Method for T Cell De-differentiation and Resulting Cells

CROSS-REFERENCE TO RELATED APPLICATIONS

[1] The present application claims priority to U.S. Provisional Application 62/768,153, filed November 16, 2018 and U.S. Provisional Application No. 62/927,061, filed October 28, 2019, the entirety of each of which is incorporated herein by reference.

BACKGROUND

[2] 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. However, 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. Approaches can be taken to alleviate this limitation, including the isolation (purification) of more naive T cell subsets at the time of culture initiation; however, this approach is restricted in part by the small number of naive T cells present in the adult human peripheral blood. Accordingly, a great need exists for isolated T cells in primitive differentiation states.

[3] It is well known that even highly 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. 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. [4] However, the ability to convert somatic cells to iPS cells is inefficient and dependent in part upon the degree of somatic cell differentiation. As one example, 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. Over the past decade, investigators have also characterized transcription factors associated with the earliest stages of T cell differentiation. However, 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.

[5] Although the biology of de-differentiation is becoming increasingly characterized, a great deal remains unknown in terms of the specific transcription factors and transcription factor kinetics associated with de-differentiation. It is also important to recognize that gene transfer methods of achieving de-differentiation are laborious and associated with complications such as teratoma generation that must be addressed through additional genetic interventions such as cell fate suicide gene programming. As a potential alternative, various pharmacologic interventions can be utilized to achieve some degree of de-differentiation. As one example, calcineurin- inhibition during cell culture by use of the immune suppressive agent cyclosporine resulted in molecular alterations that replaced the need for gene delivery of Sox2 transcription factor for the promotion of murine iPS cells. In addition, rapamycin, which is an immune suppression drug that inhibits the mammalian target of rapamycin (mTOR), can result in a de-differentiation effect on end-stage effector T cells through starvation-induced up-regulation of the transcription factor KLF2, which in turn increases the T central-memory molecules CD62L and CCR7. In addition, 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. It is important to note that 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). Inhibition of both mTORCl and mTORC2 has been associated with an increase in memory T cell promotion and maintenance. Of note, 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). In further studies, inhibition of the 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. In further experimental model research, 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.

[6] It has been demonstrated that blockade of mTOR through ex vivo culture of human T cells in rapamycin reduces T cell expression of molecules associated with effector

differentiation, such as cytokine secretion molecules and cytolytic effector molecules.

[7] In addition, the 1, 25-hydroxylated form of Vitamin D (“Vitamin D” as used herein) 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. However, previous research indicated that the inhibitory effect of Vitamin D on T cell effectors was relatively specific for Thl-type molecules rather than Th2- type molecules. Furthermore, Vitamin D was shown to promote the immune suppressive regulatory T (TREG) cell population.

[8] In a somewhat contradictory finding, it was determined that human CD8 ⁺ T cells express high levels of the Vitamin D receptor, and individuals with the highest values tended to have high levels of T cell effector function and immune senescence.

[9] In more recent research, using a murine model of Mycobacterium tuberculosis infection, it was demonstrated that Vitamin D was critical for macrophage elimination of intracellular pathogens through a mechanism that involved IFN-g production and autophagy. In addition, in human non-small cell cancer cell lines, Vitamin D signaling can promote a cytotoxic form of autophagy that contributes to an anti-tumor effect when combined with radiation. Finally, 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. In spite of this evidence linking Vitamin D to autophagy in innate immunity

(macrophage context), there exists a paucity of data relating to Vitamin D effects on autophagy in T cells during adaptive immunity, and in this context, whether the potential effects of Vitamin D and rapamycin on T cell autophagy are redundant.

[10] These somewhat conflicting results pertaining to the potential role of Vitamin D in T cell biology likely relates to the recently discovered, wide-spread effects of Vitamin D on the entire genome, both at the mRNA level and at the microRNA level. As such, the effects of Vitamin D on immunity need to be evaluated in a context-dependent framework.

SUMMARY

[11] The present disclosure is directed to methods for de-differentiation of T cells and the resulting de-differentiated T cells.

[12] In some embodiments, 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.

[13] In any of the foregoing embodiments, the method may further comprise harvesting said de-differentiated T cells.

[14] In any of the foregoing embodiments, 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.

[15] In any of the foregoing embodiments, 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. [16] In any of the foregoing embodiments, 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.

[17] In any of the foregoing embodiments, 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.

[18] The present disclosure is also directed to a de-differentiated T cell produced by the methods of any of the foregoing embodiments.

[19] The present disclosure is also directed to 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.

[20] The present disclosure is also directed to 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.

[21] The present disclosure is also directed to 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. As one, example, 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.

[22] The present disclosure is also directed to a de-differentiated T cell and populations thereof produced by the methods of the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

[23] FIGURE 1 A depicts normalized GAPDH mRNA expression for the control cells and cells treated under various conditions.

[24] FIGURE IB depicts normalized granzyme B mRNA expression for the control cells and cells treated under various conditions.

[25] FIGURE 1C depicts normalized IL-10 mRNA expression for the control cells and cells treated under various conditions.

[26] FIGURE ID depicts normalized IFN-g mRNA expression for the control cells and cells treated under various conditions.

[27] FIGURES 1 A-1D illustrate that the combination of Vitamin D and temsirolimus reduces effector molecule expression in human CD4 ⁺ and CD8 ⁺ T cells. [28] FIGURE 2A depicts normalized NANOG mRNA expression for the control cells and cells treated under various conditions.

[29] FIGURE 2B depicts normalized KLF4 mRNA expression for the control cells and cells treated under various conditions.

[30] FIGURE 2C depicts normalized KLF10 mRNA expression for the control cells and cells treated under various conditions.

[31] FIGURE 2D depicts normalized IL-7 receptor mRNA expression for the control cells and cells treated under various conditions.

[32] 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.

[33] FIGURE 3 A depicts normalized T-BET mRNA expression for the control cells and cells treated under various conditions.

[34] FIGURE 3B depicts normalized STAT1 mRNA expression for the control cells and cells treated under various conditions.

[35] FIGURE 3C depicts normalized HIF-1-a mRNA expression for the control cells and cells treated under various conditions.

[36] 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.

[37] 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. [38] 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.

[39] 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.

[40] 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.

[41] FIGURE 8 illustrates the effect of culture components during the de-differentiation interval on subsequent T cell yield (at day 13 of culture).

[42] FIGURE 9A depicts the percent of CD4 cells that are CD45RA ⁺ for cells treated under various conditions.

[43] FIGURE 9B depicts the percent of CD4 cells that are CD62L ⁺ and CCR7 ⁺ for cells treated under various conditions.

[44] FIGURE 9C depicts the percent of CD4 cells that are CD62L ⁺ , CCR7 ⁺ , and CD127 ⁺ for cells treated under various conditions.

[45] 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).

[46] FIGURE 10A depicts the percent of CD8 cells that are CD62L ⁺ and CCR7 ⁺ for cells treated under various conditions.

[47] FIGURE 10B depicts the percent of CD8 cells that are CD62L ⁺ , CCR7 ⁺ , and CD127 ⁺ for cells treated under various conditions. [48] FIGURES 10A-10B illustrate the effect of culture components during the de- differentiation interval on CD8 ⁺ T cell expression of memory markers.

[49] FIGURES 11 A-l ID depict the inflammatory Thl/Thl7 cytokine analysis of cultured de differentiated T cells in polarization-neutral media.

[50] FIGURE 11 A depicts the IFN-g secretion for cells treated under various conditions.

[51] FIGURE 1 IB depicts the GM-CSF secretion for cells treated under various conditions.

[52] FIGURE 11C depicts the TNF-a secretion for cells treated under various conditions.

[53] FIGURE 1 ID depicts the IL-17 secretion for cells treated under various conditions.

[54] FIGURES 12A-12D depict the IL-2 and Th2-type cytokine analysis of cultured de differentiated T cells in polarization-neutral media.

[55] FIGURE 12A depicts the IL-2 secretion for cells treated under various conditions.

[56] FIGURE 12B depicts the IL-4 secretion for cells treated under various conditions.

[57] FIGURE 12C depicts the IL-5 secretion for cells treated under various conditions.

[58] FIGURE 12D depicts the IL-13 secretion for cells treated under various conditions.

[59] FIGURE 13 depicts an exemplary workflow of a de-differentiation method of the present disclosure.

DETAILED DESCRIPTION

[60] The present disclosure provides methods for T cell de-differentiation and the resulting cells.

[61] As used herein, the singular forms“a”,“an” and“the” include plural referents unless the context clearly dictates otherwise. [62] The use of the term“or” in the claims and the present disclosure is used to mean“and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.

[63] Use of the term“about”, when used with a numerical value, is intended to include +/- 10%. For example, if a number of amino acids is identified as about 200, this would include 180 to 220 (plus or minus 10%).

[64] As used herein, 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. In certain aspects, 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.

[65] As used herein“control Thl/Tcl cells,” unless otherwise noted, 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. It should also be understood that where a control population of cells (or control T cell) is referred to as having been treated without culture additives, such as without temsirolimus, vitamin D and the IL-2 signaling inhibitor, this population (or T cell) 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.”

[66] As used herein, the term“anti-CD3/anti-CD28” should be understood to refer to anti- CD3/anti-CD28 antibodies. For example,“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. [67] It should also be understood that, in the present disclosure, where co-stimulation by anti- CD3/anti-CD28 antibodies is performed, this co-stimulation can be provided in any form of anti- CD3/anti-CD28 antibodies. By way of example, but not limitation, where co-stimulation is indicated as being performed by using anti-CD3/anti-CD28 beads, anti-CD3/anti-CD28 nanoparticles or microparticles can be used in an amount sufficient to achieve the equivalent effect.

[68] The present disclosure provides a 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.

[69] As shown in FIGURE 13, 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).

[70] In an embodiment, 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, manufacture of T cells without any 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.

[71] In any of the foregoing embodiments, said culture medium can not contain IL-2 and no IL-2 can added to said culture medium.

[72] In any of the foregoing embodiments, said cell density can be about 1.5 x 10 ⁶ T cells per mL to 18 x 10 ⁶ T cells per mL. By way of example but not limitation, 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. In some embodiments, by way of example but not limitation, it is anticipated that it may be preferable to initiate cell culture at higher densities such at 9 x 10 ⁶ T cells per mL or 18 x 10 ⁶ T cells per mL.

[73] In any of the foregoing embodiments, said temsirolimus can be present at a

concentration of about 0.3 mM to about 10 pM. By way of example but not limitation, 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 pM, 0.3 pM to about 0.5 pM, 0.5 pM to about 1 pM, 0.75 pM to about 1 pM, 0.5 pM to about 0.75 pM, 0.3 pM to about 10 pM, 0.3 pM to about 5 pM, 0.3 pM to about 3.3 pM, 1 pM to about 3.3 pM, 5 pM to about 10 pM, 3.3 pM to about 10 pM, 3.3 pM to about 5 pM, or, by way of example but not limitation, at a concentration of about 0.3 pM, 0.4 pM, 0.5 pM, 0.6 pM, 0.7 pM, 0.8 pM, 0.9 pM, or 1 pM, 2 pM, 3 pM, 3.3 pM, 4 pM, 5 pM, 6 pM, 7 pM,

8 pM, 9 pM, or 10 pM.

[74] In any of the foregoing embodiments, said IL-2 signaling inhibitor can be an anti-IL-2 receptor antibody or fragment thereof. By way of example but not limitation, said IL-2 signaling inhibitor can be basiliximab or daclizumab. By way of example but not limitation, 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 30 pg/mL, 20 to 30 pg/mL, or, by way of example but not limitation, at a concentration of about 5 pg/mL, 10 pg/mL, 15 pg/mL, 20 pg/mL, 25 pg/mL, 30 pg/mL, 35 pg/mL, 40 pg/mL, 45 pg/mL, or 50 pg/mL.

[75] In any of the foregoing embodiments, by way of example but not limitation, 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. In some embodiments, the level of mTORCl and mTORC2 reduction may be used as a guide to determine optimal culture interval. In some embodiments other molecular signatures of the de-differentiated cells can be used to determine optimal culture interval, including but not limited to: RNA expression of T cell effector molecules (i.e., decreased IFN-g); RNA expression of transcription factors (e.g, increased KLF4); evidence of an autophagy signature (e.g, increased p62); and up-regulation of markers present on naive T cell subsets (e.g, increased CD127), where the respective increases or decreases are with respect to a control population of T cells or control T cell.

[76] In any of the foregoing embodiments, by way of example but not limitation, said bead:T cell ratio can be 1 :3 or no co-stimulation can be performed. By way of example but not limitation, said bead:T cell ratio can be between 1 : 1 and 1 : 12, 1 : 1 and 1 :3, 1 :3 to 1 : 12. By way of further example but not limitation, 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. Finally, in the most extreme example, no anti-CD3/anti-CD28 co stimulation can be utilized, i.e. in some embodiments, no anti-CD3/anti-CD28 co-stimulation is performed.

[77] In any of the foregoing embodiments, 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. By way of example, but not limitation, 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. IX, about 0.01X to about 0.075X, about 0.01X to about 0.05X, about 0.01X to about 0.025X, about 0.025X to about 0.075X, about 0.025X to about 0.05X, about 0.05X to about 0.075X, or about 0.01X, about 0.025X, about 0.05X, about 0.075X, or about 0.01X the recommended dose. By way of example but not limitation, a reagent such as Miltenyi ^® T Cell TransAct™ 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,

1.1 pL (a nine-fold decrease) or about 0.1 IX. Finally, in the most extreme example, 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.

[78] Alternatively, if anti-CD3/anti-CD28 co-stimulation is to be used for producing manufactured T cells, the source of co-stimulation can be provided by dissolvable anti-CD3/anti- CD28 microparticles. By way of example, but not limitation, the dissolvable anti-CD3/anti- CD28 microparticles can be used at 20% of the strength recommended by the manufacturer (e.g. Cloudz ^® ; Bio-Techne). By way of further example, the dissolvable anti-CD3-anti-CD28 microparticles can be used at 5%, 10%, 15%, 20%, 25% or 30% of the manufacturer’s recommended strength.

[79] In any of the foregoing embodiments, the anti-CD3/anti-CD28 stimulation, if performed, can performed using anti-CD3/anti-CD28 in an amount sufficient to achieve the desired de differentiated cell properties.

[80] In any of the foregoing embodiments, said culture medium can further comprise 5% human serum. By way of example but not limitation 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%,

19% or 20% human serum and any range comprising values therebetween.

[81] In any of the foregoing embodiments, said culture medium can comprise X-Vivo 20 medium. Any appropriate culture medium for culturing T cells can be used.

[82] In any of the foregoing embodiments, by way of example but not limitation, 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.

[83] By way of example and not limitation, 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). It is also envisioned that 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.

[84] In any of the foregoing embodiments, 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. In some embodiments, 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. By way of example, but not limitation, 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%, 99% 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.

[85] In any of the foregoing embodiments, 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.

[86] 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.

[87] In any of the foregoing embodiments, 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 EQF-1 alpha.

[88] In any of the foregoing embodiments, the period of time of the initial de-differentiation culture lasts 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. As one, example, 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. 2017;18(9): 1865.

[89] In any of the foregoing embodiments, the culture medium may not contain human serum, temsirolimus, Vitamin D or the IL-2 signaling inhibitor at the time of culture initiation.

In such embodiments, human serum, temsirolimus, Vitamin D, the IL-2 signaling inhibitor or any combination thereof can be absent from the culture medium and 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.

[90] In some embodiments, a de-differentiated T cell that can be obtained by any of the methods of the present disclosure is provided. In some embodiments, a composition comprising a population of de-differentiated T cells is provided. In some embodiments, 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. In some embodiments, 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. By way of example, but not limitation, 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.

[91] In some embodiments 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. Such 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.

[92] In some embodiments 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.

[93] In some embodiments 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. Such 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. By way of example, but not limitation, the HIF-1-a expression can be within about 20%, 15%, 10% or 5% or the control T cell population.

[94] In some embodiments 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 T cell population 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.

[95] The foregoing properties can apply to a de-differentiated T cell to the extent that they can be applied as a property an individual de-differentiated T cell.

EXAMPLES

[96] The following examples are provided to better illustrate the methods of the present disclosure and the resultant de-differentiated T cells. These examples are not intended to be limiting or to otherwise alter the scope of the methods, cells and compositions disclosed in the present disclosure.

Example 1: Combination Vitamin D and Temsirolimus Reduces T Cell Effector Molecules

[97] We directly evaluated the individual effect of Vitamin D, mTOR inhibition (using the parenteral form of rapamycin, temsirolimus), and the combination of Vitamin D plus

temsirolimus on human T cell effector molecule expression (see FIGURE 1 A-1D).

[98] FIGURES 1 A-1D illustrate that the combination of Vitamin D and temsirolimus reduces effector molecule expression in human CD4 ⁺ and CD8 ⁺ T cells. For columns #2 through #5, 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 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. At the end of the de-differentiation interval, 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.

[99] A 3 -day culture interval was used that included a low level of T cell co-stimulation

(anti-CD3/anti-CD28 bead-to-T cell ratio of 1 :3; typical ratio used in literature is the inverse, 3: 1), temsirolimus (1 mM), Vitamin D at doses of either 0.1 or 1.0 nM, or the combination of temsirolimus and the higher-dose of Vitamin D. After culture, RNA was harvested and the level of effector molecule expression was compared to the control culture.

[100] As FIGURE 1 A shows, the various cultures had similar RNA expression of

housekeeping control genes, including GAPDH. In marked contrast, relative to the control Thl/Tcl cell culture that did not receive Vitamin D or temsirolimus, the culture addition of temsirolimus, Vitamin D, or the combination of temsirolimus plus Vitamin D resulted in the reduction in the RNA expression of T cell effector molecules, including the cytotoxic molecule Granzyme B (FIGURE IB) and the cytokine molecules IL-10 (a Th2 cytokine; FIGURE 1C) and IFN-g (a Thl cytokine; FIGURE ID). Thus, Granzyme B and IFN-gamma as markers of de- differentiation indicate that Vitamin D is effective at concentrations from 0.1 to 1.0 nM.

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).

[101] As such, using low-level co-stimulation (1 :3 ratio of anti-CD3/anti-CD28 beads to T cells) and a short 3-day culture interval, the addition of temsirolimus, Vitamin D, or the combination of temsirolimus plus Vitamin D can be utilized to reduce both Thl and Th2 cytokine effectors and cytotoxic effector mechanisms.

Example 2: Combination Vitamin D and Temsirolimus Alters Key Transcription Factors Associated With De-Differentiation

[102] We also evaluated the effect of Vitamin D, temsirolimus, or the combination on the expression of key transcription factors after a low-level of co-stimulation. [103] 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. For columns #2 through #5, 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. At the end of the de-differentiation interval, 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.

[104] As FIGURE 2 A 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. Previously, in human fibroblasts, 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.

[105] As such, using a low-level of co-stimulation, 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.

[106] By comparison, neither temsirolimus nor Vitamin D alone increased the RNA

expression of the KLF4 molecule, which is also one of the classical transcription factors associated with the iPSC state. However, the combination of temsirolimus plus Vitamin D (1.0 nM) increased KLF4 RNA expression. As such, it is preferable to include both temsirolimus and Vitamin D in T cell de-differentiation attempts. As 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 mIUΊ) and Vitamin D (1.0 nM) synergistically up- regulate KLF4.

[107] A related transcription factor, KLF10, was also up-regulated when the combination of temsirolimus plus Vitamin D (1.0 nM) was utilized. As 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).

[108] Finally, we evaluated cultured cells for RNA expression of IL-7 receptor alpha, which is up-regulated in T cells having a reduced differentiation status. Importantly, temsirolimus alone but not Vitamin D alone was capable of up-regulating IL-7 receptor alpha. Nonetheless, the combination of Vitamin D (1.0 nM) plus temsirolimus resulted in IL-7 receptor alpha up- regulation as shown in FIGURE 2D.

[109] In sum, these data indicate that low-level co-stimulation combined with temsirolimus can be used to enforce T cell de-differentiation; preferably, culture should include temsirolimus plus Vitamin D for a more complete pattern of de-differentiation.

Example 3: Combination Vitamin D and Temsirolimus Reduces Key Transcription Factors Associated With Thl Differentiation While Maintaining HIF-1 -a expression

[110] We also evaluated the effect of Vitamin D, temsirolimus, or the combination on the expression of key transcription factors associated with Thl-type differentiation, namely T-BET and STATE

[111] 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. For columns #2 through #5, 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 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. At the end of the de-differentiation interval, 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.

[112] Importantly each agent or the combination of agents down-regulated both T-BET RNA (FIGURE 3 A) and STAT1 RNA (FIGURE 3B). As shown in FIGURES 3A-3C, 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. Yet, temsirolimus at a dose of 1 mM 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).

[113] In marked contrast, temsirolimus at a dose of 1 pM, Vitamin D at a dose of 0.1 to 1.0 nM, or the combination did not down-regulate the key transcription factor HIF-1 -a (FIGURE 3C), which is important as a T cell survival factor that is critical for anti-tumor effects.

[114] In sum, these data indicate that the combination of low-level co-stimulation, temsirolimus, and Vitamin D can be used to reduce transcription factors required for Thl generation without inhibiting a key transcription factor required for overall T cell survival, HIF- 1-a. Example 4: The Combination Vitamin D, Temsirolimus, and an Anti-IL-2 Receptor Monoclonal Antibody Increases An Autophagy Signature

[115] We also evaluated the effect of Vitamin D, temsirolimus, or the combination on the process of autophagy, which is critical for promotion of a stem-like, de-differentiated state. The level of autophagy can be determined in-part by the subsequent up-regulation of the autophagy substrate, p62, by western blot analysis.

[116] 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). After the 3-day culture interval, 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.

[117] As FIGURE 4 demonstrates, inclusion of Vitamin D 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. That is, in FIGURE 4, culture #6 (fifth column), there was very little p62 expression on western blot analysis, consistent with a low level of autophagy; as the figure legend indicates, this culture condition received a low-level of co-stimulation, temsirolimus, the anti-IL-2 receptor monoclonal antibody daclizumab, but did not receive Vitamin D.

[118] In marked contrast, the other culture conditions each received Vitamin D

supplementation and each had increased p62 expression (effective dose range of Vitamin D, 0.01 nM to 1.0 nM). This 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. [119] In sum, these data indicate that inclusion of 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

temsirolimus.

Example 5: The Combination Vitamin D, Temsirolimus, and an Anti-IL-2 Receptor

Monoclonal Antibody Results In Optimal Disruption of the mTORCl Complex

[120] We also evaluated the effect of the various T cell culture conditions on expression of Raptor, which is a critical component of the mTORCl signaling complex. Importantly, inhibition of mTORCl has recently been discovered to be critical for somatic cell reprogramming to an iPSC state.

[121] 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;

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). After the 3-day culture interval, the 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.

[122] As 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).

[123] As FIGURE 5 demonstrates, 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. Thus, anti-IL-2 receptor monoclonal antibody Daclizumab (dose, 50 pg/ml) plays a beneficial role in suppressing the mTORCl sub-unit molecule, Raptor (column 2). [124] As FIGURE 5 demonstrates, the optimal inhibition of mTORCl sub-unit molecule Raptor by 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. Thus, 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. Furthermore, 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.

[125] Furthermore, as FIGURE 5 indicates, 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

[126] We also evaluated the mTORC2 complex, which is not directly sensitive to the inhibitory effects of rapamycin but can be influenced by conditions that result in prolonged mTORCl blockade. Importantly, inhibition of mTORC2 can promote a stem-like state.

[127] 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). After the 3-day culture interval, 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. [128] As 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. And, temsirolimus at a concentration of 1 mM was effective during combination agent de-differentiation for down-regulation of the mTORC2 sub-unit, Rictor. Moreover, 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.

[129] As such, 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

[130] As a result of autophagy at the level of the mitochondria (mitophagy), the quality of the mitochondrial proteins can be altered; in particular, with mitophagy, there can be an

advantageous shift in the bcl2-family member genes from pro-apoptotic family members such as BIM towards anti-apoptotic family member genes. In addition, culture methods that reduce the apoptotic tendency are associated with an increased capacity for de-differentiation.

[131] 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). After the 3-day culture interval, 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.

[132] To assess this, we measured BIM levels in the T cells cultured with a low-strength of co stimulation (1 :3 bead-to-T cell ratio) and presence of the various inhibitors. As 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.

[133] As such, we conclude that 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

Subsequent Proliferative Capacity After Removal of Inhibitors

[134] To demonstrate that the 3-day regimen comprised of a low-level of co-stimulation, temsirolimus, Vitamin D, and anti-IL-2 receptor monoclonal antibody resulted in a state of de- differentiation capable of re-differentiation, we performed experiments to re-stimulate the cells on day 3 of culture using a high-level of co-stimulation (3 : 1 bead-to-T cell ratio) after removal of the inhibitors from culture. After 10 days (13 days of total culture), the T cells were harvested, enumerated, and evaluated by flow cytometry.

[135] 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 second column represents the culture with no anti-IL-2R antibody; the fourth column represents the culture with no serum supplementation; the fifth column represents low-dose Vitamin D whereas the sixth column represents results using high-dose Vitamin D; the seventh column represents results using low- dose temsirolimus; and the eighth column represents the culture with no temsirolimus; and the ninth column represents results using the higher ratio of beads. After the 3 -day interval, 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. At day 13 of culture, viable T cells were enumerated and the overall yield is shown relative to day 0 input number.

[136] 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).

[137] In marked contrast, a very low yield was observed in the culture represented in column #4, which did not receive serum supplementation during the initial 3-day culture interval; as such, this data demonstrates that the initial day 3 culture interval must include X-Vivo 20 culture media supplemented with 5% AB serum.

[138] Also, reduction in the Vitamin D concentration to 0.01 nM or increase in the Vitamin D concentration to 1.0 nM resulted in a very low yield (data shown in column #5 and #6, respectively). As such, the preferable concentration of Vitamin D is 0.1 nM.

[139] Furthermore, reduction of the temsirolimus concentration to 0.1 mM reduced the resultant T cell yield (column #7). As such, the preferable concentration of temsirolimus is 1.0 mM.

[140] Finally, if the co-stimulation level was increased in the de-differentiation interval

(change in bead-to-T cell ration from 1 :3 to 1 : 1; results shown in last column, column #9), then the resultant T cell count was very low. As shown in FIGURE 8, it is necessary to use a low- level of co-stimulation during de-differentiation (1 :3 co-stimulation bead-to-T cell ratio) as increasing ratio to 1 : 1 results in a greatly reduced ability to manufacture T cells from the de- differentiated state (last column). As such, the preferable bead-to-T cell ratio during the de- differentiation stage of culture is 1 :3.

Example 9: The Initial Three-Component Culture Interval Results in the Generation of CD4 ⁺ T Cells Expressing Cell Surface Molecules Consistent With Reduced Differentiation

[141] At various times during the re-differentiation stage of culture, the resultant CD4 ⁺ T cells were evaluated for expression of memory markers by flow cytometry.

[142] 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). 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. After the 3-day interval, 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. 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 (last column in FIGURES 9A-9C;“Day 0 Input Value”).

[143] As shown in FIGURE 9A, relative to values from Day 0 input T cells, 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). In marked contrast, absence of these three molecules during the initial culture interval resulted in depletion of the naive T cell population (culture #1). Furthermore, elimination of temsirolimus during the initial culture interval resulted in depletion of the naive T cell population (culture #6).

[144] As shown in FIGURE 9B, 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.

[145] Finally, as shown in FIGURE 9C, 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 T cells that were triple-positive for CD62L, CCR7, and IL-7 receptor alpha (CD127). 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. In addition, reducing or eliminating only

temsirolimus from the initial culture interval also greatly reduced the frequency of triple-positive T cells (columns #5 and 6).

[146] In sum, these data indicate that the three-drug initial culture interval results in the conversion of CD4 ⁺ T cells from primarily an end-stage, effector memory population towards a less-differentiated T cell population, including co-expression of CD62L, CCR7, and CD 127, which is a state of very limited T cell differentiation.

Example 10: The Initial Three-Component Culture Interval Results in the Generation of CD8 ⁺ T Cells Expressing Cell Surface Molecules Consistent With Reduced Differentiation

[147] At various times during the re-differentiation stage of culture, the resultant CD8 ⁺ T cells were evaluated for expression of memory markers by flow cytometry.

[148] FIGURES 10A-10B illustrate the effect of culture components during the de- differentiation interval on CD8 ⁺ T cell expression of memory markers. Human CD4 ⁺ and CD8 ⁺

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. After the 3-day interval, 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. 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”).

[149] As shown in FIGURE 10 A, 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.

[150] Finally, as shown in FIGURE 10B, 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. In addition, reducing or eliminating only temsirolimus from the initial culture interval also greatly reduced the frequency of triple-positive T cells (columns #5 and 6).

[151] In sum, these data indicate that the three-drug initial culture interval results in the conversion of CD8 ⁺ T cells from primarily an end-stage, effector memory population towards a less-differentiated T cell population, including co-expression of CD62L, CCR7, and CD 127, which is a state of very limited T cell differentiation.

Example 11: De-differentiated T Cells Have an Inherent Bias Towards Low Cytokine Potential

[152] 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.

[153] 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. After 3-days, 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. After 10 days in culture (total, day 13 of culture), 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.

[154] To assess whether the de-differentiated T cell state manifested an inherent bias towards a particular cytokine secretion pattern, we cultured the de-differentiated T cells using a high-level of co-stimulation (bead-to-T cell ratio, 3: 1) and maintenance in media that did not contain any inhibitor and only contained the T cell growth cytokines IL-2 and IL-7.

[155] As FIGURES 11A-11D detail, 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). Of note, 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.

[156] Notably, inclusion of a low concentration of vitamin D (0.01 nM) during the de- differentiation interval also resulted in somewhat higher levels IFN-g and TNF-a relative to use of a higher concentration of vitamin D (0.1 nM). As such, with respect to moderating resultant T cell secretion of the inflammatory cytokine IFN-g, it is preferable to use a concentration of vitamin D that is approximately 0.1 nM.

[157] Furthermore, as shown in FIGURES 12A-12D, 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.

[158] 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. After 3-days, 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. After 10 days in culture (total, day 13 of culture), 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.

[159] 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). However, 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.

[160] In sum, these data indicate that re-differentiation of T cells in media that contains T cell growth cytokines (IL-2 and IL-7) without strong polarization signals (no addition of IFN-a, IL-4, or TGF-b) after a step 1 de-differentiation process has an inherent bias towards T cells of low cytokine potential; in particular, we demonstrated low levels of the deleterious cytokines IFN-g, TNF-a, and IL-17. This observation is particularly strong if 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.