were less than 20 pg/ml per 1 x 10 6 cells/ml/24 hr.
  • T cell re-differentiation in the TREG-T1I2 polarizing condition is favorable because it results in T cells with a low level of capacity for secretion of the Thl- and Thl7-type cytokines associated with inflammatory disease.
  • Inclusion of pemetrexed to the hybrid TREG-T1I2 polarizing condition is advantageous because it results in an increased capacity for Th2 cytokine production, which will further provide a hedge against differentiation plasticity towards the Thl- and Thl7-type subsets.
  • Example 17 Use of Select anti-TNF- a reagents prior to lymphocyte collection by apheresis to beneficially alter the input T cell TCR repertoire
  • FIGURES 23 A & 23B depict the use of RNA-based T cell receptor sequencing to detect a widespread up- and down-regulation of T cell TCR specificities after therapy with the TNF-a inhibitor, etanercept.
  • RNA was isolated from peripheral blood mononuclear cells from an ALS patient pre- and post-therapy with etanercept therapy. The RNA was subjected to TCR repertoire profiling, as previously described by Rosati E, Dowds CM, Liaskou E, Henriksen EKK, Karlsen TH, Franke A. Overview of methodologies for T-cell receptor repertoire analysis. BMC Biotechnol. 2017;17(1):6T.
  • FIGURE 23A it is demonstrated that approximately 25% of TCR specificities were up-regulated in the post-therapy sample (as indicated in red); in marked contrast, approximately 25% of TCR specificities were down-regulated in the post-therapy sample (as indicated in blue).
  • etanercept therapy resulted in marked T cell clonal expansion, as several T cell clones increased from frequencies of 0.01 pre-etanercept (near the detection limit of the assay) to post-treatment values ranging from 247 to 486, thereby consistent with a more than 4-log T cell expansion.
  • etanercept therapy resulted in marked T cell clonal contraction, as several T cell clones decreased from frequencies of 259 to 598 pre-etanercept to post-treatment values of 0.01, thereby consistent with a more than 4-log T cell clonal contraction.
  • FIGURES 23A-B indicates that anti-TNF-a therapy with etanercept, which preferentially inhibits the serum, cell-free form of TNF-a that promotes TNFR1 -expressing Thl-type cells, is associated with widespread changes in T cell receptor up- and down-regulation.
  • pre-treatment of a subject with etanercept or any other anti-TNF-a therapeutic that preferentially inhibits the serum, cell-free form of TNF-a can be utilized to shift the T cell receptor repertoire away from T cells of Thl-type phenotype on an antigen-specific basis, thereby enriching for T cells of a TREG phenotype on an antigen-specific basis.
  • Example 18 Characterization of the TREG-Th2 hybrid population as a cell product enriched for expression of CD25, CD27, 2B4, BTLA, and CTLA4.
  • FIGURE 24 illustrates that the manufactured iTREG/Th2 hybrid population has increased expression of CD25, CD27, 2B4, BTLA, and CTLA4 relative to control Thl/Tcl cultures.
  • the iTREG/Th2 hybrid population was generated by the method previously detailed using an initial phase of T cell de-differentiation followed by re-differentiation in media containing IL-2, TGF-b, and IL-4.
  • the cells were harvested and subjected to flow cytometry for assessment of CD4 + and CD8 + T cell expression of molecules of relevance, namely CD25, CD27, 2B4, BTLA, and CTLA4; comparison was made to three separate control conditions evaluating Thl/Tcl polarization.
  • FIGURE 24 indicates that hybrid TREG-UTZ cells manufactured according to the described conditions have increased expression of the following cell surface molecules by flow cytometry relative to control Thl/Tcl cells: CD25, CD27, 2B4, BTLA, and CTLA4.
  • the ITREG/UTZ hybrid cell product has CD4 + and CD8 + T cells that express at least 10% and more preferably 50% higher levels of CD25, CD27, 2B4, BTLA, and CTLA4 relative to control Thl/Tcl cells.
  • CD25 the IL-2 receptor
  • CD25 is critical for the ability of TREG cells to control autoimmunity, in particular CD8 + T cell driven responses. Therefore, expression of CD25 on the iTREG/Th2 manufactured cell product is a desirable characteristic.
  • CD27 a co-stimulatory molecule with increased expression on TREG cells, has been shown to contribute to the inhibitory function of TREGS. Therefore, expression of CD27 on the iTREG/Th2 manufactured cell product is a desirable characteristic.
  • 2B4 (CD244) has recently been shown to inhibit CD8+ T cell responses by attenuation of glycolysis and cell division. Therefore, expression of 2B4 on the iTREG/Th2 manufactured cell product is a desirable characteristic.
  • BTLA (CD272) is a co-inhibitory receptor, and the ligation of BTLA with the herpesvirus-entry mediator HVEM promotes TREG cell induction and inhibition of effector immune responses. Therefore, expression of BTLA on the iTREG/Th2 manufactured cell product is a desirable characteristic.
  • CTLA4 is a critical effector molecule of TREG cells, as recently evidenced by its ability to limit immunity to malarial infection. Therefore, expression of CTLA4 on the iTREG/Th2 manufactured cell product is a desirable characteristic.
  • Example 19 Characterization of the TKEO- ⁇ I2 hybrid population as a cell product enriched for expression of 7/67/7 ’ 7 M3, ICOS, LAIR1, and 0X40.
  • FIGURE 25 illustrates that the manufactured iTREG/Th2 hybrid population has increased expression of TIGIT, TIM3, ICOS, LAIR1, and 0X40 relative to control Thl/Tcl cultures.
  • the iTREG/Th2 hybrid population was generated by the method previously detailed using an initial phase of T cell de-differentiation followed by re-differentiation in media containing IL-2, TGF-b, and IL-4.
  • FIGURE 25 indicates that hybrid TREG-Th2 cells manufactured according to the described conditions have increased expression of the following cell surface molecules by flow cytometry relative to control Thl/Tcl cells: TIGIT, TIM3, ICOS, LAIR1, and 0X40.
  • the iTREG/Th2 hybrid cell product has CD4 + and CD8 + T cells that express at least 10% and more preferably 50% higher levels of TIGIT, TIM3, ICOS, LAIR1, and 0X40 relative to control Thl/Tcl cells.
  • TIGIT is a cell surface co-inhibitory receptor molecule that associates with regulatory T cell function. Therefore, expression of TIGIT on the ITREG/TITZ manufactured cell product is a desirable characteristic.
  • TIM3 is a co-inhibitory receptor that mediates an inhibitory effect of TREG cells. Therefore, expression of TIM3 on the ITREG/TITZ manufactured cell product is a desirable characteristic.
  • ICOS is a co-stimulatory molecule that was recently determined to help maintain immune suppression by regulatory T cells for control of immune reactivity in the central nervous system. Therefore, expression of ICOS on the ITREG/TITZ manufactured cell product is a desirable characteristic.
  • LAIRl (CD305) is a multi-faceted inhibitory molecule that can block inflammation at multiple steps, including the suppression of activated, effector memory T cells. Therefore, expression of LAIRl on the ITREG/TITZ manufactured cell product is a desirable characteristic.
  • 0X40 is a co-stimulatory molecule. Therefore, expression of 0X40 on the ITREG/TITZ manufactured cell product is a desirable characteristic.
  • a steady-state apheresis sample was obtained and enriched for lymphocytes by a Ficoll gradient and then plated in a G-Rex culture vessel and incubated in complete media containing Vitamin D (0.3 nM), temsirolimus (3.0 mM) and basiliximab (30 pg/mL). After an initial de- differentiation interval, the T cells were co-stimulated at a 3: 1 bead-to-T cell ratio with anti- CD3/anti-CD28-coated magnetic beads and cytokines were added (IL-4 (1000 IU/mL), IL-2 (10,000 IU/mL) and TGF-b (100 ng/mL)).
  • FIGURES 26A-B show the FOXP3 and GATA3 expression for CD4 + and CD8 + T cells at the start of culture and after culture (T1I2/TREG) as measured by flow cytometry.
  • the percentages provided indicate the amount of cells considered positive for CD4 + or CD8 + and the intracellular marker (shown in boxes).
  • FIGURES 26A-B show results that are indicative of the phenotype of the manufactured T1I2/TREG cell product.
  • T cells of type II cytokine phenotype can be characterized in part by their expression of the transcription factor GATA3 whereas regulatory T cell populations can be identified in part by their expression of FoxP3 transcription factor.
  • GATA3 the transcription factor for T1I2/TREG cells
  • FoxP3 the transcription factor for T1I2/TREG cell product
  • the T cell product manufactured in the T1L2/TREG culture conditions expressed a high frequency of T cells that were either single-positive for GATA3, single-positive for FOXP3, or double-positive for both GATA3 and FOXP3 (not shown); importantly, as shown, this transcription factor profile was expressed in both manufactured CD4 + (top panels) and CD8 + (bottom panels) T cells.
  • a control manufacturing culture that did not include IL-4 resulted in a greatly reduced frequency of GAT A3 -positive T cells, thereby demonstrating the important role of IL-4 in the manufacture of the T1I2/TREG hybrid population (not shown).
  • the TREG/T1I2 cells in the post-thaw state can be characterized by the following relative to control Thl/Tcl cells: (a) increased expression of CD25, CD27, 2B4, BTLA, CTLA4, TIGIT, TIM3, ICOS, LAIRl, and 0X40 by flow cytometry; (b) reduced IFN-g and TNF-a and increased secretion of IL-4 by Luminex cytokine secretion analysis; and (c) altered expression of T cell fate transcription factors, namely reduced TBET and increased FOXP3 and GAT A3.
  • Example 21 Characterization of CD73 and CD103 Expression of the TREc/Th2 hybrid population
  • a steady-state apheresis sample was obtained and enriched for lymphocytes by a Ficoll gradient and then plated in a G-Rex culture vessel and incubated in complete media containing Vitamin D (0.3 nM), temsirolimus (3.0 mM) and basiliximab (30 pg/mL). After an initial de- differentiation interval, the T cells were co-stimulated at a 3: 1 bead-to-T cell ratio with anti- CD3/anti-CD28-coated magnetic beads and cytokines were added (IL-4 (1000 IU/mL), IL-2 (10,000 IU/mL) and TGF-b (100 ng/mL)).
  • FIGURES 27A-B show the CD73 and CD 103 expression for CD4 + and CD8 + T cells at the start of culture and after culture (T1I2/TREG) as measured by flow cytometry.
  • the percentages provided indicate the amount of cells considered positive for CD4 + or CD8 + and the ectonucleotidase molecule or integrin molecule, respectively (shown in boxes).
  • Regulatory T cell populations can suppress pathogenic effector T cell populations by several defined mechanisms, including through expression of CD39 and CD73 ectonucleotidase molecules, which act to hydrolyse pro-inflammatory ATP towards the immune suppressive adenosine substrate.
  • TREG cells that express CD39 possess increased suppressive function and have been associated with resolution of inflammatory bowel disease.
  • suppressive function of human TREG cells is mediated in part by CD73.
  • T cells manufactured in the T1I2/TREG culture condition can have an increase in expression of the TREG-associated effector molecule, CD73; CD39 was also highly expressed on the TREG/Th2-manufactured T cells (not shown).
  • TREG cell function has also been correlated with expression of CD103, which is an integrin that dictates epithelial lymphocyte localization
  • CD 103 and IL-2 receptor signaling cooperate to maintain immune tolerance in the gut mucosa; furthermore, CD 103- expressing TREG cells are critical for amelioration of experimental chronic GVHD.
  • T cells manufactured in the T1I2/TREG culture condition can have an increase in expression of the TREG-associated effector molecule, CD! 03.
  • Example 22 Characterization of CD 150 and CD27/CD95 Expression of the TnEG/Th2 hybrid population
  • a steady-state apheresis sample was enriched for lymphocytes by a Ficoll gradient, plated into a G-Rex culture vessel, and incubated in complete media containing Vitamin D (0.3 nM), temsirolimus (3.0 mM) and basiliximab (30 pg/mL). After this initial de-differentiation interval, the T cells were co-stimulated at a 3: 1 bead:T cell ratio with anti-CD3/anti-CD28- coated magnetic beads and cytokines were added (IL-4 (1000 IU/mL), IL-2 (10,000 IU/mL) and TGF-b (100 ng/mL)).
  • T cells were harvested, stained for surface markers, and subjected to multi-color flow cytometric analysis for CD4, CD8, CD 150, CD27, CD95, CD45RA, CD62L, and CCR7. Results are shown in FIGURES 28A-B.
  • T cell cultured in the TREG (RAPA-501) condition were compared to the culture input T cells (“Day 0”) and also compared to control culture T cells that were propagated without mTOR inhibitors (“Control”).
  • both CD4+ and CD8+ T cell subsets contained within the RAPA-501 cell product had greatly increased expression of the stem cell marker CD 150 relative to culture input T cells and relative to control cultured T cells.
  • the RAPA-501 cell product was also enriched for a T stem cell memory (TSCM) phenotype relative to culture input cells; the control culture was devoid of this population, as the resultant T cells in this condition were effector memory, CD45RO + (not shown).
  • TSCM T stem cell memory
  • the left panel (culture input T cells) and the right panel (RAPA-501 cells) show expression of the TSCM markers CD95 and CD27 after gating on the TSCM markers CD45RA, CD62L, and CCR7; a similar difference in expression of these TSCM markers was observed for CD8 + T cells (not shown).
  • TCM T central memory
  • T cells with reduced differentiation relative to the T effector memory (TEM) population have increased in vivo persistence and mediate increased in vivo effects, including the TCM subset, the naive T cell subset, and more recently, the T stem cell memory (TSCM) subset.
  • This relationship between T cell differentiation status and in vivo T cell function is operational relative to TREG cells, as: (1 ) TREG cells of TCM phenotype were more effective at reducing experimental GVHD relative to TREG cells of TEM phenotype; and (2) TREG cells that expressed the stem cell marker CD 150 were highly effective for the prevention of stem cell graft rejection.
  • T cells manufactured in the T1I2/TREG culture condition were enriched for cells a reduced differentiation state consistent with a T stem cell subset, including expression of the CD 150 marker.
  • a steady-state apheresis sample was enriched for lymphocytes by a Ficoll gradient, plated into a G-Rex culture vessel, and incubated in complete media containing Vitamin D (0.3 nM), temsirolimus (3.0 mM) and basiliximab (30 pg/mL). After an initial de-differentiation interval, the T cells were co-stimulated at a 3: 1 bead:T cell ratio with anti-CD3/anti-CD28- coated magnetic beads and cytokines were added (IL-4 (1000 IU/mL), IL-2 (10,000 IU/mL) and TGF-b (100 ng/mL)).
  • Condition“A” This culture is termed Condition“B.”
  • Condition“B” was the same culture condition but without IL-4 addition.
  • Condition“C” reflects the standard TREG culture condition of rapamycin (1 pM), IL-2 (100 IU/mL), and TGF-b (10 ng/mL).
  • Condition“D” reflects a Thl-type control culture manufactured in the presence of IFN-a without mTOR inhibitors. After culture, the T cells were harvested, stimulated with anti-CD3/anti-CD28 beads, and the resultant supernatant was tested for cytokine content by Luminex assay.
  • T1I2/TREG cells It can be important to assess cytokine secretion of the manufactured T1I2/TREG cells. First, it is critical that the cell product can secrete IL-4, which is the driver cytokine for subsequent Th2 differentiation. Second, it is desirable that an adoptively transferred T cell population is capable of secreting IL-2, as this capacity indicates a progenitor function that permits T cells to expand more readily in vivo without the need for exogenous IL-2. Finally, it is important that the T1I2/TREG cell population has reduced secretion of the Thl- or Thl7-type cytokines IFN-a, TNF- a, IL-17, and GM-CSF. As FIGURE 29 illustrates, the manufactured T1I2/TREG cell product secreted IL-4 and IL-2 with minimal secretion of Thl- or Thl7-type cytokines.
  • Example 24 Characterization of Thl/Tcl Suppression by the TREc/Th2 hybrid population
  • a steady-state apheresis sample was enriched for lymphocytes by a Ficoll gradient, plated into a G-Rex culture vessel, and incubated in complete media containing Vitamin D (0.3 nM), temsirolimus (3.0 mM) and basiliximab (30 pg/mL). After an initial de-differentiation interval, the T cells were co-stimulated at a 3: 1 bead:T cell ratio with anti-CD3/anti-CD28- coated magnetic beads and cytokines were added (IL-4 (1000 IU/mL), IL-2 (10,000 IU/mL) and TGF-b (100 ng/mL)) for ex vivo manufacture of Th2/TREG cells.
  • Thl/Tcl cells were cultured in the presence of the type I polarizing cytokine IFN-a to generate effector Thl/Tcl cells; the Thl/Tcl culture was generated from the same donor as the RAPA-501 cell culture (autologous;“AUTO”) or from an unrelated donor (allogeneic;“ALLO”).
  • the Thl/Tcl effector T cells were plated in the bottom chamber of a transwell plate and co stimulated with anti-CD3/anti-CD28 coated beads at a bead-to-T cell ratio of 3 : 1.
  • RAPA-501 cells were added to the top chamber of the transwell plate at a Thl/Tcl-to-RAPA 501 ratio of 1 : 1.
  • FIGURES 30A-B illustrate that the RAPA-501 cells modulate effector T cells in a contact-independent manner (experiments performed in a transwell vessel).
  • RAPA-501 cells acted in a T cell receptor independent manner to suppress the cytokine secretion capactiy of effector T cells; that is, because no co-stimulation beads were added to the transwell chamber containing the RAPA-501 cells, RAPA-501 cells did not require co stimulation in order to modulate inflammatory cytokine levels, including IL-2, IFN-g, GM-CSF, and TNF-a (FIGURE 30A).
  • the ability of TREG cells to consume IL-2 is a commonly described phenomenon, although previous studies identified the requirement of cell-to-cell contact for IL-2 consumption. As such, RAPA-501 cells appear to be somewhat uniquely capable of modulating the level of multiple inflammatory cytokines in a contact-independent manner.
  • RAPA-501 cells represent a suitable candidate for neutralization of cytokines.
  • RAPA-501 cells modulated additional aspects of effector T cell biology in a contact-independent manner (use of transwell experiments), namely, the promotion of programmed death-1 (PD-1) checkpoint molecule expression on the effector T cells.
  • PD-1 programmed death-1
  • FIGURE 30B RAPA-501 cells up-regulated PD1 expression on both autologous and allogeneic Thl/Tcl cells, thereby further clarifying that one mechanism of RAPA-501 cell suppressive function occurs in a TCR-independent manner by soluble mediators.
  • a method for de-differentiation of T cells comprising: inoculating a culture input population of cells comprising T cells from a subject at a cell density in a culture medium comprising vitamin D, temsirolimus and an IL-2 signaling inhibitor; adding anti-CD3/anti-CD28 coated magnetic beads to said T cells and culture medium at a bead:T cell ratio of 1 : 1 to 1 : 12; incubating said culture input population of cells and culture medium for a period of time to yield de-differentiated T cells.
  • a de-differentiated T cell produced by the method of any one of embodiments 1-22.
  • a composition comprising a population of de-differentiated T cells, wherein at least a portion of said population of said de-differentiated T cells express less than 50% of both RAPTOR or RICTOR as compared to a control population of T cells, wherein the control population of T cells is manufactured under the same conditions as the culture input population of cells without temsirolimus, IL-2 signaling inhibitor and Vitamin D.
  • a method for de-differentiation of T cells comprising: inoculating a culture input population of cells comprising T cells from a subject at a cell density in a culture medium comprising vitamin D and temsirolimus; adding anti-CD3/anti-CD28 coated magnetic beads to said T cells and culture medium at a bead:T cell ratio of 1 : 1 or less to stimulate said T cells; incubating said culture input population of cells and culture medium for a period of time to yield de-differentiated T cells.
  • any one of embodiments 25-32 and 34-38 further comprising: measuring an expression level of RAPTOR or RICTOR in said culture input population of cells, wherein said period of time lasts until the expression level of RAPTOR or RICTOR in said culture input population of cells is reduced by at least 50% relative to a control population of T cells manufactured under the same conditions as the culture input population of cells without temsirolimus, IL-2 signaling inhibitor and Vitamin D.
  • any one of embodiments 25-32 and 34-38 further comprising: measuring an expression level of RAPTOR, RICTOR and a housekeeping protein in said culture input population of cells, wherein said period of time lasts until the expression level of RAPTOR or RICTOR in the culture input population of cells is reduced by 50% or more preferably by 90% relative to a control population of T cells manufactured under the same conditions as the culture input population of cells without temsirolimus, IL-2 signaling inhibitor and Vitamin D after normalizing for housekeeping protein expression.
  • a de-differentiated T cell produced by the method of any one of embodiments 25-43.
  • a de-differentiated T cell population characterized by at least a 10% reduction and more preferably a 50% reduction in expression of RNA for the following T cell differentiation molecules relative to a control population of T cells cultured without the culture additives specified in these methods: cytolytic molecules, including but not limited to granzyme B; and cytokine molecules, including but not limited to IFN-g.
  • a de-differentiated T cell population characterized by at least a 10% increase and more preferably a 50% increase in expression of RNA for the following T cell differentiation molecules relative to a control population of T cells cultured without the culture additives specified in these methods: transcription factors associated with induced pluripotent stem cells, including but not limited to Nanog, KLF4, and KLF10; and molecules associated with naive T cells, including but not limited to the IL-7 receptor, CD 127.
  • a de-differentiated T cell population characterized by at least a 10% decrease and more preferably a 50% decrease in expression of RNA for the following T cell differentiation molecules relative to a control population of T cells cultured without the culture additives specified in these methods: transcription factors associated with Thl effector T cells, including but not limited to T-Bet and STAT1; however, concomitantly, the manufactured T cells will have equivalent expression of transcription factors associated with cell survival, including but not limited to HIF-1 -alpha.
  • a de-differentiated T cell population characterized by at least a 10% increase and more preferably a 50% increase in expression of molecular markers of autophagy relative to a control population of T cells cultured without the culture additives specified in these methods, including but not limited to: an increase in protein level by Western Blot analysis of the autophagy-related molecule, p62.
  • HIF-1-a expression within about 20% of a control T cell population incubated under the same conditions without temsirolimus, vitamin D and the IL-2 signaling inhibitor;
  • an expression level of RAPTOR or RICTOR reduced by at least 50% and more preferably by 90% relative to a control population of T cells manufactured under the same conditions as the culture input population of cells without temsirolimus, IL-2 signaling inhibitor and Vitamin D; an expression level of RAPTOR or RICTOR normalized by a housekeeping protein is reduced by at least 50% and more preferably by 90% relative to a control population of T cells manufactured under the same conditions as the culture input population of cells without temsirolimus, IL-2 signaling inhibitor and Vitamin D; and
  • a de-differentiated T cell characterized by one or more of the following properties: at least a 10% decrease, and more preferably, a 50% decrease in mRNA expression of one or more of granzyme B, IL-10, and IFN-g relative to a control T cell incubated under the same conditions without temsirolimus, vitamin D and the IL-2 signaling inhibitor;
  • HIF-1-a expression within about 20% of a control T cell incubated under the same conditions without temsirolimus, vitamin D and the IL-2 signaling inhibitor;
  • an expression level of RAPTOR or RICTOR reduced by at least 50% and more preferably by 90% relative to a control T cell manufactured under the same conditions as the culture input population of cells without temsirolimus, IL-2 signaling inhibitor and Vitamin D; an expression level of RAPTOR or RICTOR normalized by a housekeeping protein is reduced by at least 50% and more preferably by 90% relative to a control T cell manufactured under the same conditions as the culture input population of cells without temsirolimus, IL-2 signaling inhibitor and Vitamin D; and

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Abstract

La présente invention concerne des procédés de production de lymphocytes hybrides TREG et TREG/TH2 à partir de lymphocytes T dédifférenciés, lesdits lymphocytes hybrides TREG et TREG/TH2, des populations et des compositions de ceux-ci. La présente invention concerne des procédés de production de lymphocytes T dédifférenciés, lesdits lymphocytes T dédifférenciés, des populations et des compositions de ceux-ci.
PCT/US2019/061811 2018-11-16 2019-11-15 Procédé de fabrication de lymphocytes t/th2 régulateurs hybrides humains (lymphocytes treg/th2 hybrides) à partir de lymphocytes t dédifférenciés Ceased WO2020102726A1 (fr)

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WO2012143516A1 (fr) * 2011-04-20 2012-10-26 Genzyme Polyclonals S.A.S. Amplification de lymphocytes t régulateurs au moyen d'immunoglobulines anti-thymocyte avec une cytokine, un inhibiteur de protéine kinase tor et/ou un agent de différenciation
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WO2016138091A2 (fr) * 2015-02-24 2016-09-01 Board Of Regents, The University Of Texas System Procédés de sélection de lymphocytes t génétiquement modifiés
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WO2012143516A1 (fr) * 2011-04-20 2012-10-26 Genzyme Polyclonals S.A.S. Amplification de lymphocytes t régulateurs au moyen d'immunoglobulines anti-thymocyte avec une cytokine, un inhibiteur de protéine kinase tor et/ou un agent de différenciation
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WO2016138091A2 (fr) * 2015-02-24 2016-09-01 Board Of Regents, The University Of Texas System Procédés de sélection de lymphocytes t génétiquement modifiés

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COPSEL ET AL.: "Very Low Numbers of CD 4+ FoxP3+Tregs Expanded in Donors via TL1A-lg and Low-Dose IL -2 Exhibit a Distinct Activation/Functional Profile and Suppress GVHD in a Preclinical Model", BIOL BLOOD MARROW TRANSPLANT, vol. 24, no. 9, 8 May 2018 (2018-05-08), pages 1788 - 1794, XP085487864, DOI: 10.1016/j.bbmt.2018.04.026 *
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