Using inducible regulatory T (iT REG ) cells for ALS treatment

Abstract

The present disclosure provides methods for treating ALS using pentostatin and cyclophosphamide followed by TREG and / or TREG / Th2 hybrid cells from dedifferentiated T cells. The present disclosure further provides methods for producing TREG and TREG / Th2 hybrid cells from dedifferentiated T cells, the TREG and TREG / Th2 hybrid cells, groups thereof, and compositions thereof. Methods for producing dedifferentiated T cells, the dedifferentiated T cells, groups thereof, and compositions thereof are also provided.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 62 / 768,176, filed on November 16, 2018, and U.S. Provisional Application No. 62 / 927,075, filed on October 28, 2019, the entire contents of each of which are incorporated herein by reference. Background Art

[0003] Adoptive T cell therapy is an emerging intervention for the effective treatment of cancer and infectious diseases, autoimmune and neurodegenerative diseases. It is becoming increasingly clear that the transfer of T cells with a more primitive differentiation state that is converted into higher proliferation potential and other key attributes is associated with improved in vivo effects after adoptive transfer. However, most forms of adoptive T cell therapy require ex vivo manufacturing steps, which usually lead to further cell differentiation; this is particularly problematic because T cells from adults are already mainly in a late state of differentiation (called effector memory cells) and often exist in a senescent state under the control of checkpoint inhibitory molecules. Methods to alleviate this limitation can be taken, including separating (purifying) more naive T cell subsets at the beginning of culture; however, this method is partially limited to the small number of naive T cells present in the peripheral blood of adults. Therefore, separated T cells in a primitive differentiation state are very much needed.

[0004] It is well known that even highly differentiated cells have an inherent ability to dedifferentiate toward a more primitive state. In fact, in the most extreme examples, differentiated cells can be manipulated to reach an induced pluripotent stem cell (iPSC) state, whereby such iPS cells share key properties with embryonic stem cells and can then be further regulated to redifferentiate into different tissue fates; cell therapies using such iPSC methods have many potential clinical applications. The generation of iPS cells from differentiated somatic cells was initially demonstrated by transferring key transcription factors by means of viral or non-viral mediated methods, including Sox2, Oct3 / 4, KLF4 and c-myc or Sox2, Oct3 / 4, Nanog and Lin28.

[0005] However, the ability to convert somatic cells into iPS cells is low and depends in part on the degree of somatic cell differentiation. As an example, the ability to convert mature mouse immune T cells into iPS cells is 300 times lower than that of converting mouse hematopoietic stem cells into iPS cells. However, using gene transfer methods, it has been demonstrated that mature human peripheral blood T cells retain the ability to convert into an iPS cell state. In the past decade, researchers have also characterized transcription factors associated with the earliest stages of T cell differentiation. However, redifferentiating T cells from various types of stem cell precursors is a relatively inefficient process that typically takes one to two months.

[0006] Although the biology of dedifferentiation is increasingly characterized, there are still many unknowns in terms of specific transcription factors and transcription factor dynamics associated with dedifferentiation. It is also important to recognize that the gene transfer method for achieving dedifferentiation is laborious and is associated with complications such as teratoma generation, which must be resolved by additional genetic interventions (such as cell fate suicide gene programming). As a potential alternative, various drug interventions can be used to achieve a certain degree of dedifferentiation. As an example, during cell culture, molecular changes are caused by inhibiting calcineurin by using the immunosuppressant cyclosporine, which replaces the need for gene delivery of Sox2 transcription factors for promoting mouse iPS cells. In addition, rapamycin is an immunosuppressive drug that inhibits mammalian rapamycin target (mTOR), which can produce dedifferentiation effects on terminal effector T cells by upregulating the transcription factor KLF2 induced by starvation, which in turn increases T central memory molecules CD62L and CCR7. In addition, rapamycin and the resulting inhibition of mTOR signaling are essential for maintaining cell quiescence in naive T cells with reduced differentiation states. It is important to note that the mTOR pathway includes both the mTORC1 complex (containing the Raptor subunit) and the mTORC2 complex (containing the Rictor subunit). The inhibition of both mTORC1 and mTORC2 is associated with increased promotion and maintenance of memory T cells. It is worth noting that rapamycin can only directly inhibit mTORCl; however, downstream inhibition of mTORC2 may occur with the extension of rapamycin-mediated mTORC1 inhibition. It is also known that reducing T cell growth factor signaling by mTOR inhibition or other pathway inhibition will upregulate another key molecule associated with T cells with a more primitive differentiation state, i.e., IL-7 receptor α (CD127). In further studies, inhibition of the T cell mTOR pathway by the pharmacological agent rapamycin or the Wnt-β-catenin signaling activator TWS11 promoted the dedifferentiation of human naive T cells into a less differentiated T stem cell memory population previously identified and characterized in murine and human T cells. In further experimental model studies, pharmacological inhibition of the AKT 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 state and enhanced T cell function upon adoptive transfer.

[0007] It has been demonstrated that blocking mTOR by culturing human T cells ex vivo in the presence of rapamycin reduces T cell expression of molecules associated with effector differentiation, such as cytokine secretion molecules and cytolytic effector molecules.

[0008] Additionally, the 1,25-hydroxylated form of vitamin D (as used herein, "vitamin D") can inhibit human T cell effector function. The inhibitory effects of vitamin D on human T cell proliferation can be synergistic with immunosuppressive drug exposure using agents such as cyclosporine A or rapamycin. However, previous studies have shown that the inhibitory effects of vitamin D on T cell effectors are relatively specific for Th1-type molecules rather than Th2-type molecules. In addition, vitamin D has been shown to promote immunosuppressive regulatory T (T REG ) cell population.

[0009] In a somewhat paradoxical finding, it was determined that human CD8 + T cells expressed high levels of the vitamin D receptor, and individuals with the highest values ​​tended to have high levels of T cell effector function and immunosenescence.

[0010] In more recent studies, using a murine model of Mycobacterium tuberculosis infection, it was demonstrated that vitamin D is essential for macrophage elimination of intracellular pathogens through a mechanism involving IFN-γ production and autophagy. Additionally, in human non-small cell carcinoma cell lines, vitamin D signaling can promote a cytotoxic form of autophagy that, when combined with radiation, contributes to anti-tumor effects. Finally, vitamin D receptor signaling promotes autophagy in normal human breast tissue; loss of such vitamin D receptor signaling is associated with an increased risk of breast cancer. Although there is evidence that vitamin D is associated with autophagy in innate immunity (macrophage context), there is a lack of data on the role of vitamin D on autophagy in T cells during adaptive immunity, and in this context, the potential effects of vitamin D and rapamycin on T cell autophagy are redundant.

[0011] These somewhat contradictory results regarding the potential role of vitamin D in T cell biology may be related to the recent discovery of broad effects of vitamin D on the entire genome at the mRNA level and at the microRNA level. Thus, the effects of vitamin D on immunity need to be evaluated within a contextually relevant framework.

[0012] Regulatory T(T REG ) cells are essential for maintaining immune tolerance. REG A decrease in the number or quality of T cells is the underlying cause of many primary autoimmune diseases, including type 1 diabetes mellitus (T1DM), multiple sclerosis, rheumatoid arthritis, and systemic lupus erythematosus, to name a few. REG Deficiency is associated with an acceleration of the natural history of primary neurodegenerative diseases. REGDeficiency is associated with severe complications in the setting of solid organ and hematopoietic cell transplantation, most notably increased rates of transplant rejection and graft-versus-host disease (GVHD). REG Due to the critical role of T cells in maintaining immune homeostasis, many experimental methods have been developed to promote REG One such promising approach is adoptive transfer of T cells. REG cells, the T REG T cells exist in two main subtypes: (1) natural (n) T REG Cells, which originate from the thymus ("nT REG ” or “Natural T REG ”), the thymus degenerates with age, thus reducing the number of nT cells available for adoptive transfer. REG The number of cells; and (2) the inducible (i) T REG cells, which are transformed in the periphery from the more abundant effector T cell pool. REG The number of cells is limited, so nT REG Attempts to use cells for adoptive T cell therapy have relied on ex vivo manufacturing methods to isolate and subsequently expand nT cells. REG Cells. nT for adoptive cell therapy REG Clinical trials of iT cells are still in the early stages of implementation, mainly in phase I / II clinical trials for the prevention of GVHD and the treatment of T1DM. REG Cells (ex vivo generated T cells of the present disclosure) REG cells, including T REG and T REG There are other challenges in the identification of iT / Th2 cells, namely: (1) although peripheral effector T cells are relatively abundant, they exist mainly in a mature state of effector memory with limited replication 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 Th1 and Th17 subsets. REG If cell therapy is to become highly feasible, it will be necessary to develop an in vitro manufacturing method that simultaneously: (1) dedifferentiates effector T cells toward a less differentiated memory phenotype that increases proliferation potential and significantly improves T REG cell therapy potential; and (2) eliminate pathogenic Th1 and Th17 pathways while promoting T cells to T REG Phenotypic differentiation.

[0013] iT is initiated by collecting peripheral blood mononuclear cells containing lymphocytes from the subject to be treated (in the case of autologous therapy) or from a normal donor (in the case of allogeneic therapy).REG Cell production. Typically, this collection is performed under steady state, that is, without the administration of any growth factors; however, in an 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. "Plerixafor and G-CSF versus placebo and G-CSF to mobilize hematopoietic stem cells for autologous stem cell transplantation in patients with multiple myeloma". Blood. 2009; 113(23): 5720-5726. In the present disclosure, a method that can be used in the collection of cells for iT REG Prior to administration of anti-TNF-α therapeutic agents, lymphocytes were produced to enrich for T REG That is, it was demonstrated that the anti-TNF-α agent etanercept (a recombinant receptor that preferentially inhibits serum, cell-free TNF-α and relatively preserves the membrane-bound form of TNF-α on the cell surface) induces global changes in the T cell receptor (TCR) repertoire when measured by RNA sequencing. Since membrane-bound TNF-α is delivered to T cells via the TNFR2 receptor, REG cells provide positive signals, so our method provides a method for making iT REG Cells were previously enriched for T REG Other therapeutic agents that preferentially inhibit serum, cell-free TNF-α may also be used in this intervention, including but not limited to the anti-TNF-α monoclonal antibody adalimumab.

[0014] Amyotrophic lateral sclerosis (ALS) is a primary neurodegenerative disease involving the cerebral cortex, brainstem, and spinal cord that causes progressive disability and death, usually due to respiratory failure. ALS is a familial disease that occurs in 10% of patients due to various genetic events; the remainder of patients have sporadic ALS, which has an unknown etiology but may involve environmental factors. The most recent registry data (2013) indicate that the prevalence of ALS in the United States is approximately 16,000 cases; these data also indicate that ALS disproportionately affects whites, males, and individuals in the 60 to 69 age group. Veterans and possibly professional American football players appear to be at increased risk for ALS, thus suggesting that chemical exposure or head trauma may increase the risk of developing the disease. ALS is a heterogeneous disease with a variety of clinical manifestations and rates of progression. Although the average survival of patients with ALS ranges from two to four years from diagnosis, survival can be as short as a few months or more than a decade. Prognosis for patients with ALS is difficult to estimate because disease scoring systems, such as the patient-reported ALSFRS-R (ALS Functional Score, Revised), fail to account for both linear and nonlinear aspects of disease progression. This difficulty in estimating the rate of disease progression represents a limitation of ALS clinical trials and suggests that potential disease biomarkers, including already developed immunological monitoring, should be emphasized as a component of treatment protocols. The clinical onset of ALS is insidious, with most patients presenting with weakness of the upper or lower limbs or difficulty speaking or swallowing (bulbar onset). ALS remains a diagnosis of exclusion because there are no definitive blood, spinal fluid, or radiological tests; therefore, ALS is often a diagnosis of exclusion after other conditions have been ruled out. This process of ruling out other conditions can often take up to a year, delaying treatment attempts and clinical trial accrual; this delay in referral can have an impact because up to 50% of the motor neurons may no longer be functioning at the time of the final ALS diagnosis. Given this context, it is generally recommended that ALS patients be enrolled in research trials at a relatively early time point after diagnosis.

[0015] ALS is a primary neurodegenerative disease in which neuroinflammation is a secondary propagating factor. Evidence for this conclusion stems in part from the observation that TAR DNA-binding protein 43 (TDP-43) is dysfunctional in the vast majority of patients with both familial and sporadic ALS. TDP-43, which is restricted to the nucleus in the healthy state, is an RNA- and DNA-binding protein susceptible to aggregation, thus explaining the cytoplasmic inclusions seen in neurons of ALS patients. The exact mechanisms leading to alterations in the TDP-43 pathway remain to be fully elucidated but appear to involve various cellular stress events or the amplification of genomic elements (retrotransposable elements, RTEs) that replicate themselves via RNA intermediates. Ultimately, such events lead to multifaceted programmed cell death in neurons, including programmed necrosis. Notably, the necrotic mode of cell death that occurs in ALS patients has been shown to be particularly immunogenic relative to the more orderly apoptotic cell death. Indeed, TNF-α, a known molecular mediator of motor neuron death in ALS, can produce a necrotic form of cell death. Necrotic cell death can lead to the release of autoantigens, which can then be presented to the adaptive immune system to induce autoimmunity; in addition, since protein aggregates themselves can be immunogenic, protein aggregates (including but not limited to TDP-43; SOD-1; p62) that appear in ALS patients may be targets of autoimmune responses that arise following neurodegeneration. In fact, it has recently been shown that monocytes from ALS patients develop an inflammatory phenotype after being pulsed with exosomes containing TDP-43.

[0016] In response to primary neurodegeneration, there is extensive evidence that the innate inflammasome and the adaptive peripheral immune system work together to further inappropriately promote ALS disease progression. In a superoxide dismutase 1 (SOD1) transgenic mouse model of ALS, CD3 + T cell infiltration and microglial activation are considered pro-inflammatory factors that promote disease progression. Moreover, in the PU.1 knockout mouse model of ALS, the transfer of wild-type microglia with reduced inflammatory propensity relative to host microglia reduced neurodegeneration and improved survival. In addition, CD4 +The protective effect of T cells indicates the double-edged sword nature of the peripheral immune T cell pool in ALS (acting as a propagation factor or a protective factor). In ALS patients, direct evidence of the harmful effects of peripheral adaptive immune system T cells can be determined by confirming that T cells infiltrating the spinal cord express oligoclonal T cell receptor (TCR) lineages. In addition, professional antigen presenting cells (dendritic cells) derived from the peripheral immune system can be isolated in the spinal cord tissue of ALS patients that are closely related to inflammatory peripheral-derived monocytes and resident CNS microglia. In addition, in ALS patients, purified monocytes express a pro-inflammatory RNA expression profile, including an increase in the innate inflammatory molecule IL-1-β, which can then drive the promotion of CD4 + T-helper-1 (Th1), CD8 + T-cytotoxic-1 (Tc1) and CD4 + IL-23 pathway of Th17-mediated neurodegenerative immunity. In subsequent studies, protective CD4 + The phenotype of T cell subsets has been characterized as regulatory T (T REG ) cell population that reduces inflammation through a mechanism mediated in part by the counter-regulatory Th2-type cytokines IL-4 and IL-10.

[0017] This biology is consistent with a large body of data from neuroinflammation research, which indicates that: microglia are key cellular components in the brain that drive neurodegeneration; and that microglia and CNS-infiltrating peripheral CD4 + T cells interact and influence the pathogenesis of the disease. Consistent with the results of mouse modeling, patients with enriched FoxP3 + T REG Patients with peripheral immune systems that have Th2-type T cells and Th2-type T cells have a reduced rate of progression of ALS. In addition, it has recently been found that T cells in ALS patients REG A clinical trial is currently evaluating multiple infusions of nT REG The use of cells plus low-dose IL-2 administration to treat ALS (ClinicalTrials.gov; NCT03241784); IL-2 is a stimulatory STAT5 signaling pathway and can thereby promote nT REG Cytokines for in vivo expansion of cells.

[0018] Inducible (i)T REG Cells are not like nT REG In contrast, iT REGIt is a group transformed from other pathogenic post-thymic T cell subsets (such as Th1 cells). REG and iT REG Both play important and non-redundant roles in reducing inflammatory responses, but developing iT cells is more feasible in terms of regulatory T cell potency and ease of manufacturing. REG Furthermore, if used in combination with the immune monitoring techniques and host therapy regimens described here (pentostatin, cyclophosphamide, lamivudine), adoptive iT therapy for ALS may be promising. REG Therapy will be particularly effective.

[0019] Riluzole, the first drug approved for the treatment of ALS in 1995 It is only moderately effective in reducing the morbidity and mortality of ALS. Despite extensive clinical research (whereby more than 60 molecules have been investigated for ALS treatment), only two additional molecules have achieved some clinical success, the antioxidant edaravone and the tyrosine kinase inhibitor masitinib. Edaravone, which was recently approved by the FDA for the treatment of ALS, Provides minimal clinical benefit, is expensive, and requires continuous daily intravenous infusion for 2 weeks with 2 weeks of rest; masitinib is not FDA-approved. A Phase II trial of rapamycin for the treatment of ALS is just beginning (Clinical Trials Registry Identifier: NCT03359538). Because rapamycin has the ability to promote T REG Given the tendency of rapamycin to reconstitute cells, it is possible that rapamycin could also be a favorable agent for use in ALS. However, long-term treatment with rapamycin has significant toxicity, requires pharmacological monitoring, may have paradoxical effects in terms of actual exacerbation of ALS in some models, and can limit the expansion of adoptively transferred T cell populations.

[0020] Thus, given the current state of very limited therapeutic options, there is a great need to evaluate new strategies for treating ALS. In this application, a method for inducing (i) regulatory T (T REG ) New ALS treatment methods centered on cell therapy. Summary of the invention

[0021] The present disclosure relates to methods for dedifferentiating T cells and differentiating such cells into T REG or T REG / Th2 cell approach.

[0022] In some embodiments, the initial dedifferentiation method may comprise starting a culture with an input cell population harvested at steady state (without administration of drug).

[0023] In some embodiments, the method comprises starting a dedifferentiation culture with an input cell population harvested from a subject (in an autologous context) or a normal donor (in an allogeneic context) that has been or is being treated with an anti-TNF-α therapeutic agent that has a preferential selectivity for inhibiting serum, cell-free forms of TNF-α and relatively retains membrane-bound TNF-α. Such therapeutic agents include, but are not limited to: recombinant receptor etanercept, which can be administered by subcutaneous injection at a conventional dose of 25 or 50 mg per week; or monoclonal antibody adalimumab, which can be administered by intravenous injection at a conventional dose of 40 mg per week or 40 mg every other week. In all of these cases, the dose of the anti-TNF-α therapeutic agent can be adjusted according to the desired biomarker changes, which can include, but are not limited to, changes in the TCR repertoire analyzed by RNA sequencing and a shift to type 2 TNF receptor (TNFR2) and a shift from type 1 TNF receptor (TNFR1) as measured by flow cytometry.

[0024] In some embodiments, the method includes: in a culture medium including vitamin D, temsirolimus and an IL-2 signaling inhibitor, inoculating a culture input cell population including T cells from a subject at a certain cell density; adding anti-CD3 / anti-CD28 coated magnetic beads to the T cells and culture medium at a bead:T cell ratio of 1:1 or lower to stimulate the T cells, or in the most extreme example, not adding anti-CD3 / anti-CD28 co-stimulation; incubating the culture input cell population and culture medium for a certain period of time to produce dedifferentiated T cells. This dedifferentiation process can also be performed in the absence of any bead co-stimulation.

[0025] In any of the preceding embodiments, the method may further comprise harvesting the dedifferentiated T cells.

[0026] In any of the foregoing embodiments, the method may further comprise, after harvesting the dedifferentiated T cells: packaging at least a portion of the dedifferentiated T cells in a package; and freezing the package containing the portion of the dedifferentiated T cells.

[0027] In any of the foregoing embodiments, the method can further comprise, prior to inoculating the culture input cell population into the culture medium: harvesting the culture input cell population from the subject.

[0028] In any of the foregoing embodiments, the method may further include measuring the expression level of RAPTOR or RICTOR in the culture input cell population, wherein the time period continues until the expression level of RAPTOR or RICTOR in the culture input cell population is reduced by at least 50%, and more preferably by 90%, respectively, relative to a control T cell population, and wherein the control T cell population is manufactured under the same conditions as the culture input cell population in the absence of temsirolimus, an IL-2 signaling inhibitor, and vitamin D.

[0029] In any of the foregoing embodiments, the method may further include measuring the expression levels of RAPTOR or RICTOR and a housekeeping protein in the culture input cell population, wherein the time period continues until the expression level of RAPTOR or RICTOR in the manufactured T cells (normalized by the housekeeping protein) is at least 50% lower, and more preferably 90% lower, than the expression level of RAPTOR or RICTOR in the control T cell population (normalized by the housekeeping protein), respectively, and the control T cell population is manufactured under the same conditions as the culture input cell population in the absence of temsirolimus, IL-2 signaling inhibitor and vitamin D.

[0030] The present disclosure also relates to a dedifferentiated T cell produced by the method described in any of the preceding embodiments.

[0031] The present disclosure also relates to a composition comprising a dedifferentiated cell population, wherein at least a portion of the dedifferentiated cell population expresses at least 50% less RAPTOR or RICTOR, and more preferably 90% less, compared to a control T cell population produced under the same conditions as the culture input cell population in the absence of temsirolimus, an IL-2 signaling inhibitor, and vitamin D.

[0032] In any of the foregoing embodiments, the method may further include measuring at least a portion of the dedifferentiated cell population, wherein relative to a control T cell population, the RNA expression of the following molecules expressed by at least a portion of the dedifferentiated cell population changes by at least 10%, and more preferably changes by 50%, namely: T cell effector molecules (including but not limited to granzyme B, IL-10, and IFN-γ) are reduced; transcription factors associated with cells with a reduced differentiation state (including but not limited to Nanog, KLF4, and KLF10) are increased; the expression of molecules preferentially expressed on naive T cell subsets (including but not limited to CD 127, IL-7 receptor α chain) is increased; transcription factors associated with Th1 type differentiation (including but not limited to T-BET and STAT1) are reduced; and transcription factors that promote cell survival (including but not limited to HIF-1α) are relatively retained.

[0033] In any of the foregoing embodiments, the method may further include measuring at least a portion of the dedifferentiated cell population, wherein the at least a portion of the dedifferentiated cell population expresses at least a 10% change in the expression of a molecule indicating that the cell has undergone autophagy, and more preferably a 50% change. As an example, the dedifferentiated cells have increased p62 expression by Western blot analysis relative to control T cells. Other standard methods for measuring 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.

[0034] The present disclosure also relates to a dedifferentiated T cell produced by the method described in any of the preceding embodiments.

[0035] The present disclosure also relates to a composition, comprising a dedifferentiated cell population, wherein relative to a control T cell population, at least a portion of the dedifferentiated cell population expresses RNA expression of the following molecules that is altered by at least 10%, and more preferably by 50%, namely: T cell effector molecules (including but not limited to granzyme B, IL-10, and IFN-γ) are reduced; transcription factors associated with cells with a reduced differentiation state (including but not limited to Nanog, KLF4, and KLF10) are increased; the expression of molecules preferentially expressed on naive T cell subsets (including but not limited to CD 127, IL-7 receptor α chain) is increased; transcription factors associated with Th1 type differentiation (including but not limited to T-BET and STAT1) are reduced; and transcription factors that promote cell survival (including but not limited to HIF-1α) are relatively retained.

[0036] The present disclosure also relates to a composition comprising a dedifferentiated cell population, as defined by a change in expression of a molecule indicating cells that have undergone autophagy of at least 10%, and more preferably a change in 50% of the dedifferentiated cells. As an example, the dedifferentiated cells have increased p62 expression by Western blot analysis relative to control T cells. Other methods of measuring autophagy may 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.

[0037] The present disclosure also relates to a composition comprising a dedifferentiated cell population, wherein at least a portion of the dedifferentiated cell population expresses less than 50% of both RAPTOR and RICTOR compared to a control T cell population.

[0038] The present disclosure relates to methods for differentiating dedifferentiated T cells into T REG or T REG / Th2 cell approach.

[0039] In some embodiments, the method comprises: culturing dedifferentiated T cells of the present disclosure or otherwise dedifferentiated T cells in a medium comprising IL-2, IL-4, and TGF-β; adding anti-CD3 / anti-CD28 coated magnetic beads at a ratio of 3:1 (bead:T cell ratio); and incubating the dedifferentiated T cells for a period of time to produce T cells. REG / Th2 cells.

[0040] In some embodiments, the method comprises: culturing dedifferentiated T cells in a medium comprising IL-2, IL-4, and TGF-β, the dedifferentiated T cells having at least 50% less RAPTOR or RICTOR expression relative to a control T cell population made under the same conditions as the culture input cell population in the absence of temsirolimus, an IL-2 signaling inhibitor, and vitamin D; adding anti-CD3 / anti-CD28 coated magnetic beads at a ratio of 3:1 (bead:T cell ratio); and incubating the dedifferentiated T cells for a period of time to produce T cells. REG / Th2 cells.

[0041] In some embodiments, the method comprises: culturing dedifferentiated T cells in a medium comprising IL-2 and TGF-β, the dedifferentiated T cells having at least 90% less RAPTOR or RICTOR expression relative to a control T cell population made under the same conditions as the culture input cell population in the absence of temsirolimus, an IL-2 signaling inhibitor, and vitamin D; adding anti-CD3 / anti-CD28 coated magnetic beads at a ratio of 3:1 (bead:T cell ratio); and incubating the dedifferentiated T cells for a period of time to produce T cells. REG cell.

[0042] In any of the preceding embodiments, the culture medium may further comprise pemetrexed.

[0043] The present disclosure also relates to a T REG or T REG / Th2 cells, which are produced by any of the aforementioned methods.

[0044] The present disclosure also relates to methods for treating amyotrophic lateral sclerosis in a subject in need thereof.

[0045] In some embodiments, the method comprises subjecting the subject to one or more primary treatment cycles, each of the one or more primary treatment cycles comprising: administering pentostatin to the subject; and / or administering cyclophosphamide to the subject; and subjecting the subject to one or more immunotherapy treatment cycles, the one or more immunotherapy treatment cycles comprising: administering to the subject a therapeutically effective amount of a manufactured T REG The composition of cells.

[0046] In some embodiments, a method comprises a first treatment cycle, a second treatment cycle, optionally one or more additional treatment cycles, and one or more immunotherapy treatment cycles, wherein the first treatment cycle comprises: administering pentostatin to the subject and / or administering cyclophosphamide to the subject; the second treatment cycle comprises: administering pentostatin to the subject and / or administering cyclophosphamide to the subject; each of the one or more additional treatment cycles comprises: administering pentostatin to the subject and / or administering cyclophosphamide to the subject, and each of the one or more immunotherapy treatment cycles comprises: administering pentostatin to the subject and / or administering cyclophosphamide to the subject, and wherein the manufactured T REG The cells are administered to the subject.

[0047] In some embodiments, a method comprises one or more treatment cycles comprising: administering to the subject a therapeutically effective amount of a manufactured T REG cell.

[0048] In some embodiments, a method may include administering to the subject a therapeutically effective amount of a manufactured T REG cell. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1A Normalized GAPDH mRNA expression of control cells and cells treated under various conditions is depicted.

[0050] Figure 1B Normalized granzyme B mRNA expression of control cells and cells treated under various conditions is depicted.

[0051] Figure 1C Normalized IL-10 mRNA expression of control cells and cells treated under various conditions is depicted.

[0052] Figure 1D Normalized IFN-γ mRNA expression of control cells and cells treated under various conditions is depicted.

[0053] Figures 1A-1D demonstrated that the combination of vitamin D and temsirolimus reduced the expression of effector molecules in human CD4+ and CD8+ T cells.

[0054] Figure 2A Normalized NANOG mRNA expression of control cells and cells treated under various conditions is depicted.

[0055] Figure 2BNormalized KLF4 mRNA expression of control cells and cells treated under various conditions is depicted.

[0056] Figure 2C Normalized KLF10 mRNA expression of control cells and cells treated under various conditions is depicted.

[0057] Figure 2D Normalized IL-7 receptor mRNA expression of control cells and cells treated under various conditions is depicted.

[0058] Figures 2A-2D demonstrated that the combination of vitamin D and temsirolimus increased the expression of stem cell-associated transcription factors and the naive T cell molecule IL-7 receptor-α in human CD4+ and CD8+ T cells.

[0059] Figure 3A Normalized T-BET mRNA expression of control cells and cells treated under various conditions is depicted.

[0060] Figure 3B Normalized STAT1 mRNA expression of control cells and cells treated under various conditions is depicted.

[0061] Figure 3C Normalized HIF-1-α mRNA expression of control cells and cells treated under various conditions is depicted.

[0062] Figures 3A-3C demonstrated that the combination of vitamin D and temsirolimus reduced the expression of transcription factors associated with effector Th1 / Tc1 cells, but did not reduce the expression of HIF-1-α, a transcription factor associated with T cell survival.

[0063] Figure 4 P62 expression normalized by actin expression is depicted for cells treated under various conditions and demonstrates that the combination of vitamin D, temsirolimus, and anti-IL-2 receptor blockade induces expression of the autophagy-related molecule p62.

[0064] Figure 5 Raptor expression normalized by actin expression is depicted for cells treated under various conditions and demonstrates that the combination of vitamin D, temsirolimus, and anti-IL-2 receptor blockade reduces expression of the mTORC1-associated molecule Raptor.

[0065] Figure 6Western blots depict the expression of GAPDH, p70S6K, SGK1, Raptor, and Rictor of cells treated under various conditions and demonstrate that the combination of vitamin D, temsirolimus, and anti-IL-2 receptor blockade reduces the expression of the mTORC1-associated molecule Raptor and the mTORC2-associated molecule Rictor.

[0066] Figure 7 BIM expression normalized by actin expression is depicted for cells treated under various conditions and it is demonstrated that the combination of vitamin D, temsirolimus, and anti-IL-2 receptor blockade reduces expression of the pro-apoptotic molecule BIM. Figure 8 The effects of culture components during the dedifferentiation interval on subsequent T cell yield (at day 13 of culture) are shown.

[0067] Figure 8 The effects of culture components during the dedifferentiation interval on subsequent T cell yield (at day 13 of culture) are shown.

[0068] Fig. 9A Depicted are the percentages of CD45RA+CD4 cells for cells treated under various conditions.

[0069] Fig. 9B Depicted are the percentages of CD4+ and CCR7+ cells for cells treated under various conditions.

[0070] Fig. 9C Depicted are the percentages of CD4+, CD62L+, CCR7+, and CD127+ cells for cells treated under various conditions.

[0071] Figures 9A-9C The effects of culture components on CD4+ T cell expression of memory markers during the dedifferentiation interval (on day 13 of culture) are shown.

[0072] Fig. 10A Depicted are the percentages of CD62L+ and CCR7+ CD8 cells for cells treated under various conditions.

[0073] Fig. 10B Depicted are the percentages of CD62L+, CCR7+, and CD127+ CD8 cells among cells treated under various conditions.

[0074] Figures 10A-10B The effects of culture components on CD8+ T cell expression of memory markers during the dedifferentiation interval are demonstrated.

[0075] Figures 11A-11DDepicted are inflammatory Th1 / Th17 cytokine analysis of dedifferentiated T cells cultured in polarizing neutral medium.

[0076] Fig.11A IFN-γ secretion from cells treated under various conditions is depicted.

[0077] Fig. 11B GM-CSF secretion by cells treated under various conditions is depicted.

[0078] Fig. 11C TNF-α secretion by cells treated under various conditions is depicted.

[0079] Fig.11D IL-17 secretion by cells treated under various conditions is depicted.

[0080] Figures 12A-12D Depicted are IL-2 and Th2-type cytokine analysis of dedifferentiated T cells cultured in polarizing neutral medium.

[0081] Fig. 12A IL-2 secretion by cells treated under various conditions is depicted.

[0082] Fig. 12B IL-4 secretion by cells treated under various conditions is depicted.

[0083] Fig. 12C IL-5 secretion by cells treated under various conditions is depicted.

[0084] Fig.12D IL-13 secretion by cells treated under various conditions is depicted.

[0085] Fig.13 Depicted is the favorable expansion of dedifferentiated T cells under hybrid Th2 / TREG polarizing conditions relative to Th1 polarizing conditions.

[0086] Fig.14A Depicted are the percentages of CD4+CD45RA+ cells to total CD4+ cells for cells treated under various conditions.

[0087] Fig. 14B Depicted are the percentages of CD4+CD62L+CCR7+ cells to total CD4+ cells for cells treated under various conditions.

[0088] Fig. 14C Depicted are the percentages of CD4+CD62L+CCR7+CD127+ cells to total CD4+ cells for cells treated under various conditions.

[0089] Figure 14-A14C shows that culture of dedifferentiated T cells under hybrid Th2 / TReg polarizing conditions leads to the generation of naive and triple positive T central memory CD4+ T cells.

[0090] Fig.15A Depicted are the percentages of CD8+CD62L+CCR7+ cells to total CD8 cells for cells treated under various conditions.

[0091] Fig.15A Depicted are the percentages of CD8+CD62L+CCR7+CD127+ cells to total CD8+ cells for cells treated under various conditions.

[0092] Figures 15A-15B We show that culture of dedifferentiated T cells under hybrid Th2 / TReg polarization conditions leads to the generation of triple-positive T central memory CD8+ T cells.

[0093] Fig.16A IL-2 secretion by cells treated under various conditions is depicted.

[0094] Fig. 16B IL-4 secretion by cells treated under various conditions is depicted.

[0095] Fig. 16C IL-5 secretion by cells treated under various conditions is depicted.

[0096] Figures 16A-16C We show that culture of dedifferentiated T cells under hybrid Th2 / TReg polarizing conditions results in the generation of T cells with a primitive Th2 cell cytokine phenotype: IL-2, IL-4, and IL-5 secretion.

[0097] Fig.17A IL-10 secretion by cells treated under various conditions is depicted.

[0098] Fig. 17B IL-13 secretion by cells treated under various conditions is depicted.

[0099] Fig. 17C IL-17 secretion by cells treated under various conditions is depicted.

[0100] Figures 17A-17C We show that culture of dedifferentiated T cells under hybrid Th2 / TReg polarizing conditions results in the generation of T cells with a primitive Th2 cell cytokine phenotype: IL-10, IL-13, and IL-17 secretion.

[0101] Fig.18A IFN-γ secretion from cells treated under various conditions is depicted.

[0102] Fig.18B TNF-α secretion by cells treated under various conditions is depicted.

[0103] Fig.18C GM-CSF secretion by cells treated under various conditions is depicted.

[0104] Figures 18A-18C We show that culture of dedifferentiated T cells under hybrid Th2 / TREG polarizing conditions results in the generation of T cells with a primitive Th2 cell cytokine phenotype: IFN-γ, TNF-α, and GM-CSF secretion.

[0105] Fig.19A The percentage of CD4+ T cells in culture according to day and culture inhibitor is depicted.

[0106] Fig.19B The percentage of CD4+FOXP3+ T cells in cultures according to day and culture inhibitor is depicted.

[0107] Fig.19C The percentage of CD4+Tbet+ T cells in the cultures according to day and culture inhibitor is depicted.

[0108] Fig.19D The percentage of CD4+GATA3+ T cells in cultures according to day and culture inhibitor is depicted.

[0109] Figures 19A-19D We show that extended culture of dedifferentiated T cells under hybrid Th2 / TREG polarizing conditions in the presence of pemetrexed leads to the generation of CD4+ T cells expressing FOXP3 and GATA3 transcription factors.

[0110] Fig. 20A The percentage of CD8+ T cells in culture according to day and culture inhibitor is depicted.

[0111] Fig. 20B The percentage of CD8+FOXP3+ T cells in cultures according to day and culture inhibitors are depicted.

[0112] Fig. 20C The percentage of CD8+Tbet+ T cells in cultures according to day and culture inhibitor is depicted.

[0113] Fig.20D The percentage of CD8+GATA3+ T cells in cultures according to day and culture inhibitor is depicted.

[0114] Figures 20A-20DWe show that extended culture of dedifferentiated T cells under hybrid Th2 / TREG polarizing conditions in the presence of pemetrexed leads to the generation of CD8+ T cells expressing FOXP3 and GATA3 transcription factors.

[0115] Fig.21A IL-4 secretion by cells in culture according to day and culture inhibitor is depicted.

[0116] Fig. 21B IL-5 secretion by cells in culture according to day and culture inhibitor is depicted.

[0117] Fig. 21C IL-13 secretion by cells in culture according to day and culture inhibitor is depicted.

[0118] Figures 21A-21C We show that extended culture of dedifferentiated T cells under hybrid Th2 / TREG polarizing conditions results in the generation of T cells expressing the major Th2 cytokine phenotype: IL-4, IL-5, and IL-13 secretion.

[0119] Fig.22A IL-2 secretion by cells in culture according to day and culture inhibitor is depicted.

[0120] Fig. 22B IFN-γ secretion by cells in culture according to day and culture inhibitor is depicted.

[0121] Fig. 22C GM-CSF secretion by cells in culture according to day and culture inhibitor is depicted.

[0122] Figures 22A-22C We show that extended culture of dedifferentiated T cells under hybrid Th2 / TREG polarizing conditions results in the generation of T cells expressing the predominant Th2 cytokine phenotype: IL-2, IFN-γ, and GM-CSF secretion.

[0123] Fig.23A and 23B We demonstrate that treatment with the anti-TNF-α therapy etanercept significantly alters the TCR repertoire when measured by RNA sequencing, representing a novel approach for treatment of subjects prior to lymphocyte collection by apheresis.

[0124] Fig.24 It is demonstrated that extended culture of dedifferentiated T cells under hybrid Th2 / TREG polarizing conditions results in the generation of T cells expressing increased levels of the following molecules: CD25, CD27, 2B4, BTLA, and CTLA relative to control Th1 / Tc1 cells.

[0125] Fig.25It was demonstrated that extended culture of dedifferentiated T cells under hybrid Th2 / TREG polarizing conditions resulted in the generation of T cells expressing increased levels of the following molecules: TIGIT, TIM3, ICOS, LAIR1, and OX40 relative to control Th1 / Tc1 cells.

[0126] Fig.26A Depicted is FOXP3 expression in CD4+ and CD8+ T cells at the beginning and after culture as measured by flow cytometry.

[0127] Fig.26B Depicted is GATA3 expression in CD4+ and CD8+ T cells at the beginning and after culture as measured by flow cytometry.

[0128] Fig.27A Depicted is the expression of CD73 in CD4+ and CD8+ T cells at the beginning and after culture as measured by flow cytometry.

[0129] Fig.27B Depicted is the expression of CD103 in CD4+ and CD8+ T cells at the beginning and after culture as measured by flow cytometry.

[0130] Fig.28A Depicted are CD150 frequencies measured by flow cytometry in CD4+ and CD8+ T cells at the start of culture, after culture, and on control T cells not exposed to mTOR inhibitors measured by flow cytometry.

[0131] Fig.28B Depicted are the expression of CD27 versus CD95 by CD4+ T cells at the beginning and after culture as measured by flow cytometry.

[0132] Fig.29 IL-4, IL-2, IFN-γ, TNF-α, IL-17, and GM-CSF of the different cultured cells and control cells are depicted.

[0133] Fig. 30A Depicted are the cytokine content of transwell assays of Th1 / Tc1 cells with or without RAPA-501 cells.

[0134] Fig. 30B Depicted are flow cytometry results for CD4 and PD1 assays in Example 24.

[0135] Fig.31A IL-6, IP-10, and IFN-γ secretion by human microglia with or without exposure to RAPA-501 cells is depicted.

[0136] Fig.31B IL-6, IP-10, and IFN-γ secretion by human microglia with or without exposure to RAPA-501 cells is depicted.

[0137] Fig.32 The PC protocol and overall treatment approach are schematically depicted.

[0138] Fig.33 Schematic depiction of lymphocyte collection by apheresis before and after the PC regimen.

[0139] Fig.34 Schematic depiction of iT REG The PC regimen was performed before each repeated administration of cells.

[0140] Fig.35 Schematic diagram of the use of iT REG Monitoring of patients undergoing cell therapy.

[0141] Figures 36A-36B We demonstrate that treatment with the anti-TNF-α therapy etanercept significantly alters the TCR repertoire when measured by RNA sequencing, representing a novel approach for treatment of subjects prior to lymphocyte collection by apheresis.

[0142] Fig.37 showed that in hybrid Th2 / T REG Extended culture of dedifferentiated T cells under polarizing conditions results in the generation of T cells that express increased levels of the following molecules relative to control Th1 / Tc1 cells: CD25, CD27, 2B4, BTLA, and CTLA.

[0143] Fig.38 showed that in hybrid Th2 / T REG Extended culture of dedifferentiated T cells under polarizing conditions resulted in the generation of T cells expressing increased levels of the following molecules relative to control Th1 / Tc1 cells: TIGIT, TIM3, ICOS, LAIR1, and OX40.

[0144] Fig.39 Alternative designs are depicted.

[0145] Fig.40A Depicted are CD4 + RAPA-501GATA3 and FOXP3 in cells.

[0146] Fig.40B Depicted are CD8 + RAPA-501GATA3 and FOXP3 in cells.

[0147] Fig.41 An exemplary workflow of the dedifferentiation method of the present disclosure is depicted.

[0148] Fig.42 Depicts the hybrid T of the present disclosure REG / Presumed mechanism of action of Th2 cells. DETAILED DESCRIPTION

[0149] The present invention provides a method for T cell dedifferentiation and the resulting cells, a method for producing human hybrid regulatory T / Th2 cells (hybrid T REG / Th2 cells) and a method for using inducible regulatory T (iT REG ) cells for ALS treatment.

[0150] definition

[0151] As used herein, the singular forms "a", "an" and "the" may include plural referents unless the context clearly indicates otherwise.

[0152] In the claims and this disclosure the term "or" is used to mean "and / or" unless explicitly stated to refer to only alternatives or the alternatives are mutually exclusive.

[0153] When used with numerical values, the use of the term "about" is intended to include + / - 10%. For example, if the number of amino acids identified is about 200, this would include 180 to 220 (plus or minus 10%).

[0154] The terms "patient," "individual," and "subject" are used interchangeably herein and refer to a mammalian subject to be treated, with human patients being preferred. In some cases, the methods of the invention can be used in experimental animals, veterinary applications, and development of animal models of disease, including but not limited to rodents including mice, rats, and hamsters, and primates.

[0155] "Sample" is used herein in its broadest sense. Samples including cells, polynucleotides, polypeptides, peptides, antibodies, etc. may include body fluids; soluble fractions of cell preparations, or culture medium in which cells are grown; chromosomes, organelles, or membranes isolated or extracted from cells; genomic DNA, RNA or cDNA, polypeptides or peptides in solution or bound to a substrate; cells; tissues; tissue prints; fingerprints, skin or hair; etc.

[0156] "Treatment" is an intervention intended to prevent the development of a disease or to alter the pathology or symptoms of a disease. Thus, "treatment" can refer to both therapeutic treatment and prophylactic or preventive measures. Patients in need of treatment include those who already have the condition as well as those in need of prevention of the condition. In tumor (e.g., cancer) treatment, therapeutic agents can directly reduce the pathology of tumor cells, or make tumor cells more susceptible to treatment with other therapeutic agents (e.g., radiation and / or chemotherapy).

[0157] As used herein, a "treatment cycle" may generally refer to any primary treatment cycle, a first treatment cycle, a second treatment cycle, or one or more additional treatment cycles.

[0158] As used herein, "immune cell" is intended to include any cell of the immune system that can be assayed, including but not limited to B lymphocytes (also known as B cells), T lymphocytes (also known as T cells), natural killer (NK) cells, natural killer T (NKT) cells, lymphokine-activated killer (LAK) cells, monocytes, macrophages, neutrophils, granulocytes, mast cells, platelets, Langerhans cells, stem cells, dendritic cells, peripheral blood mononuclear cells, tumor infiltrating (TIL) cells, genetically modified immune cells (including hybridomas), drug-modified immune cells, and derivatives, precursors or progenitor cells of the above cell types.

[0159] "T cells" or "T lymphocytes" are a subpopulation of lymphocytes that are derived from the thymus and have heterodimeric receptors associated with proteins of the CD3 complex (e.g., rearranged T cell receptors, heterodimeric proteins on the surface of T cells that are responsible for the antigen / MHC specificity of the cell). T cell responses can be detected by measuring the effects of the T cell response on other cells (e.g., target cell killing, activation of other immune cells such as B cells) or the cytokines produced by the T cell response.

[0160] As used herein, the term "dedifferentiated T cells" refers to T cells that have been dedifferentiated by any of the methods disclosed herein. In certain aspects, dedifferentiated T cells have reduced RAPTOR or RICTOR expression relative to a control T cell population made under the same conditions without temsirolimus, an IL-2 signaling inhibitor, and vitamin D. "Dedifferentiated T cells" do not include T cells collected from patients, i.e., naturally occurring T cells.

[0161] As used herein, the term "anti-CD3 / anti-CD28" is understood to refer to anti-CD3 / anti-CD28 antibodies. For example, "anti-CD3 / anti-CD28 magnetic beads" are understood to refer to magnetic beads having anti-CD3 / anti-CD28 antibody moieties associated therewith. Where it is disclosed that anti-CD3 / anti-CD28 co-stimulation is not provided even by a particular format such as anti-CD3 / anti-CD28 magnetic beads, it is understood that this may also exclude the use of other formats of anti-CD3 / anti-CD28 co-stimulation.

[0162] It should also be understood that in the present disclosure, where costimulation by anti-CD3 / anti-CD28 antibodies is performed, this costimulation can be provided by any form of anti-CD3 / anti-CD28 antibodies. By way of example and not limitation, where costimulation is indicated by using anti-CD3 / anti-CD28 beads, anti-CD3 / anti-CD28 nanoparticles or microparticles can be used. Where it is disclosed that anti-CD3 / anti-CD28 costimulation is not provided even by a specific form such as anti-CD3 / anti-CD28 magnetic beads, it should be understood that this can also exclude the use of other forms of costimulation of anti-CD3 / anti-CD28.

[0163] As used herein, unless otherwise indicated, the terms "human hybrid TREG / Th2 cells", "iTREG" and "TREG / Th2 cells" refer to cells that have been differentiated by the methods of the present disclosure. The "human hybrid TREG / Th2 cells", "iTREG" and "TREG / Th2 cells" of the present disclosure do not include T cells collected from patients, i.e., naturally occurring T cells.

[0164] As used herein, the term "manufactured TREG cells" refers to cells produced by the dedifferentiation and redifferentiation methods of the present disclosure, and unless otherwise specified, should be understood to include TREG cells and human hybrid TREG / Th2 cells.

[0165] As used herein, unless otherwise indicated, "control Th1 / Tc1 cells" refer to cells that have not been treated with vitamin D, temsirolimus, or an IL-2 signaling inhibitor, but have been co-stimulated using anti-CD3 / anti-CD28 coated magnetic beads at a 3:1 ratio (beads:T cells) in medium supplemented with 20 IU / mL IL-2 and 20,000 IU / mL IFN-α and otherwise cultured identically to the cells to which they are being compared. It should also be understood that where a control cell population (or control T cells) is referred to as being untreated with culture additives (including temsirolimus, vitamin D and an IL-2 signaling inhibitor) or in the context of dedifferentiated cells, this population (or T cells) has been further co-stimulated using anti-CD3 / anti-CD28 coated magnetic beads at a 3:1 ratio (beads:T cells) in medium supplemented with 20 IU / mL IL-2 and 20,000 IU / mL IFN-α and otherwise cultured identically to the cells to which they are compared (i.e., they are "control Th1 / Tc1 cells").

[0166] The present disclosure provides novel methods for the ex vivo generation of T cells in a reduced differentiation state, which are based on the use of novel pharmacological combinations and defined T cell co-stimulatory conditions to convert differentiated effector memory T cells into less differentiated central memory-type T cells.

[0167] like Fig.41 As shown, the dedifferentiated T cells of the present disclosure may 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, RORγ, FOXP3 and GATA3). Redifferentiated T cells may have a hybrid fate characterized by expression of GATA3 and FOXP3, and stem cell memory characterized by expression of CD45RA and CD150 and no expression of checkpoint proteins.

[0168] like Fig.42 As shown, the hybrid T REG The putative mechanism of action of Th2 cells, in which hybrid T REG Th2 cells can be activated through inflammation via CD39 or CD73 receptors and by TNF-α, which enables the cells to modulate pathogenic T cells to prevent killing.

[0169] Methods for T cell dedifferentiation and resulting cells

[0170] A novel method for the ex vivo generation of T cells in a reduced differentiation state is presented, which is based on the conversion of differentiated effector memory T cells into less differentiated central memory-type T cells using a novel pharmacological combination and defined T cell co-stimulatory conditions.

[0171] In one embodiment, the method includes: inoculating a culture input cell group including T cells from a subject at a certain cell density in a culture medium including vitamin D, temsirolimus and an IL-2 signaling inhibitor; adding anti-CD3 / anti-CD28 coated magnetic beads to the T cells and culture medium at a bead:T cell ratio of 1:1 or lower to stimulate the T cells, or without adding any co-stimulatory beads; incubating the culture input cell group and culture medium for a certain period of time to produce dedifferentiated T cells. In some embodiments, the subject has been treated with anti-TNF-α therapy before collecting the culture input cell group. In some embodiments, the anti-TNF-α therapy is etanercept or adalimumab. In some embodiments, co-stimulation using anti-CD3 / anti-CD28 is not performed.

[0172] In any of the foregoing embodiments, the culture medium may be free of IL-2 and no IL-2 may be added to the culture medium.

[0173] In any of the foregoing embodiments, the cell density can be about 1.5 x 106 T cells per mL to 18 x 106 T cells per mL. By way of example and not limitation, 6×106 T cells per mL to 18×106 T cells per mL, 12×106 T cells per mL to 18×106 T cells per mL, 1.5×106 T cells per mL to 12×106 T cells per mL, 1.5×106 T cells per mL to 6×106 T cells per mL, 6×106 T cells per mL to 12×106 T cells per mL, or 1.5×106 T cells per mL, 3×106 T cells per mL, 6×106 T cells per mL, 9×106 T cells per mL, 12×106 T cells per mL, 15×106 T cells per mL, or 18×106 T cells per mL. In some embodiments, by way of example and not limitation, it is contemplated that it may be preferable to initiate cell culture at a higher density, such as 9×10 6 T cells per mL or 18×10 6 T cells per mL.

[0174] In any of the foregoing embodiments, the temsirolimus can be present at a concentration of about 0.3 μM to about 10 μM. By way of example and not limitation, the temsirolimus can be present at a concentration of about 0.3 μM to about 1 μM, 0.3 μM to about 0.75 μM, 0.3 μM to about 0.5 μM, 0.5 μM to about 1 μM, 0.75 μM to about 1 μM, 0.5 μM to about 0.75 μM, 0.3 μM to about 10 μM, 0.3 μM to about 5 μM, 0.3 μM to about 3.3 μM, 1 μM to about 5 μM, To about 10 μM, 3.3 μM to about 10 μM, 3.3 μM to about 5 μM, or by way of example and not limitation, at a concentration of about 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, or 1 μM, 2 μM, 3 μM, 3.3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM or 10 μM.

[0175] In any of the foregoing embodiments, the IL-2 signaling inhibitor may be an anti-IL-2 receptor antibody or a fragment thereof. By way of example and not limitation, the IL-2 signaling inhibitor may be basiliximab or daclizumab. By way of example and not limitation, the IL-2 signaling inhibitor may be 5 to 50 μg / mL, 5 to 40 μg / mL, 5 to 30 μg / mL, 5 to 20 μg / mL, 5 to 10 μg / mL, 10 to 50 μg / mL, 20 to 50 μg / mL, 30 to 50 μg / mL, 40 to 50 μg / mL, 30 to 40 μg / mL, 20 to 40 μg / mL, 10 to 40 μg / mL, 5 to 40 μg / mL, 5 to 30 In some embodiments, the present invention relates to a concentration of at least 5 μg / mL, 10 μg / mL, 15 μg / mL, 20 μg / mL, 25 μg / mL, 30 μg / mL, 35 μg / mL, 40 μg / mL, 45 μg / mL or 50 μg / mL.

[0176] In any of the foregoing embodiments, by way of example and not limitation, the time period may 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 mTORC1 and mTORC2 reduction can be used as a guide to determine the optimal culture interval. In some embodiments, other molecular markers of dedifferentiated cells can be used to determine the optimal culture interval, including but not limited to: RNA expression of T cell effector molecules (ie, IFN-γ decreases); RNA expression of transcription factors (ie, KLF4 increases); Evidence of autophagy markers (ie, p62 increases); and upregulation of markers present on naive T cell subsets (ie, CD127 increases).

[0177] In any of the foregoing embodiments, by way of example and not limitation, the bead: T cell ratio can be 1: 3, or costimulation may not be performed. By way of example and not limitation, the bead: T cell ratio can be between 1: 1 and 1: 12, between 1: 1 and 1: 3, between 1: 3 and 1: 12. By way of further example and not limitation, the 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 examples, anti-CD3 / anti-CD28 costimulation cannot be utilized, i.e., in some embodiments, anti-CD3 / anti-CD28 costimulation is not performed during the initial dedifferentiation process.

[0178] In any of the foregoing embodiments, the co-stimulation of the culture input cell population can be achieved using nanoparticles containing anti-CD3 / anti-CD28, which can be used at a concentration lower than the recommended concentration. By way of example and not limitation, such nanoparticles can be used at about 0.01 times to about 0.1 times, about 0.025 times to about 0.1 times, about 0.05 times to about 0.1 times, about 0.075 times to about 0.1 times, about 0.01 times to about 0.075 times, about 0.01 times to about 0.05 times, about 0.01 times to about 0.025 times, about 0.025 times to about 0.075 times, about 0.025 times to about 0.05 times, about 0.05 times to about 0.075 times, or about 0.01 times, about 0.025 times, about 0.05 times, about 0.075 times, or about 0.01 times, about 0.05 times, about 0.075 times, or about 0.01 times, about 0.025 times, about 0.05 times, about 0.075 times, or about 0.01 times. By way of example and not limitation, compared to the recommended dose (10 μL per 1×10 6 T cells), the drug may be used at a reduced dose, by way of example and not limitation, such as 1.1 μL (a nine-fold reduction) or about 0.11-fold. Finally, in the most extreme example, anti-CD3 / anti-CD28 co-stimulation cannot be used, ie, in some embodiments, anti-CD3 / anti-CD28 co-stimulation is not performed during the initial dedifferentiation process.

[0179] Alternatively, if anti-CD3 / anti-CD28 co-stimulation is to be used to generate manufactured T cells, the source of co-stimulation can be provided by soluble anti-CD3 / anti-CD28 microparticles. By way of example and not limitation, the manufacturer (e.g., The soluble anti-CD3 / anti-CD28 microparticles can be used at 20% of the strength recommended by Bio-Techne. By way of further example, the soluble anti-CD3 / anti-CD28 microparticles can be used at 5%, 10%, 15%, 20%, 25% or 30% of the manufacturer's recommended strength.

[0180] In any of the foregoing embodiments, if anti-CD3 / anti-CD28 stimulation is performed, it may be performed using anti-CD3 / anti-CD28 in an amount sufficient to achieve the desired dedifferentiated cell properties.

[0181] In any of the foregoing embodiments, the culture medium can further include 5% human serum. By way of example and not limitation, the culture medium can include 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 ranges including values ​​therebetween.

[0182] In any of the foregoing embodiments, the culture medium may include X-Vivo 20 medium. Any suitable culture medium for culturing T cells may be used.

[0183] In any of the foregoing embodiments, by way of example and not limitation, the vitamin D can be present in the culture medium at about 0.03nM to about 1nM, 0.03nM to about 0.5nM, 0.03nM to about 0.1nM, 0.03nM to about 0.05nM, 0.05nM to about 0.1nM, 0.05nM to about 0.5nM, 0.05nM to about 1nM, 0.1nM to about 1nM, 0.1nM to about 0.5nM or 0.5nM to about 1nM, or by way of example and not limitation, the vitamin D is present at a concentration of about 0.03nM, 0.05nM, 0.1nM, 0.5nM or 1nM.

[0184] In any of the foregoing embodiments, the method may further include measuring the expression level of RAPTOR or RICTOR and a housekeeping protein in the culture input cell population, wherein the time period lasts until the expression level of RAPTOR or RICTOR in the manufactured T cells is reduced by at least 50% relative to a control T cell population, respectively, wherein the control T cell population is manufactured under the same conditions as the culture input cell population without temsirolimus, an IL-2 signaling inhibitor, and vitamin D. In some embodiments, the time period lasts until the expression level of RAPTOR or RICTOR in the manufactured T cells is reduced by 50% or more relative to a control T cell population, respectively, wherein the control T cell population is manufactured under the same conditions as the culture input cell population without temsirolimus, an IL-2 signaling inhibitor, and vitamin D. By way of example and not limitation, the time period can continue until the expression level of RAPTOR or RICTOR is reduced by at least 50%, 60%, 70%, 80%, 90%, 95%, 99% or more, respectively, relative to a control T cell population produced under the same conditions as the culture input cell population in the absence of temsirolimus, an IL-2 signaling inhibitor, and vitamin D.

[0185] In any of the foregoing embodiments, the housekeeping protein may be actin. In some embodiments, the housekeeping protein may be GAPDH. In any of the foregoing embodiments, the step of measuring expression levels may be performed by Western blot analysis.

[0186] In any of the foregoing embodiments, the time period can be continued until the expression level of RAPTOR or RICTOR in the culture input cell population is reduced by at least 50% relative to a control T cell population, the control T cell population being manufactured under the same conditions as the culture input cell population without temsirolimus, an 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 a control T cell population.

[0187] In any of the foregoing embodiments, the time period of initial dedifferentiation culture can be continued until the RNA expression pattern differs by at least 10%, and more preferably 50%, relative to control T cells cultured under the same conditions in the absence of temsirolimus, vitamin D, and an IL-2 signaling inhibitor, i.e., T cell effector molecules (including but not limited to granzyme B, IL-10, and IFN-γ) are reduced; transcription factors associated with cells with a reduced differentiation state (including but not limited to Nanog, KLF4, and KLF10) are increased; expression of molecules preferentially expressed on naive T cell subsets (including but not limited to CD 127, IL-7 receptor α chain) is increased; transcription factors associated with Th1 type differentiation (including but not limited to T-BET and STAT1) are reduced; and transcription factors that promote cell survival (including but not limited to HIF-1α) are relatively retained.

[0188] In any of the foregoing embodiments, the initial dedifferentiation culture period can be continued until the RNA expression pattern differs by at least 10%, and more preferably 50%, relative to control T cells cultured under the same conditions without temsirolimus, vitamin D, and IL-2 signaling inhibitors, i.e., wherein the expression of molecules indicating cells that have undergone autophagy changes by at least 10%, and more preferably 50%. As an example, the dedifferentiated cells have increased p62 expression by Western blot analysis relative to control T cells; by way of example and not limitation, other methods of measuring 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.

[0189] In any of the foregoing embodiments, the culture medium may be free of human serum, temsirolimus, vitamin D, IL-2 signaling inhibitors, or any combination thereof, which may not be present in the culture medium at the start of the culture. In such embodiments, human serum, temsirolimus, vitamin D, or IL-2 signaling inhibitors may be added to the culture medium at about the same time as the culture input cell population is inoculated or at a subsequent time.

[0190] By way of example and not limitation, an intravenous formulation of 1,25-vitamin D ("Calcitriol") may be used. This formulation is preferred because it is completely soluble in the culture medium and has a 1,25 hydroxylation that occurs naturally in the kidney and therefore must be present when vitamin D is added to the culture. Trade names for calcitriol include Rocaltrol, Calcijex, and Decostriol. It is also contemplated 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; 2016, Vol. 26, No. 11.

[0191] In some embodiments, dedifferentiated T cells that can be obtained by any method of the methods disclosed herein are provided. In some embodiments, compositions comprising dedifferentiated T cell populations are provided. In some embodiments, at least a portion of the dedifferentiated T cells express less than 50% RAPTOR or RICTOR relative to a control T cell population made under the same conditions as the culture input cell population in the absence of temsirolimus, an IL-2 signaling inhibitor, and vitamin D. In some embodiments, the dedifferentiated T cells express less than 50% RAPTOR or RICTOR relative to a control T cell population made under the same conditions as the culture input cell population in the absence of temsirolimus, an IL-2 signaling inhibitor, and vitamin D. By way of example and not limitation, the dedifferentiated T cells or population of dedifferentiated T cells may express 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1% or less RAPTOR or RICTOR relative to a control T cell or a control T cell population made under the same conditions as the culture input cell population, respectively, but in the absence of temsirolimus, an IL-2 signaling inhibitor, and vitamin D.

[0192] In some embodiments, the dedifferentiated T cell population or dedifferentiated T cells can be characterized by reduced RNA expression of cytolytic molecules (including but not limited to granzyme B) and / or cytokine molecules (including but not limited to IFN-γ) relative to a control T cell population incubated under the same conditions without temsirolimus, vitamin D, and an IL-2 signaling inhibitor. By way of example and not limitation, such reduction can be at least 10%, at least 20%, at least 30%, at least 40%, at least 50% or more.

[0193] In some embodiments, relative to a control T cell population incubated under the same conditions without temsirolimus, vitamin D, and an IL-2 signaling inhibitor, a dedifferentiated T cell population or a dedifferentiated T cell can be characterized by an increase in RNA expression of transcription factors associated with iPSCs (including but not limited to Nanog, KLF4, and KLF10) and / or molecules associated with naive T cells (including but not limited to IL-7 receptor CD127). By way of example and not limitation, such an increase can be at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or more.

[0194] In some embodiments, relative to a control T cell population incubated under the same conditions without temsirolimus, vitamin D, and an IL-2 signaling inhibitor, a dedifferentiated T cell population or dedifferentiated T cells can be characterized by a reduction in RNA expression of transcription factors associated with Th1 effector T cells (including but not limited to T-Bet and STAT1), wherein approximately the same amount of HIF-1-α expression is maintained. By way of example and not limitation, such reduction can be at least 10%, at least 20%, at least 30%, at least 40%, at least 50% or more. By way of example and not limitation, HIF-1-α expression can be within about 20%, 15%, 10%, or 5% of a control T cell population.

[0195] In some embodiments, the dedifferentiated T cell population or dedifferentiated T cells can be characterized by increased protein expression of p62 relative to a control T cell population incubated under the same conditions without temsirolimus, vitamin D, and an IL-2 signaling inhibitor. By way of example and not limitation, such an increase can be at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or more.

[0196] For the production of human hybrid regulatory T / Th2 cells (hybrid T REG / Th2 cells) and T REG Methods

[0197] In the present disclosure, there is provided an in vitro manufacturing method for producing iTREG cells, the early differentiation state of the iTREG cells and the exhaustion of Th1 type and Th17 type polarization are enhanced. This method requires a two-step process, the first step is composed of T cell dedifferentiation, and the second step is iTREG cell manufacturing. Thus dedifferentiated T cell substrate manufacturing human iTREG cells can use a new combination of cytokines (standard iTREG uses IL-2 and TGF-β cytokines, and additionally uses cytokine IL-4 related to Th2 differentiation classics) and optional new agents (such as pemetrexed as described herein) to carry out. In some embodiments, iTREG cells can be produced in the absence of pemetrexed. Since such cells express TREG and Th2 molecules simultaneously, the cells produced by this method are called "human hybrid TREG / Th2 cells".

[0198] In some embodiments, the method includes: culturing the dedifferentiated T cells of the present disclosure in a culture medium including IL-2, IL-4 and TGF-β; adding anti-CD3 / anti-CD28 coated magnetic beads at a ratio of 3: 1 (bead: T cell ratio); and incubating the dedifferentiated T cells for a period of time to produce TREG / Th2 cells. In some embodiments, the method includes culturing the dedifferentiated T cell population of the present disclosure. The ratio of anti-CD3 / anti-CD28 beads can be changed as long as the costimulation is sufficient to differentiate the cells.

[0199] In some embodiments, the method includes: culturing dedifferentiated T cells in a medium including IL-2, IL-4 and TGF-β, relative to a control T cell group manufactured under the same conditions without temsirolimus, IL-2 signaling inhibitors and vitamin D, the dedifferentiated T cells have reduced RAPTOR or RICTOR expression; adding anti-CD3 / anti-CD28 coated magnetic beads at a ratio such as 3: 1 (bead: T cell ratio); and incubating the dedifferentiated T cells for a certain period of time to produce TREG / Th2 cells. In some embodiments, the method includes culturing a dedifferentiated T cell group of the present disclosure. The ratio of anti-CD3 / anti-CD28 beads can be changed as long as costimulation is sufficient to differentiate cells. In some embodiments, the expression of RAPTOR or RICTOR is normalized by housekeeping proteins (such as, for example and not limitation, actin or GAPDH).

[0200] In any of the foregoing embodiments, IL-2 can be present in the 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.

[0201] In any of the foregoing embodiments, the culture medium may further include IL-4. In any of the foregoing embodiments, IL-4 may be present in the culture medium at a concentration of about 100 IU / mL to 1000 IU / mL, 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. In some embodiments, by way of example and not limitation, if it is desired to achieve reduced Th2 polarization, lower concentrations such as 100 IU / mL may be used.

[0202] In any of the foregoing embodiments, TGF-β can be present in the culture medium at a concentration of about 10 ng / mL. By way of example and not limitation, the concentration of TGF-β can be about 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, or 10 ng / mL.

[0203] In any of the foregoing embodiments of differentiation, the beads: T cell ratio can be 3: 1. In some embodiments, an equivalent amount of anti-CD3 / anti-CD28 alternatives with the same effect can be used. In some embodiments, the amount of costimulation is sufficient to saturate the cells. In any of the foregoing embodiments, the amount of costimulation can be sufficient to increase the expression of GATA3 and FOXP3 in human hybrid TREG / Th2 cells.

[0204] In any of the foregoing embodiments, the culture medium may further include pemetrexed. By way of example and not limitation, pemetrexed may be present in the culture medium at a concentration of 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 a concentration of 5 nM, 10 nM, 25 nM, 50 nM, 75 nM or 100 nM equivalents. In certain embodiments, the culture medium does not include pemetrexed, and pemetrexed is not added to the culture medium.

[0205] In any of the foregoing embodiments, by way of example and not limitation, the time period for incubating the dedifferentiated T cells can be 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. In some embodiments, by way of example and not limitation, if the differentiation state of the hybrid Th2 / TREG cells is very limited, a shorter culture interval, such as 3 days to 10 days, can be considered.

[0206] The present disclosure also relates to methods and TREG cells produced by any of the aforementioned methods without the use of IL-4.

[0207] In some embodiments, by flow cytometry, relative to control Th1 / Tc1 cells, TREG or TREG / Th2 cells produced by the methods of the present disclosure may have increased expression of at least one of CD25, CD27, 2B4, BTLA, CTLA4, TIGIT, TIM3, ICOS, LAIR1, and OX40. In some embodiments, by way of example and not limitation, this increase may be 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.

[0208] In some embodiments, the TREG or TREG / Th2 cells produced by the methods of the present disclosure may have reduced secretion of inflammatory cytokines relative to control Th1 / Tc1 cells. By way of example and not limitation, such cytokines may include IFN-γ and TNF-α. In some embodiments, by way of example and not limitation, this reduction may be 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.

[0209] In some embodiments, the TREG or TREG / Th2 cells produced by the methods of the present disclosure may have reduced TBET expression and increased FOXP3 expression relative to control Th1 / Tc1 cells and / or have increased IL-4 secretion and increased GATA3 expression relative to control Th1 / Tc1 cells. In some embodiments, by way of example and not limitation, this reduction or increase may be 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.

[0210] In some embodiments, a TREG or TREG / Th2 cell population may have at least 5% of CD4+ or CD8+ cells expressing GATA3. By way of example and not limitation, a TREG or TREG / Th2 cell population may 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 expressing GATA3. In some embodiments, whether a cell expresses GATA3 is determined by flow cytometry. In some embodiments, relative to producing T REG or T REG The T cells of the Th2 cell group are the control T cell group with characteristics, T REG or T REG Th2 cell populations can express GATA3-expressing CD4 + or CD8 + The frequency of the increase of T cells. In some embodiments, the frequency of the increase can be an increase of 50% or more. By way of example and not limitation, the increase can be 50%, 100%, 200%, 300%, 500%, 1000%, 2000%, 3000% or more.

[0211] In some embodiments, a TREG or TREG / Th2 cell population may have at least 5% CD4+ or CD8+ cells expressing FoxP3. By way of example and not limitation, a TREG or TREG / Th2 cell population may have at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, or at least 45% CD4+ or CD8+ T cells expressing FoxP3. In some embodiments, whether a cell expresses FoxP3 is determined by flow cytometry. In some embodiments, relative to producing T REG or T REG The T cells of the Th2 cell group are the control T cell group with characteristics, T REG or T REG Th2 cell populations can express FOXP3 in CD4 + or CD8 + The frequency of the increase of T cells. In some embodiments, the frequency of the increase can be an increase of 50% or more. By way of example and not limitation, the increase can be 50%, 100%, 200%, 300%, 500%, 1000%, 2000%, 3000% or more.

[0212] In some embodiments, a TREG or TREG / Th2 cell population may have at least 10% of CD4+ or CD8+ cells expressing CD73. By way of example and not limitation, a TREG or TREG / Th2 cell population may have at least 10%, at least 15%, at least 20%, or at least 25% of CD4+T cells expressing CD73. By way of further example and not limitation, a TREG or TREG / Th2 cell population may 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 expressing CD73. In some embodiments, whether a cell expresses CD73 is determined by flow cytometry. In some embodiments, relative to producing T REG or T REG The T cells of the Th2 cell group are the control T cell group with characteristics, T REG or T REG Th2 cell populations can express CD4 + or CD8 + The frequency of the increase of T cells. In some embodiments, the frequency of the increase can be an increase of 50% or more. By way of example and not limitation, the increase can be 50%, 100%, 200%, 300%, 500%, 1000%, 2000%, 3000% or more.

[0213] In some embodiments, a TREG or TREG / Th2 cell population may have at least 10% of CD4+ or CD8+ cells expressing CD103. By way of example and not limitation, a TREG or TREG / Th2 cell population may 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 expressing CD103. In some embodiments, whether a cell expresses CD103 is determined by flow cytometry. In some embodiments, relative to producing T REG or T REG The T cells of the Th2 cell group are the control T cell group with characteristics, T REG or T REG Th2 cell populations can express CD4 + or CD8 + The frequency of the increase of T cells. In some embodiments, the frequency of the increase can be an increase of 50% or more. By way of example and not limitation, the increase can be 50%, 100%, 200%, 300%, 500%, 1000%, 2000%, 3000% or more.

[0214] In some embodiments, T is measured by flow cytometry. REG or T REG The Th2 cell population may have at least 5% CD4 expressing both FOXP3 and GATA3 + or CD8 + By way of example and not limitation, T REG or T REG The Th2 cell population may have at least 5%, 10%, 20%, 30%, 40% or 50% of CD4+ T cells expressing both FOXP3 and GATA3. + or CD8 + cell.

[0215] In some embodiments, a TREG or TREG / Th2 cell population may have at least 20% of CD4+ or CD8+ T cells expressing CD150. By way of example and not limitation, a TREG or TREG / Th2 cell population may 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 expressing CD150. In some embodiments, a TREG or TREG / Th2 cell population may have an increased frequency of cells expressing CD150 relative to a control T cell population incubated without exposure to an mTOR inhibitor. In some embodiments, whether a cell expresses CD150 is determined by flow cytometry. In some embodiments, relative to producing T REG or T REG The T cells of the Th2 cell group are the control T cell group with characteristics, T REG or T REG Th2 cell populations can express CD4 + or CD8 + The frequency of the increase of T cells. In some embodiments, the frequency of the increase can be an increase of 50% or more. By way of example and not limitation, the increase can be 50%, 100%, 200%, 300%, 500%, 1000%, 2000%, 3000% or more.

[0216] In some embodiments, after costimulation with anti-CD3 / anti-CD28 beads at a 3:1 bead:T cell ratio, the TREG or TREG / Th2 cell population can express at least 5 pg / mL / 1×106 cells / day of IL-4. By way of example and not limitation, the TREG or TREG / Th2 cell population 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 / 1×106 cells / day of IL-4.

[0217] In some embodiments, a TREG or TREG / Th2 cell population may express at least 100 pg / mL / 1×10 6 cells / day of IL-2 following co-stimulation using anti-CD3 / anti-CD28 beads at a 3:1 beads:T cell ratio.

[0218] In some embodiments, the TREG or TREG / Th2 cell population may express less than 100 pg / mL / 1×10 6 cells / day of IFN-γ or GM-CSF following co-stimulation using anti-CD3 / anti-CD28 beads at a 3:1 beads:T cell ratio.

[0219] In some embodiments, the TREG or TREG / Th2 cell population may express less than 10 pg / mL / 1×10 6 cells / day of IFN-α or IL-17 following co-stimulation using anti-CD3 / anti-CD28 beads at a 3:1 beads:T cell ratio.

[0220] In some embodiments, TREG or TREG / Th2 cells may express both GATA3 and FOXP3. In some embodiments, TREG or TREG / Th2 cells may express GATA3, FOXP3, CD103, and CD73. In some embodiments, a TREG or TREG / Th2 cell population may be characterized by at least 5% of T cells expressing GATA3, at least 5% of T cells expressing FOXP3, at least 5% of T cells expressing CD103, and at least 5% of T cells expressing CD73, as measured by flow cytometry.

[0221] In any of the foregoing embodiments, the TREG or TREG / Th2 cells or populations thereof may have at least one of the properties or any combination thereof to the extent that the foregoing properties are not incompatible.

[0222] For use with inducible regulatory T (iT REG ) cells for the treatment of ALS

[0223] In this protocol, pentostatin plus cyclophosphamide regimen will be used as an immunodepletion and immunosuppression method that will enhance iT in ALS. REG Cell therapy. This PC regimen may have direct beneficial effects because it can deplete and suppress Th1-type immune cells associated with the pathogenesis of ALS. In addition, the PC regimen will serve as host conditioning, which will increase the immune T cell space, thereby achieving more effective iT REGCell therapy. Specifically, for this regimen, the dose of the PC regimen has been reduced to help mitigate any potential adverse effects of the regimen in the new ALS patient population. For this regimen: The starting dose of pentostatin was increased from 4 mg / m 2 Reduce to 1 mg / m 2 ; the number of infusions of pentostatin was reduced from the previous value of four infusions per cycle, compared to the current regimen value of one infusion per cycle; and the initial dose of cyclophosphamide was reduced from 200 mg per day to 100 mg per day. Second, the intensity of the PC regimen has been reduced in accordance with the established goals regarding the depth of immune depletion to be achieved with the PC regimen. Because a more cautious approach must be taken in the ALS patient population, the current regimen, the PC regimen, attempts to achieve a more modest reduction in ALC counts, i.e., to values ​​just below 750 cells per microliter. Generally, this level of immune depletion is not associated with long-term poor immune function in terms of a high incidence of opportunistic infections.

[0224] Without being bound by theory, it is expected that the PC regimen will deplete and inhibit the Th1 / Tc1 type adaptive immune subsets associated with the progression of ALS pathogenesis. However, this therapy does not address the underlying major events in ALS, namely: misfolded RNA species, insufficient autophagy to clear harmful RNA / DNA products, and subsequent RNA / DNA activation of innate inflammation at the level of the NLRP3 inflammasome driven by the P2X7 receptor. This inflammasome activation, which has been shown to be effective in ALS models, drives subsequent IL-1-β activation as one of the earliest and most potent pro-inflammatory signals, which then drives adaptive T cell inflammation mediated primarily by the Th1 / Tc1 subset. In fact, it has recently been proposed that NLRP3 inhibition represents a novel approach for the treatment of a variety of neurodegenerative diseases. Nucleoside reverse transcriptase inhibitors (NRTIs) are antiviral agents approved for the treatment of HIV disease, which may also play a role in the treatment of ALS. ALS patients may have increased levels of human endogenous retrovirus K (HERV-K), which in part drive disease pathogenesis in model systems through regulation of TDP-43 deposits, a key mechanistic component in ALS; in order to translate this biology clinically, a clinical trial evaluating an HIV antiviral cocktail of Darunavir, Ritonavir, Raltegravir, and Zidovudine has been initiated (NCT02437110). The NRTI molecule lamivudine (3TC) has also been described to inhibit the P2X7 receptor, which drives NLRP3 activation that occurs in ALS. Based in part on these observations, a Phase II clinical trial (NCT02363452) has been initiated to evaluate the ability of a three-drug regimen of lamivudine, zidovudine, and abacavir to reduce inflammation in patients with Aicardi-Goutieres Syndrome (AGS), a disease that mimics the innate inflammatory events in ALS, namely: accumulation of intracellular RNA species, activation of inflammasome pathways, and resulting systemic Th1-driven inflammation. Because lamivudine has been characterized as a potent inhibitor of the NLRP3 inflammasome, and because of the desire to produce a treatment regimen that is well tolerated in the ALS patient population, single-agent NRTI therapy with lamivudine was chosen in this regimen.

[0225] Thus, without being bound by theory, it is expected that a sequential strategy in which Th1 / Tc1 responses are first depleted and suppressed (by a PC regimen) and then secondarily controlling the driving force inflammasome activation will represent a new approach for providing sustainable modulation of the complex neuroinflammatory networks involved in ALS. It may also be beneficial to use lamivudine in an ALS therapeutic platform as a next step, which is to integrate iT REG Cell therapy was further incorporated into the platform. That is, three treatment modalities (pentostatin / cyclophosphamide; lamivudine; and iT REG Each of these treatment modalities in glioma cells operates at least in part by diverting the transport of ATP from the P2X7-driven NLRP3-mediated inflammasome to the immunosuppressive molecule adenosine. First, pentostatin increases adenosine by inhibiting adenosine deaminase, thereby preventing adenosine from being converted to inosine; second, lamivudine is a known P2X7 inhibitor, thereby directly inhibiting the inflammasome. Third, iT REG The cells provide CD39- and CD73-mediated ectonucleotidase activity that processes ATP into adenosine. Regarding this final process, it is important to note that microglia intrinsically utilize CD39 and CD73 to reduce neuroinflammation.

[0226] Taken together, these data provide evidence that the primary neurodegenerative process in ALS induces a secondary inflammatory response that drives disease progression and points to multistep therapeutic interventions, including control of inflammasome activation, depletion and inhibition of Th1 / Tc1 subsets, and T cell death. REG Given this information, there is great interest in evaluating immunomodulatory therapies for patients with ALS. It is hypothesized that optimal control of neuroinflammation in patients with ALS will require a three-pronged approach that addresses each of the components mentioned above, namely: (1) control of innate inflammasome activation (via lamivudine administration, as described below); (2) reduction of pre-existing Th1-type inflammatory cells (via the pentostatin / cyclophosphamide regimen, as further described below); and (3) promotion of iT through adoptive T cell transfer. REG There are several reasons why this combined approach may be necessary. First, if underlying inflammasome activation cannot be addressed by maintenance therapy, host conditioning therapy and T REG Any immunological and therapeutic benefits achieved during cell therapy will likely be undermined by the underlying neurodegenerative process. Second, even if optimized T cells are REG Infusion of independent cell populations into a host with uncontrolled Th1-driven inflammation also represents a serious immunological challenge: pre-existing Th1-type cells can be recruited in the adoptively transferred T cells. REGcells show differentiation plasticity, thereby converting protective T REG Third, the efficacy of adoptively transferred T cell populations is intricately linked to the extent of the immune T cell space, which can be largely defined by the presence of T cell growth factors such as IL-7 and IL-15; the creation of this immune space is created by the host preparation regimen (including the pentostatin plus cyclophosphamide (PC) regimen) that has been used for decades in allogeneic hematopoietic stem cell transplantation and is now used in the field of cancer therapy using adoptive T cell transfer. Notably, T cells previously implicated in neurodegenerative diseases or autoimmune diseases REG Clinical trials of cell therapies have not yet incorporated host preparation protocols such as the PC regimen.

[0227] In some embodiments, the method comprises subjecting the subject to one or more primary treatment cycles, each of the one or more primary treatment cycles comprising: administering pentostatin to the subject; and / or administering cyclophosphamide to the subject; and subjecting the subject to one or more immunotherapy treatment cycles, the one or more immunotherapy treatment cycles comprising: administering to the subject a therapeutically effective amount of a manufactured T REG The therapeutically effective amount can be determined by a person skilled in the art by methods known in the art and disclosed herein.

[0228] In the foregoing embodiments, each of the one or more immunotherapy treatment cycles may further include administering a nucleoside reverse transcriptase inhibitor to the subject. The nucleoside reverse transcriptase inhibitor may be an inhibitor of the NLRP3 inflammasome; or the nucleoside reverse transcriptase inhibitor may be lamivudine. Each of the one or more immunotherapy treatment cycles may further include: administering pentostatin to the subject; and / or administering cyclophosphamide to the subject. The step of administering pentostatin to the subject during each of the one or more immunotherapy treatment cycles may be performed on day 1 and day 4 of each of the one or more immunotherapy treatment cycles. The step of administering pentostatin to the subject during each of the one or more immunotherapy treatment cycles may be performed on day 1, day 2, day 3, day 4, and day 5 of each of the one or more immunotherapy treatment cycles. The method may include two or more immunotherapy treatment cycles, and by way of example and not limitation, each of the two or more immunotherapy treatment cycles is separated by 0 to 4 weeks, 0 to 3 weeks, 0 to 2 weeks, 0 to 1 week, and by way of example and not limitation, any value between 0 weeks, 1 week, 2 weeks, 3 weeks, or 4 weeks. Each of the one or more immunotherapy treatment cycles may be 18 weeks long. Each of the one or more immunotherapy treatment cycles may further include administering an adenosine receptor modulator to the subject. Other nucleoside reverse transcriptase inhibitors may be used, including but not limited to those disclosed in U.S. Pat. No. 9,326,983 (which is incorporated herein by reference), lamivudine, zidovudine, stavudine, cordycepin, azidothymidine, abacavir, structure (Formula I) Compound, structure Compounds of (Formula II), chemical derivatives thereof, pharmaceutically acceptable salts thereof, and combinations thereof.

[0229] In the foregoing embodiments, by way of example and not limitation, the one or more primary treatment cycles may be between 2 weeks and 5 weeks, 2 weeks and 4 weeks, 2 weeks and 3 weeks, 3 weeks and 4 weeks, or 4 weeks and 5 weeks, and by way of example and not limitation, any value between, for example, 2 weeks, 3 weeks, 4 weeks, or 5 weeks. The method may include two or more primary treatment cycles, wherein each of the two or more primary treatment cycles is separated by 0 to 2 weeks. By way of example and not limitation, each of the two or more primary treatment cycles is separated by 0 to 1 week or 1 to 2 weeks, and by way of example and not limitation, any value between, for example, 1 day, 1 week, or 2 weeks.

[0230] In the aforementioned embodiment, the method may further include, before the one or more primary treatment cycles: harvesting peripheral lymphocytes from the subject. The method may further include, after harvesting peripheral lymphocytes from the subject: culturing the peripheral lymphocytes to produce the manufactured T cells. REG The method may further include, after the one or more primary treatment cycles: harvesting peripheral lymphocytes from the subject. The method may further include, after the one or more primary treatment cycles: culturing the peripheral lymphocytes to produce the manufactured T cells. REG Cells. The method may further include, after each of the one or more primary treatment cycles: measuring the subject's absolute lymphocyte count (ALC), and if the ALC is < 750 per μl, proceeding to the one or more immunotherapy treatment cycles. In some embodiments, the target ALC value may vary, and by way of example and not limitation, may be 0, 250, 500, 750, 1000, 1250, or 1500 cells per microliter.

[0231] In the aforementioned embodiments, the first immunotherapy treatment cycle in the one or more immunotherapy treatment cycles is separated from the last primary treatment cycle in the one or more primary treatment cycles by 0 to 2 weeks. By way of example and not limitation, each of the two or more immunotherapy treatment cycles is separated by 0 to 1 week or 1 to 2 weeks and by way of example and not limitation, any value between 0 weeks, 1 week or 2 weeks.

[0232] In the foregoing examples, by way of example and not limitation, the dose of pentostatin may be 0.5 mg / m 2 To 4 mg / m 2 , 1mg / m 2 To 4 mg / m 2 , 2mg / m 2 To 4 mg / m 2and by way of example and not limitation, between 0.5 mg / m 2 , 1mg / m 2 , 1.5mg / m 2 , 2mg / m 2 , 2.5mg / m 2 , 3mg / m 2 , 3.5mg / m 2 and 4 mg / m 2 The pentostatin may be administered on any day of each of the one or more primary treatment cycles. By way of example and not limitation, pentostatin may be administered to a subject on day 1 or day 1 and day 4 of each of the one or more primary treatment cycles.

[0233] In the aforementioned embodiments, cyclophosphamide can be administered to a subject at a dosage of 50 mg to 400 mg. By way of example and not limitation, the dosage of cyclophosphamide can be a dosage between any combination of 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg or 400 mg and 400 mg, 350 mg, 300 mg, 250 mg, 200 mg, 150 mg, 100 mg or 50 mg and by way of example and not limitation, a dosage of any value between such as 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg or 400 mg. By way of example and not limitation, cyclophosphamide can be administered on the 1st day, the 2nd day and the 3rd day or the 1st day, the 2nd day, the 3rd day, the 4th day and the 5th day of each primary treatment cycle in the one or more primary treatment cycles.

[0234] In the aforementioned embodiments, the pentostatin and cyclophosphamide can be administered to the subject in the form of a single composition. The single composition can be administered to the subject intravenously. The step of administering pentostatin and cyclophosphamide to the subject may include: administering a first composition comprising pentostatin to the subject; and administering a second composition comprising cyclophosphamide to the subject.

[0235] In the foregoing embodiments, the lamivudine may be administered to the subject at a dosage between 150 mg per day and 150 mg twice per day.

[0236] In the aforementioned embodiment, the manufactured T REG The cells may be present in an amount of, by way of example and not limitation, between 1×10 cells per kg of the subject's body weight during each of the one or more immunotherapy treatment cycles. 6 5×10 cells per kg of subject's body weight 6cells, 2×10 per kg of subject's body weight 6 5×10 cells per kg of subject's body weight 6 cells, 3×10 per kg of subject's body weight 6 5×10 cells per kg of subject's body weight 6 cells, 4×10 per kg of subject's body weight 6 5×10 cells per kg of subject's body weight 6 cells, 1×10 per kg of subject's body weight 6 4×10 cells per kg of subject's body weight 6 cells, 1×10 per kg of subject's body weight 6 3×10 cells per kg of subject's body weight 6 cells, 1×10 per kg of subject's body weight 6 2×10 cells per kg of subject's body weight 6 The dosage range is between 1×10 cells per kg of the subject's body weight and by way of example and not limitation, between 1×10 cells per kg of the subject's body weight. 6 cells, 2×10 per kg of subject's body weight 6 cells, 3×10 per kg of subject's body weight 6 cells, 4×10 per kg of subject's body weight 6 cells or 5×10 per kg of subject’s body weight 6 Any value between 1×10 cells is administered to the subject. By way of example and not limitation, about 1×10 6 To about 200×10 6 T cells / infusion REG By way of further example and not limitation, approximately 1×10 6 To about 200×10 6 cells / infusion, 10×10 6 To about 200×10 6 cells / infusion, 50×10 6 To about 200×10 6 cells / infusion, 100×10 6 To about 200×10 6 cells / infusion, at least 1×10 6 cells / infusion, 10×10 6 cells / infusion, 50×10 6 cells / infusion, 100×10 6 cells / infusion or 200×10 6 T cells / infusion REG In some embodiments, about 40×106 In some embodiments, about 120×10 6 cells / infusion. REG The cells may include a ratio of central memory cells to effector memory cells selected from 1:1, 3:1, 10:1, 1:3 and 1:10. REG The composition of cells may further include normal T REG The T cells produced REG The cells can be administered to the subject on day 8 of each of the one or more immunotherapy treatment cycles. REG and nT REG are administered to a subject in combination.

[0237] In some embodiments, a method may include a first treatment cycle, a second treatment cycle, optionally one or more additional treatment cycles, and one or more immunotherapy treatment cycles, wherein the first treatment cycle includes: administering pentostatin to the subject and / or administering cyclophosphamide to the subject; the second treatment cycle includes: administering pentostatin to the subject and / or administering cyclophosphamide to the subject; each of the one or more additional treatment cycles includes: administering pentostatin to the subject and / or administering cyclophosphamide to the subject, and each of the one or more immunotherapy treatment cycles includes: administering pentostatin to the subject and / or administering cyclophosphamide to the subject, and manufacturing T REG The cells are administered to the subject.

[0238] In the foregoing embodiment, the first treatment cycle can be 14 days long. The step of administering pentostatin to the subject can be performed on day 1 of the first treatment cycle. During the first treatment cycle, pentostatin can be administered at 1 mg / m 2 The subject may be administered a dose of 100 mg to the subject. During the first treatment cycle, cyclophosphamide may be administered to the subject at a dose of 100 mg. The step of administering cyclophosphamide to the subject may be repeated during the first treatment cycle, and by way of example and not limitation, the step of administering cyclophosphamide to the subject may be performed on day 1, day 2, and day 3 of the first treatment cycle.

[0239] In the foregoing embodiment, the second treatment cycle can be 14 days long. The step of administering pentostatin to the subject during the second treatment cycle can be performed on day 1 of the second treatment cycle. During the second treatment cycle, pentostatin can be administered at 2 mg / m 2The step of administering cyclophosphamide to the subject during the second treatment cycle may be repeated during the second treatment cycle. By way of example and not limitation, the step of administering cyclophosphamide to the subject during the second treatment cycle may be performed on day 1, day 2, and / or day 3 of the second treatment cycle. During the second treatment cycle, cyclophosphamide may be administered to the subject at a dose of 100 mg.

[0240] In the foregoing embodiments, the subject may undergo the one or more additional treatment cycles. Each of the one or more additional treatment cycles may be 14 days long. The one or more additional treatment cycles may be spaced 0 to 2 weeks apart. By way of example and not limitation, each of the two or more additional treatment cycles may be spaced 0 to 1 week or 1 to 2 weeks apart and by way of example and not limitation, any value between 0 weeks, 1 week, or 2 weeks. The step of administering pentostatin to the subject during each of the one or more additional treatment cycles may be performed on day 1 and / or day 4 of the one or more additional treatment cycles. During each of the one or more additional treatment cycles, pentostatin may be administered at 2 mg / m 2 The step of administering cyclophosphamide to the subject may be repeated during each of the one or more additional treatment cycles. The step of administering cyclophosphamide to the subject during each of the one or more additional treatment cycles may be performed on day 1, day 2, day 3, day 4 and / or day 5 of each of the one or more additional treatment cycles. During each of the one or more additional treatment cycles, cyclophosphamide may be administered to the subject at a dose of 100 mg to 200 mg. The additional cycle may further include, before administering pentostatin to the subject during each of the one or more additional treatment cycles: measuring the subject's creatinine clearance (CrCl), and adjusting the dose of pentostatin administered to the subject based on the CrCl, wherein when CrCl>60 ml / min / 1.73 m2, 2 mg / m 2 Administer pentostatin at 1 mg / m2 when 60 ml / min / 1.73 m2 > CrCl > 30 ml / min / 1.73 m2 2Pentostatin is administered, and wherein pentostatin is not administered when CrCl < 30 mL / min / 1.73 m². The additional cycle(s) may further comprise, prior to administering cyclophosphamide to the subject during each treatment cycle of the one or more additional treatment cycles: measuring the absolute neutrophil count (ANC) and adjusting the dose of cyclophosphamide administered to the subject based on the ANC, wherein when ANC > 1000 per microliter, cyclophosphamide is administered at a dose of 100 mg, wherein when ANC is 500 - 999 per microliter, cyclophosphamide is administered at a dose of 50 mg, and wherein when ALC < 50 per microliter or ANC < 500 per microliter, cyclophosphamide is not administered. The one or more additional treatment cycles may comprise at least two additional treatment cycles, and the last treatment cycle of the at least two treatment cycles comprises, prior to administering cyclophosphamide to the subject during the last treatment cycle of the at least two treatment cycles: measuring the absolute lymphocyte count (ALC) and the absolute neutrophil count (ANC) and adjusting the dose of cyclophosphamide administered to the subject based on the ALC and ANC, wherein when ALC > 1250 per microliter, cyclophosphamide may be administered at a dose of 200 mg, wherein when ANC > 1000 per microliter and 750 per microliter < ALC < 1250 per microliter, cyclophosphamide may be administered at a dose of 100 mg, wherein when ANC is 500 - 999 per microliter, cyclophosphamide may be administered at a dose of 50 mg, and wherein when ANC < 500 per microliter and / or ALC < 750 per microliter, cyclophosphamide may not be administered.

[0241] In the foregoing embodiments, each of the treatment cycle, the second treatment cycle, and the one or more additional treatment cycles may be spaced 0 to 2 weeks apart. By way of example and not limitation, each of the treatment cycle, the second treatment cycle, and the one or more additional treatment cycles may be spaced 0 to 1 week or 1 to 2 weeks apart and, by way of example and not limitation, any value between, such as 0 weeks, 1 week, or 2 weeks.

[0242] In the foregoing embodiments, the method may further comprise, prior to administering pentostatin to the subject in the first treatment cycle: measuring the creatinine clearance rate (CrCl) of the subject and adjusting the dose of pentostatin administered to the subject based on the CrCl, wherein when CrCl > 60 mL / min / 1.73 m², at 1 mg / m 2Pentostatin was administered, wherein when 60 ml / min / 1.73 m2 > CrCl > 30 ml / min / 1.73 m2, pentostatin was administered at 0.5 mg / m2, and wherein when CrCl < 30 ml / min / 1.73 m2, pentostatin was not administered.

[0243] In the foregoing embodiment, the method may further include, prior to administering cyclophosphamide to the subject in the first treatment cycle: measuring an absolute neutrophil count (ANC), and adjusting the dose of cyclophosphamide administered to the subject based on the ALC and the ANC, wherein when the ANC is > 1000 per microliter, cyclophosphamide is administered at a dose of 100 mg, wherein when the ANC is 500-999 per microliter, cyclophosphamide is administered at a dose of 50 mg, and wherein when the ANC is < 500 per microliter, cyclophosphamide is not administered.

[0244] In the aforementioned embodiment, the method may further include, before administering pentostatin to the subject during the second treatment cycle: measuring the subject's creatinine clearance (CrCl), and adjusting the dose of pentostatin administered to the subject based on the CrCl, wherein when CrCl>60 ml / min / 1.73 m2, the dose of pentostatin administered to the subject is 2 mg / m 2 Administer pentostatin at 1 mg / m2 when 60 ml / min / 1.73 m2 > CrCl > 30 ml / min / 1.73 m2 2 Pentostatin was administered, and when CrCl < 30 ml / min / 1.73 m2, pentostatin was not administered.

[0245] In the foregoing embodiment, the method may further include, prior to administering cyclophosphamide to the subject during the second treatment cycle: measuring an absolute neutrophil count (ANC), and adjusting the dose of cyclophosphamide administered to the subject based on the ANC, wherein when the ANC is > 1000 per microliter, cyclophosphamide may be administered at a dose of 100 mg, wherein when the ANC is 500-999 per microliter, cyclophosphamide may be administered at a dose of 50 mg, and wherein when the ANC is < 500 per microliter, cyclophosphamide may not be administered.

[0246] In the foregoing embodiment, the method may further include, prior to each of the one or more additional treatment cycles: measuring the subject's absolute lymphocyte count (ALC), and adjusting the subject's treatment based on the ALC, wherein if the ALC < 750 per microliter, administering a maintenance treatment cycle to the subject, the maintenance treatment cycle comprising subjecting the patient to the one or more immunotherapy treatment cycles, and wherein if the ALC > 750 per microliter, subjecting the patient to the one or more additional treatment cycles.

[0247] In the foregoing embodiment, the method may further include, prior to each of the one or more additional treatment cycles: measuring the subject's absolute lymphocyte count (ALC), and adjusting the subject's treatment based on the ALC, wherein if the ALC is < 750 per microliter, administering a maintenance treatment cycle to the subject, the maintenance treatment cycle comprising administering a nucleoside reverse transcriptase inhibitor to the subject, and not further administering the one or more additional treatment cycles prior to the maintenance treatment cycle, and wherein if the ALC is > 750 per microliter, continuing to subject the patient to the one or more additional treatment cycles.

[0248] In the foregoing embodiments, by way of example and not limitation, each of the one or more immunotherapy treatment cycles can be 18 weeks long. In some embodiments, immunotherapy treatment cycles can be spaced 0 to 4 weeks apart. Immunotherapy treatment cycles can be repeated (including indefinitely). Repetition of immunotherapy cycles can be performed according to the protocol or in the event of relapse. By way of example and not limitation, immunotherapy cycles can occur 1-4 times per year. Pentostatin can be administered at 2 mg / m on Day 1 and Day 4 of each of the one or more immunotherapy treatment cycles. 2Cyclophosphamide may be administered to the subject at a dose of 100 mg on day 1, day 2, day 3, day 4, and / or day 5 of each of the one or more immunotherapy treatment cycles. Each of the one or more immunotherapy treatment cycles may further include: administering a nucleoside reverse transcriptase inhibitor to the subject. The nucleoside reverse transcriptase inhibitor may be lamivudine. The lamivudine may be administered to the subject at a dose of 150 mg per day to 150 mg twice per day during each of the one or more immunotherapy treatment cycles. Each of the one or more immunotherapy treatment cycles may further include: during each of the one or more immunotherapy treatment cycles: measuring the subject's creatinine clearance (CrCl), and adjusting the dose of lamivudine administered to the subject based on the CrCl, wherein when CrCl>50 ml / min / 1.73 m2, lamivudine is administered at 150 mg twice a day, wherein when 50 ml / min>CrCl>30 ml / min / 1.73 m2, lamivudine is administered at 150 mg per day, and wherein when CrCl<30 ml / min / 1.73 m2, lamivudine is not administered.

[0249] In the foregoing embodiments, a method may include administering to the subject a therapeutically effective amount of a manufactured T REG cell.

[0250] In the aforementioned embodiment, the manufactured T REG The cells may be administered to the subject on day 8 of each of the one or more immunotherapy treatment cycles. REG The cells can be administered to the subject on any day of the immunotherapy cycle, such as day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, day 14, or day 15.

[0251] In the aforementioned embodiment, the manufactured T REG Cells can be expressed at a rate of 1 × 10 cells per kg of subject body weight. 6 5×10 cells per kg of subject's body weight 6 The dosage range is, by way of example and not limitation, between 1×10 cells per kg of subject body weight. 6 5×10 cells per kg of subject's body weight 6 cells, 2×10 per kg of subject's body weight 6 5×10 cells per kg of subject's body weight6 cells, 3×10 per kg of subject's body weight 6 5×10 cells per kg of subject's body weight 6 cells, 4×10 per kg of subject's body weight 6 5×10 cells per kg of subject's body weight 6 cells, 1×10 per kg of subject's body weight 6 4×10 cells per kg of subject's body weight 6 cells, 1×10 per kg of subject's body weight 6 3×10 cells per kg of subject's body weight 6 cells, 1×10 per kg of subject's body weight 6 2×10 cells per kg of subject's body weight 6 The dosage range is between 1×10 cells per kg of the subject's body weight and by way of example and not limitation, between 1×10 cells per kg of the subject's body weight. 6 cells, 2×10 per kg of subject's body weight 6 cells, 3×10 per kg of subject's body weight 6 cells, 4×10 per kg of subject's body weight 6 cells or 5×10 per kg of subject’s body weight 6 Any value between 100 cells was administered.

[0252] Each of the one or more treatment cycles may further include: administering pentostatin to the subject; and / or administering cyclophosphamide to the subject. Pentostatin may be administered at a dose between 1 mg / m 2 With 2mg / m 2 The dosage range is as follows and by way of example and not limitation, between 1 mg / m 2 , 1.5mg / m 2 or 2 mg / m 2The dosage of any value between 100mg and 200mg is applied to the subject. The dosage of cyclophosphamide can be between 100mg and 200mg and, by way of example and not limitation, between any value such as 100mg, 150mg or 200mg. The pentostatin can be applied to the subject on the 1st and 4th day of each treatment cycle in the one or more treatment cycles. The cyclophosphamide can be applied to the subject on the 1st, 2nd, 3rd, 4th and / or 5th day of each treatment cycle in the one or more treatment cycles. The subject can be treated with pentostatin and cyclophosphamide in advance. Each treatment cycle in the one or more treatment cycles can further include administering a nucleoside reverse transcriptase inhibitor to the subject. The nucleoside reverse transcriptase inhibitor can be an inhibitor of the NLRP3 inflammasome. The nucleoside reverse transcriptase inhibitor can be lamivudine. Each treatment cycle in the one or more treatment cycles can be spaced 4 weeks apart.

[0253] In the aforementioned embodiment, the method may further include converting the normal T REG The cells were prepared as described REG In the aforementioned embodiment, the method may further include, before the one or more treatment cycles: harvesting peripheral lymphocytes from the subject. In the aforementioned embodiment, the method may further include, after harvesting peripheral lymphocytes from the subject: culturing the peripheral lymphocytes to produce the manufactured T cells. REG cell.

[0254] Examples

[0255] The following examples are provided to better illustrate the methods of the present disclosure and the resulting dedifferentiated T cells and iT cells. REG Or redifferentiated T cells. These examples are not intended to limit or otherwise alter the scope of the methods, cells, and compositions disclosed in this disclosure.

[0256] Example 1: Combination of vitamin D and temsirolimus reduces T cell effector molecules

[0257] The individual effects of vitamin D, mTOR inhibition (using parenteral forms of rapamycin, temsirolimus), and the combination of vitamin D plus temsirolimus on human T cell effector molecule expression were directly assessed (see Figure 1).

[0258] Figures 1A-1D Demonstrated that the combination of vitamin D and temsirolimus reduces human CD4 + and CD8 +Expression of effector molecules in T cells. For columns #2 to #5, T cells were subjected to a 3-day dedifferentiation interval containing low levels of anti-CD3 / anti-CD28 co-stimulation (bead to T cell ratio; 1:3); high doses of temsirolimus (1 μM); vitamin D (0.1 or 1.0 nM); and culture in X-Vivo 20 culture medium. The first column represents a control culture (no temsirolimus, no vitamin D, using a 3:1 bead to T cell ratio; and containing type I polarization cytokine IFN-α (20,000 IU / mL, unless otherwise stated, this amount is used for control cultures in Examples 1-11 below)). The second column represents a culture with a low bead to T cell ratio and temsirolimus but without vitamin D; in contrast, the third column represents a culture with vitamin D (0.1 nM) but without temsirolimus. The fourth column represents cultures with high dose ("HD") vitamin D (1.0 nM) but without temsirolimus. The fifth column represents cultures with both high dose vitamin D (1.0 nM) and temsirolimus. At the end of the dedifferentiation interval, cells were harvested, RNA isolated, and RNA expression analysis was performed by the Luminex Quantigene method. All results shown represent relative RNA expression, with results for Th1 / Tc1 control cultures normalized to a value of 1.0.

[0259] A 3-day culture interval was used that contained low levels of T cell co-stimulation (a 1:3 ratio of anti-CD3 / anti-CD28 beads to T cells; the typical ratio used in the literature is the inverse 3:1), temsirolimus (1 μM), 0.1 or 1.0 nM doses of vitamin D, or a combination of temsirolimus and higher doses of vitamin D. Following culture, RNA was harvested and expression levels of effector molecules were compared to control cultures.

[0260] like Figure 1A As shown, the various cultures had similar RNA expression of housekeeping control genes, including GAPDH. In sharp contrast, the addition of temsirolimus, vitamin D, or a combination of temsirolimus and vitamin D to the cultures resulted in reduced RNA expression of T cell effector molecules, including the cytotoxic molecule granzyme B ( Figure 1B ) and the cytokine molecule IL-10 (Th2 cytokine; Figure 1C ) and IFN-γ (Th1 cytokine; Figure 1D). Thus, granzyme B and IFN-γ, markers of dedifferentiation, indicate that vitamin D is effective at concentrations between 0.1 and 1.0 nM. Temsirolimus at a dose of 1 μM was beneficial as a dedifferentiation agent alone (column 2, reduction of granzyme B and IFN-γ), and did not abolish the effect of vitamin D when used in combination (column 5).

[0261] Thus, using low levels of co-stimulation (1:3 ratio of anti-CD3 / anti-CD28 beads to T cells) and a shorter 3-day culture interval, the addition of temsirolimus, vitamin D, or a combination of temsirolimus and vitamin D can be used to reduce Th1 and Th2 cytokine effectors as well as cytotoxic effector mechanisms.

[0262] Example 2: Combination of vitamin D and temsirolimus alters key transcription factors associated with dedifferentiation

[0263] The effects of vitamin D, temsirolimus, or the combination on the expression of key transcription factors after low levels of co-stimulation were also evaluated.

[0264] Figures 2A-2D Demonstrated that the combination of vitamin D and temsirolimus increases human CD4 + and CD8 + The combination of vitamin D and temsirolimus reduces the expression of stem cell-associated transcription factors and the naive T cell molecule IL-7 receptor-α in human CD4 + and CD8 + Expression of effector molecules in T cells. For columns #2 to #5, T cells were subjected to a 3-day dedifferentiation interval containing low levels of anti-CD3 / anti-CD28 co-stimulation (bead to T cell ratio; 1:3); high doses of temsirolimus (1 μM); vitamin D (0.1 or 1.0 nM); and culture in X-Vivo 20 culture medium. The first column represents a control culture (no temsirolimus, no vitamin D, using a 3:1 bead to T cell ratio; and containing type I polarization cytokines IFN-α). The second column represents a culture with a low bead to T cell ratio and temsirolimus but without vitamin D; in contrast, the third column represents a culture with vitamin D (0.1 nM) but without temsirolimus. The fourth column represents a culture with a high dose ("HD") of vitamin D (1.0 nM) but without temsirolimus. The fifth column represents cultures with both high doses of vitamin D (1.0 nM) and temsirolimus. At the end of the dedifferentiation interval, cells were harvested, RNA isolated, and RNA expression analysis was performed by the Luminex Quantigene method. All results shown represent relative RNA expression, with results for Th1 / Tc1 control cultures normalized to a value of 1.0.

[0265] like Figure 2A As shown, temsirolimus or a combination of temsirolimus and vitamin D upregulated the Nanog transcription factor, which is considered one of the few key factors required for the dedifferentiation of somatic cells to the 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.

[0266] Thus, using low levels of co-stimulation, temsirolimus increased the iPSC transcription factor Nanog; this boosting effect of temsirolimus was not abolished by vitamin D concentrations ranging from 0.1 to 1.0 nM.

[0267] By comparison, neither temsirolimus nor vitamin D alone increased 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 and vitamin D (1.0 nM) increased KLF4 RNA expression. Thus, it is preferred to include both temsirolimus and vitamin D in T cell dedifferentiation attempts. Figure 2B As shown, while neither temsirolimus nor vitamin D alone was used to beneficially upregulate the dedifferentiation molecule KLF4, the combination of temsirolimus (1 μM) and vitamin D (1.0 nM) synergistically upregulated KLF4.

[0268] When temsirolimus was used in combination with vitamin D (1.0 nM), the related transcription factor KLF10 was also upregulated. Figure 2C As shown, a 1 μM dose of temsirolimus alone beneficially upregulated the dedifferentiation molecules KLF10, Nanog, and IL-7 receptor α; although vitamin D alone was not effective in upregulating these molecules, when used in combination, vitamin D did not abrogate the effects of temsirolimus (bar 5).

[0269] Finally, the cultured cells were evaluated for RNA expression of IL-7 receptor alpha, which is upregulated in T cells with a reduced differentiation state. Importantly, temsirolimus alone, but not vitamin D alone, was able to upregulate IL-7 receptor alpha. Nevertheless, the combination of vitamin D (1.0 nM) and temsirolimus caused an upregulation of IL-7 receptor alpha, as shown in Figure 2. Figure 2D shown.

[0270] Taken together, these data suggest that low-level co-stimulation combined with temsirolimus can be used to enhance T cell dedifferentiation; preferably, for a more complete pattern of dedifferentiation, cultures should contain temsirolimus with vitamin D.

[0271] Example 3: Combination of vitamin D and temsirolimus reduces key transcription factors associated with Th1 differentiation while maintaining HIF-1-α expression

[0272] The effect of vitamin D, temsirolimus or the combination on the expression of key transcription factors associated with Th1 type differentiation, namely T-BET and STAT1, was also evaluated.

[0273] Figures 3A-3C showed that the combination of vitamin D and temsirolimus reduced the expression of transcription factors associated with effector Th1 / Tc1 cells, but did not reduce the expression of HIF-1-α, a transcription factor associated with T cell survival. + and CD8 + Expression of effector molecules in T cells. For columns #2 to #5, T cells were subjected to a 3-day dedifferentiation interval containing low levels of anti-CD3 / anti-CD28 co-stimulation (bead to T cell ratio; 1:3); high doses of temsirolimus (1 μM); vitamin D (0.1 or 1.0 nM); and culture in X-Vivo 20 culture medium. The first column represents a control culture (no temsirolimus, no vitamin D, using a 3:1 bead to T cell ratio; and containing type I polarization cytokines IFN-α). The second column represents a culture with a low bead to T cell ratio and temsirolimus but without vitamin D; in contrast, the third column represents a culture with vitamin D (0.1 nM) but without temsirolimus. The fourth column represents a culture with a high dose ("HD") of vitamin D (1.0 nM) but without temsirolimus. The fifth column represents cultures with both high doses of vitamin D (1.0 nM) and temsirolimus. At the end of the dedifferentiation interval, cells were harvested, RNA isolated, and RNA expression analysis was performed by the Luminex Quantigene method. All results shown represent relative RNA expression, with results for Th1 / Tc1 control cultures normalized to a value of 1.0.

[0274] Importantly, each agent or combination of agents downregulated T-BET RNA ( Figure 3A ) and STAT1 RNA ( Figure 3B ) Both. Figures 3A-3C As shown, vitamin D alone at doses of 0.1 to 1.0 nM was used to beneficially downregulate the differentiation molecules T-BET and STAT1. Temsirolimus at a dose of 1 μM did not deleteriously downregulate the pro-survival transcription factor HIF-1α, even when combined with 1.0 nM vitamin D. However, temsirolimus alone at a dose of 1 μM was used to beneficially downregulate the differentiation molecules T-BET and STAT1; similar results were produced in combination with vitamin D (the two agents were not antagonistic).

[0275] In stark contrast, temsirolimus at a dose of 1 μM, vitamin D at doses of 0.1 to 1.0 nM, or the combination did not downregulate the key transcription factor HIF-1-α ( Figure 3C ), the key transcription factor is very important as a T cell survival factor that is crucial for anti-tumor effects.

[0276] Altogether, these data suggest that a combination of low-level co-stimulation, temsirolimus, and vitamin D can be used to reduce transcription factors required for Th1 generation without inhibiting HIF-1-α, a key transcription factor required for overall T cell survival.

[0277] Example 4: Combination of vitamin D, temsirolimus and anti-IL-2 receptor monoclonal antibody increases autophagy markers

[0278] The effects of vitamin D, temsirolimus or the combination on the autophagic process, which is critical for promoting a stem cell-like dedifferentiated state, were also evaluated.The level of autophagy can be determined in part by the subsequent upregulation of the autophagic substrate p62 by means of Western blot analysis.

[0279] Figure 4 showed that the combination of vitamin D, temsirolimus, and anti-IL-2 receptor blockade induced the expression of the autophagy-related molecule p62. + and CD8 + T cells were subjected to a dedifferentiation protocol involving the use of low-level co-stimulation (1:3 beads to T cells ratio), temsirolimus (“TEM”, e.g. Figure 4 3 days of culture with 50 μg / ml of dapoxetine ("DAC", as indicated). After a 3-day culture interval, T cells were harvested and proteins were isolated and subjected to Western blot analysis for autophagy-related gene p62 and housekeeping gene actin.

[0280] like Figure 4 As demonstrated, inclusion of vitamin D in T cell cultures is essential for increasing autophagy, as measured by upregulated p62. Vitamin D at doses of 0.01 to 0.1 nM synergizes with temsirolimus at concentrations of 0.3 to 1.0 μM to beneficially upregulate the autophagy marker p62 during dedifferentiation. Figure 4In culture #6 (fifth column), there was very little expression of p62 in Western blot analysis, consistent with low levels of autophagy; as indicated in the legend, this culture condition received low levels of co-stimulation, temsirolimus, the anti-IL-2 receptor monoclonal antibody daclizumab, but no vitamin D.

[0281] In stark contrast, the other culture conditions each received vitamin D supplementation and each had increased p62 expression (the effective dose of vitamin D ranged from 0.01 nM to 1.0 nM). Figure 4 We also demonstrated that vitamin D without the addition of anti-IL-2 receptor monoclonal antibody and vitamin D without the addition of temsirolimus were sufficient to induce T cell autophagy.

[0282] Taken together, these data suggest that inclusion of vitamin D at low levels of co-stimulation is an effective approach for inducing T cell autophagy alone or in combination with other T cell inhibitors (ie, anti-IL-2 receptor agents or the mTOR inhibitor temsirolimus).

[0283] Example 5: Combination of vitamin D, temsirolimus and anti-IL-2 receptor monoclonal antibody induces optimal disruption of the mTORC1 complex

[0284] The effects of various T cell culture conditions on the expression of Raptor, a key component of the mTORC1 signaling complex, were also evaluated. Importantly, inhibition of mTORC1 was recently found to be essential for reprogramming somatic cells to the iPSC state.

[0285] Figure 5 demonstrated that a combination of vitamin D, temsirolimus, and anti-IL-2 receptor blockade reduced the expression of the mTORC1-associated molecule Raptor. + and CD8 + T cells were subjected to a dedifferentiation protocol involving the use of low-level co-stimulation (1:3 beads to T cells ratio), temsirolimus (“TEM”, e.g. Figure 5 3 days of culture with 50 μg / ml of 5 μM dT cells ("D", as indicated; concentrations of 1.0 or 0.3 μM), vitamin D ("D", as indicated; concentrations of 0.01, 0.03, 0.1, 0.3, or 1.0 nM), and anti-IL-2 receptor monoclonal antibody (daclizumab, 50 μg / ml; "DAC", as indicated). After a 3-day culture interval, T cells were harvested and proteins were isolated and subjected to Western blot analysis for the mTORC1 complex protein Raptor and the housekeeping gene actin.

[0286] like Figure 5As demonstrated, optimal inhibition of the mTORC1 complex, as indicated by reduced Raptor expression, occurred when T cells were co-stimulated at a low beads to T cell ratio (1:3) in combination with temsirolimus (1.0 μM), vitamin D (0.1 nM), and the anti-IL-2 receptor monoclonal antibody daclizumab (50 μg / nl) (first column shown; culture 1).

[0287] like Figure 5 As shown, omission of daclizumab resulted in a modest increase in Raptor expression, indicating a role for anti-IL-2 receptor agents for optimal mTORC1 inhibition. Thus, anti-IL-2 receptor monoclonal antibody daclizumab (at a dose of 50 μg / ml) plays a beneficial role in inhibiting the mTORC1 subunit molecule Raptor (second column).

[0288] like Figure 5 As shown, the optimal inhibition of the mTORC1 subunit molecule Raptor by vitamin D is between 0.03 and 0.1 nM of vitamin D; concentrations below or above this range result in reduced optimal inhibition of Raptor. Therefore, vitamin D levels as low as 0.03 nM are sufficient to optimally inhibit Raptor; however, reducing vitamin D levels to 0.01 nM results in suboptimal Raptor inhibition. In addition, as indicated by culture 7 (vitamin D concentration of 0.3 nM) with higher Raptor expression levels, increasing vitamin D levels to concentrations exceeding 0.1 nM may be harmful.

[0289] In addition, if Figure 5 As indicated, optimal downregulation of Raptor requires a combination of vitamin D and temsirolimus (with the optimal dose of temsirolimus being 1.0 μM), as culture 9 supplemented with temsirolimus at a concentration of 0.3 μM had a higher level of Raptor expression.

[0290] Example 6: Combination of Vitamin D, Temsirolimus, and Anti-IL-2 Receptor Monoclonal Antibody Disrupts Both the mTORC1 and mTORC2 Complexes

[0291] We also evaluated the mTORC2 complex, which is not directly sensitive to the inhibitory effects of rapamycin but is affected by conditions that lead to prolonged mTORC1 blockade. Importantly, inhibition of mTORC2 promoted a stem cell-like state.

[0292] Figure 6 showed that the combination of vitamin D, temsirolimus, and anti-IL-2 receptor blockade reduced the expression of the mTORC1-related molecule Raptor and the mTORC2-related molecule Rictor. + and CD8+ T cells were subjected to a dedifferentiation protocol involving the use of low-level co-stimulation (1:3 beads to T cells ratio), temsirolimus (“TEM”, e.g. Figure 6 ; concentration of 1.0 μM), vitamin D ("D", as indicated; concentration of 0.03, 0.1, 0.3 or 1.0 nM) and anti-IL-2 receptor monoclonal antibody (daclizumab, 50 μg / ml; "DAC", as indicated) for 3 days of culture. After the 3-day culture interval, T cells were harvested and proteins were isolated and subjected to Western blot analysis for mTORC1 complex protein Raptor; mTORC2 complex protein Rictor; mTORC1 post protein p70S6K; mTORC2 post protein SGK1; and housekeeping gene GAPDH.

[0293] like Figure 6 As shown, the mTORC1 molecule Raptor and the mTORC2 molecule Rictor of T cell cultures in medium containing temsirolimus, vitamin D and the anti-IL-2 receptor antibody daclizumab were both reduced relative to control cultures without any of the three inhibitors. The levels of the mTORC1 post-molecule p70S6K and the mTORC2 post-molecule SGK1 were relatively preserved. Therefore, vitamin D (concentrations between 0.03 and 1.0 nM) is effective for downregulating the mTORC2 subunit Rictor during the dedifferentiation period of the combined agent. Moreover, temsirolimus at a concentration of 1 μM is effective for downregulating the mTORC2 subunit Rictor during the dedifferentiation period of the combined agent. In addition, the anti-IL-2 receptor monoclonal antibody daclizumab (dose, 50 μg / ml) did not eliminate the ability of temsirolimus and vitamin D to downregulate the mTORC2 subunit Rictor.

[0294] Thus, T cell culture using low levels of co-stimulation and a three-part suppressive regimen of temsirolimus, vitamin D, and anti-IL-2 receptor monoclonal antibody represents a novel approach for depletion of both Raptor and Rictor subunits.

[0295] Example 7: The combination of vitamin D, temsirolimus and anti-IL-2 receptor monoclonal antibody reduces the expression of the pro-apoptotic Bcl2-family member gene BIM

[0296] Due to autophagy at the mitochondrial level (mitophagy), the quality of mitochondrial proteins can be changed; specifically, for mitophagy, the conversion of bcl2-family member genes from pro-apoptotic family members (such as BIM) to anti-apoptotic family members can be advantageous. In addition, the culture method that reduces the apoptotic tendency is associated with the increase of dedifferentiation ability.

[0297] Figure 7 demonstrated that a combination of vitamin D, temsirolimus, and anti-IL-2 receptor blockade reduced the expression of the pro-apoptotic molecule BIM. + and CD8 + T cells were subjected to a dedifferentiation protocol involving the use of low-level co-stimulation (1:3 beads to T cells ratio), temsirolimus (“TEM”, e.g. Figure 7 3 days of culture with 50 μg / ml of daptomycin ("DAC", as indicated). After a 3-day culture interval, T cells were harvested and proteins were isolated and subjected to Western blot analysis for pro-apoptosis-related gene BIM and housekeeping gene actin.

[0298] To assess this, we measured BIM levels in T cells cultured at low co-stimulatory intensity (1:3 beads to T cells ratio) and in the presence of various inhibitors. Figure 7 As shown, T cell cultures containing a combination of temsirolimus, vitamin D (0.1 nM), and the anti-IL-2 receptor monoclonal antibody daclizumab had the lowest levels of BIM expression. Figure 7 Still, it was shown that the anti-IL-2 receptor monoclonal antibody daclizumab (at a dose of 50 μg / ml) plays a beneficial role in inhibiting the pro-apoptotic molecule BIM (bar 2). Each of the three inhibitors appears to play a role in BIM inhibition, as none of the single inhibitors increased BIM levels.

[0299] Thus, it can be concluded that the combined inhibitor regimen represents an approach for inducing a favorable shift in the tendency of mitochondria to control apoptosis.

[0300] Example 8: Three inhibitor dedifferentiation protocols generate T cells with subsequent proliferation capacity after removal of inhibitors

[0301] To demonstrate that a 3-day regimen including low levels of co-stimulation, temsirolimus, vitamin D, and anti-IL-2 receptor monoclonal antibody produced a dedifferentiated state capable of redifferentiation, an experiment was performed to re-stimulate cells using high levels of co-stimulation (bead to T cell ratio of 3:1) on day 3 of culture after removal of inhibitors from the culture. After 10 days (13 days in culture total), T cells were harvested, counted, and evaluated by flow cytometry.

[0302] Figure 8 The effects of culture composition during the dedifferentiation interval on subsequent T cell yield (at day 13 of culture) are shown.+ and CD8 + T cells were subjected to a 3-day dedifferentiation interval containing low levels of anti-CD3 / anti-CD28 co-stimulation (1:3 or 1:1 beads:T cell ratios as indicated); temsirolimus (1 μM; or low dose ["Lo"] of 0.1 μM); vitamin D (0.1 nM; or high dose ["HD"] of 1.0 nM; or low dose of 0.01 nM); anti-IL-2 receptor monoclonal antibody (50 μg / ml of daclizumab); and culture in X-Vivo 20 medium supplemented with 5% human AB serum. The first column represents control cultures (no temsirolimus, vitamin D, or anti-IL-2R antibody). The second column represents cultures without anti-IL-2R antibodies; the fourth column represents cultures without serum supplementation; the fifth column represents low doses of vitamin D, while the sixth column represents results using high doses of vitamin D; the seventh column represents results using low doses of temsirolimus; and the eighth column represents cultures without temsirolimus; the ninth column represents results using a higher ratio of beads. After a 3-day interval, the culture medium was replaced with fresh X-Vivo20 without inhibitors, providing high levels of co-stimulation (3:1 beads: T cell ratio), and T cell growth factors IL-2 (100 IU / ml) and IL-7 (10 ng / ml) were added. On day 13 of culture, the number of viable T cells was calculated, and the total yield relative to the number of inputs on day 0 is shown.

[0303] Figure 8 T cell counts after the redifferentiation phase are shown. As shown in these data (column #3), for the first 3-day dedifferentiation interval, T cells initially maintained using low-level co-stimulation, temsirolimus, vitamin D, and anti-IL-2 receptor monoclonal antibody had satisfactory T cell yields (greater than 250% of culture input).

[0304] In stark contrast, very low yields were observed in the culture represented by column #4, which did not receive serum supplementation during the initial 3-day culture interval; thus, this data demonstrates that the initial 3-day culture interval must contain X-Vivo 20 medium supplemented with 5% AB serum.

[0305] Furthermore, decreasing the vitamin D concentration to 0.01 nM or increasing the vitamin D concentration to 1.0 nM resulted in very low yields (as shown by the data of columns #5 and #6, respectively). Thus, the preferred concentration of vitamin D is 0.1 nM.

[0306] Furthermore, decreasing the concentration of temsirolimus to 0.1 μM reduced the yield of T cells obtained (bar #7). Thus, the preferred concentration of temsirolimus is 1.0 μM.

[0307] Finally, if the level of co-stimulation is increased during the dedifferentiation interval (bead to T cell ratio changed from 1:3 to 1:1; results shown in the last column (column #9)), the resulting T cell counts will be very low. Figure 8 As shown, low levels of co-stimulation (1:3 co-stimulatory beads to T cells ratio) must be used during dedifferentiation, as increasing the ratio to 1:1 results in a greatly reduced ability to make T cells from the dedifferentiated state (last column). Thus, the preferred bead to T cell ratio during the dedifferentiation phase of culture is 1:3.

[0308] Example 9: Initial three-component culture intervals result in decreased expression of cell surface molecules CD4 + T cell generation

[0309] At various times during the redifferentiation phase of the culture, the resulting CD4 + Expression of memory markers in T cells.

[0310] Figures 9A-9C Demonstrated that CD4 memory markers were expressed in culture components during the dedifferentiation interval + Effects of T cell expression (on day 13 of culture). + and CD8 + T cells were subjected to a 3-day dedifferentiation interval, the dedifferentiation interval comprising (as above Figures 9A-9C Indicated) low level of anti-CD3 / anti-CD28 costimulation (1:3 beads to T cells ratio); temsirolimus (1 μM or 0.1 μM [low dose; "Lo"]); vitamin D (0.1 nM; or 0.01 nM [low dose; "Lo"]); anti-IL-2 receptor monoclonal antibody (50 μg / ml of daclizumab); and culture in X-Vivo 20 medium supplemented with 5% human AB serum. After a 3-day interval, the medium was changed to fresh X-Vivo 20 without inhibitors, providing high level costimulation (3:1 beads: T cells ratio), and the T cell growth factors IL-2 (100 IU / ml) and IL-7 (10 ng / ml) were added. T cells were subjected to flow cytometric analysis to assess CD4 + and CD45RA + Co-expression of markers (results shown in the upper panel; assessed on day 13 of culture); CD4 + 、CD62L + and CCR7 + Co-expression of markers (lower left panel; assessed on day 3 of culture); and CD4 + 、CD62L + 、CCR7+ and CD127 + Co-expression of markers (lower right panel; assessed on day 10 of culture). + All results of T cell values ​​( Figures 9A-9C ; “Day 0 Input Value”).

[0311] like Fig. 9A As shown, T cells initially propagated in the presence of a combination of temsirolimus, vitamin D, and anti-IL-2 receptor monoclonal antibody had relatively preserved expression of the CD45RA marker expressed on naive T cells (bar #3), relative to values ​​from day 0 infusion T cells. In stark contrast, the 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).

[0312] like Fig. 9B As shown, each of the T cell cultures that were initially propagated over a 3 day interval incorporating low levels of co-stimulation had increased T cell expression of the central memory molecules CD62L and CCR7.

[0313] Finally, if Fig. 9C As shown, T cells initially propagated under a combination of temsirolimus, vitamin D, and anti-IL-2 receptor monoclonal antibodies had greatly increased expression of T cells that were triple positive for CD62L, CCR7, and IL-7 receptor alpha (CD127) (relative to day 0 input cells). Removal of the three inhibitors during the initial 3-day culture period (column #1) eliminated the ability of the initial culture interval to promote expansion of this triple-positive cell population. In addition, reduction or removal of temsirolimus alone during the initial culture interval also greatly reduced the frequency of triple-positive T cells (columns #5 and 6).

[0314] In summary, these data suggest that the initial culture interval of the three drugs increases CD4 + T cells transform from a predominantly late effector memory population to a less differentiated T cell population, which is a very limited T cell differentiation state, involving co-expression of CD62L, CCR7, and CD127.

[0315] Example 10: Initial three-component culture intervals result in decreased expression of cell surface molecules CD8 consistent with differentiation + T cell generation

[0316] At various times during the redifferentiation phase of culture, the resulting CD8 + Expression of memory markers in T cells.

[0317] Figures 10A-10B Demonstrated that the culture components expressed memory markers CD8 during the dedifferentiation interval + Effects on T cell expression. + and CD8 + T cells were subjected to a 3-day dedifferentiation interval, the dedifferentiation interval comprising (as above Figures 10A-10B Indicated) low level of anti-CD3 / anti-CD28 costimulation (bead to T cell ratio; 1:3); temsirolimus (1 μM or 0.1 μM [low dose; "Lo"]); vitamin D (0.1 nM; or 0.01 nM [low dose; "Lo"]); anti-IL-2 receptor monoclonal antibody (50 μg / ml of daclizumab); and culture in X-Vivo 20 medium supplemented with 5% human AB serum. After a 3-day interval, the medium was changed to fresh X-Vivo 20 without inhibitors, providing high level costimulation (3:1 bead:T cell ratio), and the T cell growth factors IL-2 (100 IU / ml) and IL-7 (10 ng / ml) were added. T cells were subjected to flow cytometric analysis to assess CD8 + 、CD62L + and CCR7 + Co-expression of markers (left panel; assessed on day 10 of culture); and CD8 + 、CD62L + 、CCR7 + and CD127 + Co-expression of CD8 + All results of T cell values ​​( Figures 10A-10B ; “Day 0 Input Value”).

[0318] like Fig. 10A As shown, each of the T cell cultures that were initially propagated over a 3-day interval incorporating low-level co-stimulation had increased CD8 + T cell expression.

[0319] Finally, if Fig. 10B As shown, T cells initially propagated under the combination of temsirolimus, vitamin D, and anti-IL-2 receptor monoclonal antibody had CD8 +The expression of T cells was greatly increased (relative to the input cells on day 0). Removal of the three inhibitors during the initial 3-day culture period (column #1) eliminated the ability of the initial culture interval to promote the expansion of this triple-positive cell population. In addition, reducing or eliminating only temsirolimus during the initial culture interval also greatly reduced the frequency of triple-positive T cells (columns #5 and 6).

[0320] In summary, these data suggest that the initial culture interval of the three drugs increases CD8 + T cells transform from a predominantly late effector memory population to a less differentiated T cell population, which is a very limited T cell differentiation state, involving co-expression of CD62L, CCR7, and CD127.

[0321] Example 11: Dedifferentiated T cells have an inherent bias towards low cytokine potential

[0322] Figures 11A-11D Components of the dedifferentiation process are emphasized, including the use of: low-level co-stimulation (a 1:3 anti-CD3 / anti-CD28 bead to T cell ratio, which is reduced relative to the conventional method described in Kalamaz 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.

[0323] Figures 11A-11D Figure 3 shows the profiling of inflammatory Th1 / Th17 cytokines in dedifferentiated T cells cultured in polarizing neutral medium. + and CD8 +T cells were subjected to a 3-day dedifferentiation process comprising the culture components as indicated below: temsirolimus (Y indicates a concentration of 1 μM; Y, Lo indicates a concentration of 0.1 μM); vitamin D (Y indicates a concentration of 0.1 nM; Y, Lo indicates a concentration of 0.01 nM); anti-IL-2 receptor monoclonal antibody (50 μg / ml of daclizumab); co-stimulation with anti-CD3 / anti-CD28 coated magnetic beads at a low ratio (1:3 beads to T cells ratio) and supplemented with 5% human serum. After 3 days, dedifferentiated T cells were co-stimulated (typical beads to T cells ratio of 3:1) in culture medium supplemented with T cell growth factors rhu IL-2 (100 IU / ml) and rhu IL-7 (10 ng / ml), which are not potent in inducing T cell polarization. After 10 days of culture (total, day 13 of culture), T cells were harvested and washed, and restimulated with 3 / 28 beads (ratio 3:1) for 24 hours; the resulting supernatant was harvested and cytokine content was detected by Luminex multi-analyte method. All results shown are expressed as cytokine levels in pg / ml / 1×10 6 cells / ml / 24 hours.

[0324] To assess whether the dedifferentiated T cell state exhibits an intrinsic bias toward specific cytokine secretion patterns, dedifferentiated T cells were cultured using high levels of co-stimulation (bead to T cell ratio of 3:1) and maintenance in medium without any inhibitors but containing only the T cell growth factors IL-2 and IL-7.

[0325] like Figures 11A-11D As shown in the details of Figure 1, the resulting T cells that redifferentiated from each dedifferentiated precursor state secreted very low levels of inflammatory cytokines, including IFN-γ (most values ​​were below 1000 pg / ml), TNF-α (most values ​​were below 100 pg / ml), and IL-17 (all values ​​were below 10 pg / ml). Notably, GM-CSF was secreted at higher levels under some conditions, in some cases greater than 10,000 pg / ml. GM-CSF values ​​were moderate under dedifferentiation conditions, which included higher doses of temsirolimus (1.0 μM) and higher doses of vitamin D (0.1 nM); thus, in order to obtain moderate T cell factor secretion of GM-CSF, it is desirable to expand T cells in a dedifferentiation method that incorporates these higher concentrations of temsirolimus and vitamin D.

[0326] It is noteworthy that the inclusion of low concentrations of vitamin D (0.01 nM) during the dedifferentiation interval also produced slightly higher levels of IFN-γ and TNF-α than the use of higher concentrations of vitamin D (0.1 nM). Thus, the use of vitamin D concentrations close to 0.1 nM is preferred in terms of moderate T cell secretion of the inflammatory cytokine IFN-γ.

[0327] In addition, if Figures 12A-12D As shown, the resulting T cells redifferentiated from each of the dedifferentiated precursor state T cells secreted very low levels of IL-2, although again, the levels were lower under conditions incorporating higher concentrations of temsirolimus and vitamin D relative to the use of lower concentrations of these agents.

[0328] Figures 12A-12D Depicted is the analysis of IL-2 and Th2-type cytokines in dedifferentiated T cells cultured in polarizing neutral medium. + and CD8 + T cells were subjected to a 3-day dedifferentiation process comprising the culture components as indicated below: temsirolimus (Y indicates a concentration of 1 μM; Y, Lo indicates a concentration of 0.1 μM); vitamin D (Y indicates a concentration of 0.1 nM; Y, Lo indicates a concentration of 0.01 nM); anti-IL-2 receptor monoclonal antibody (50 μg / ml of daclizumab); co-stimulation with anti-CD3 / anti-CD28 coated magnetic beads at a low ratio (1:3 beads to T cells ratio) and supplemented with 5% human serum. After 3 days, dedifferentiated T cells were co-stimulated (typical beads to T cells ratio of 3:1) in culture medium supplemented with T cell growth factors rhu IL-2 (100 IU / ml) and rhu IL-7 (10 ng / ml), which are not potent in inducing T cell polarization. After 10 days of culture (total, day 13 of culture), T cells were harvested and washed, and restimulated with 3 / 28 beads (ratio 3:1) for 24 hours; the resulting supernatant was harvested and cytokine content was detected by Luminex multi-analyte method. All results shown are expressed as cytokine levels in pg / ml / 1×10 6 cells / ml / 24 hours.

[0329] The resulting T cells also secreted very low levels of the Th2-type cytokines IL-4 (values ​​less than 20 pg / ml) and IL-5 (values ​​less than 60 pg / ml). However, in some T cell culture conditions, IL-13 levels were increased, with less cytokine secretion detected under conditions incorporating higher concentrations of temsirolimus and vitamin D relative to conditions using lower concentrations of these agents.

[0330] Taken together, these data suggest that after the first step of the dedifferentiation process, T cell redifferentiation in medium containing T cell growth factors (IL-2 and IL-7) and without strong polarizing signals (no added IFN-α, IL-4, or TGF-β) has an inherent bias toward T cells of low cytokine potential; specifically, low levels of the deleterious cytokines IFN-γ, TNF-α, and IL-17 were demonstrated. This observation was particularly strong if the dedifferentiation step incorporated low-level co-stimulation and propagation in medium containing temsirolimus at a concentration of 1.0 μM, vitamin D at a concentration of 0.1 nM, and anti-IL-2 receptor monoclonal antibodies.

[0331] Example 12: Dedifferentiated T cells in hybrid T REG / Favorable expansion under Th2 polarization conditions and in the presence of the novel agent pemetrexed

[0332] The dedifferentiation components were evaluated in T cells and incorporated into T REG The role of polarizing cytokines IL-2 and TGF-β or Th1 polarizing cytokine IFN-α.

[0333] In fact, TBET or GATA3 has been shown to maintain T REG However, despite evidence that TBET or GATA3 play a role in T REG However, due to the close connection between TBET and the subsequent Th1-type pathway in autoimmunity, we chose to give priority to the production of T cells. REG Thus, the first step of dedifferentiation and subsequent T cell differentiation were evaluated. REG Is it possible that the second step of the redifferentiation process, which is induced by polarization signals (IL-2, TGF-β) and the major Th2 polarization signal (IL-4), could generate human “hybrid” T REG -Th2 cells. In this regard, the literature from experimental mouse models has mixed results, as REG Purposeful addition of IL-4 to cells in vitro has been shown to promote or inhibit T REG In addition to this, exogenous IL-4 in T REG Apart from the controversial mouse literature regarding the role of IL-4 in the production of T cells, there is a lack of evidence on the effects of IL-4 on human T cells. REG Although one study found that IL-4 retains the ability of human T cells to REG The functions of cells.

[0334] Human iT with hybrid Th2 components REG Cells may be beneficial for adoptive T cell therapy because iT REGCells have been characterized as having a propensity for dedifferentiation plasticity in vivo, whereby iT REG Cells can be transformed into pathogenic Th1 or Th17 subsets. REG If differentiation toward a Th2-type phenotype is encoded during manufacturing, then the Th2 bias will predictably limit plasticity toward a Th1 / Th17 phenotype.

[0335] In addition, the drug pemetrexed was evaluated for its potential to promote iT REG Cell phenotype is beneficial. Use agents to preferentially produce iT REG There are precedents in cells; most notably, the mTOR inhibitor rapamycin has been linked to iT REG However, pemetrexed has not been characterized as having an inhibitory effect on iT REG Promotional effect. As a folic acid anti-metabolite, pemetrexed has a complex mechanism of action.

[0336] Fig.13 Depicts the relative polarization of Th1 under hybrid Th2 / T REG Polarization conditions promote the expansion of dedifferentiated T cells. + and CD8 + T cells undergo a 3-day dedifferentiation process ("Step 1"). Fig.13 As indicated, this step 1 dedifferentiation intervention variably included: no inhibitor ("None"); temsirolimus alone ("T"; 1.0 μM); vitamin D alone ("D"; 0.1 nM); anti-IL-R monoclonal antibody basiliximab alone ("B"; 10 μg / ml); or different combinations of inhibitors (T, D or T, D, B). After 3 days, dedifferentiated T cells were co-stimulated (classical bead to T cell ratio of 3:1) in medium variably supplemented with: Th1 polarizing conditions (rhu IFN-α; 10,000 IU / ml); T REG polarization (rhu IL-2, 100 IU / ml; rhu TGF-β, 10 ng / ml); or hybrid Th2-T REG Polarizing conditions (IL-2, TGF-β plus rhu IL-4 addition [1000 IU / ml]). In addition, T cell cultures in the presence of different polarizing conditions were performed in the absence of the novel inhibitory molecule pemetrexed ("0") or in the presence of different concentrations of pemetrexed (as indicated, 10 nM ["10"]; 33 nM ["33"]; or 100 nM ["100"]). After a total of 10 days of culture, n = 24 cultures were harvested, and the number of viable cells was counted and plotted (y axis represents the number of cells × 10 6 ; Input cell number is 1.5×10 6 cells).

[0337] like Fig.13 As shown, the ability of T cells to redifferentiate after dedifferentiation in step 1 depends on: the specific components added during dedifferentiation; the specific cytokines added during redifferentiation; and the presence of pemetrexed during redifferentiation.

[0338] Of note, attempts to redifferentiate sufficient numbers of T cells after dedifferentiation in step 1 were unsuccessful under Th1-polarizing conditions (see Fig.13 , cultures #9 to #16; all T cell yields were below T cell input number). A greatly restricted ability to redifferentiate along a Th1-type pathway was observed if the dedifferentiation conditions included temsirolimus and vitamin D alone or in combination with an anti-IL-2 receptor agent, and if pemetrexed was not added during the step 2 cultures or was added at concentrations ranging from 10 to 100 nM.

[0339] In sharp contrast, in the hybrid T REG - Under conditions of Th2 polarization, attempts to differentiate sufficient numbers of T cells after dedifferentiation in step 1 were successful (see Fig.13 , cultures #2 and #5). Of note, under the most stringent dedifferentiation conditions (containing temsirolimus, vitamin D, and anti-IL-2 receptor monoclonal antibody), only when pemetrexed was added at a concentration of 10 nM in the culture in step 2 was the dedifferentiation condition maintained at T REG -Sufficient T cells were observed under Th2 polarization conditions.

[0340] Using this most stringent dedifferentiation step 1 condition, pure T REG Attempts to redifferentiate sufficient numbers of T cells during step 2 under polarizing conditions (IL-2 plus TGF-β, no IL-4) were unsuccessful, even in the presence of pemetrexed (cultures #20 and #21).

[0341] In summary, from a numerical perspective, successful T cell redifferentiation is achieved using T REG -Th2 hybrid polarizing conditions (IL-2, TGF-β and IL-4) and using the agent pemetrexed at a concentration of 10 nM performed optimally.

[0342] Example 13: Dedifferentiated T cells in hybrid T REG / Th2 conditions will produce CD4 + and CD8 + T cells

[0343] The effects of step 1 dedifferentiation and subsequent step 2 redifferentiation on T cell memory status under different cytokine polarization conditions / different pemetrexed conditions were also evaluated. That is, the study suggests that T cells with limited differentiation states have improved therapeutic utility in adoptive cell therapy; therefore, limited T cell differentiation would be a favorable feature of step 1 / step 2 T cell manufacturing methods.

[0344] Figures 14A-14C showed that in hybrid Th2 / T REG Cultivation of dedifferentiated T cells under polarizing conditions leads to naive and triple positive T central memory CD4 + T cell production. Make human CD4 + and CD8 + T cells were subjected to a 3-day dedifferentiation process and then cultured in media containing different polarizing culture conditions and the presence of pemetrexed, such as Fig.13 As described. Of the total n=24 culture conditions, only cultures with good cell yields were further evaluated; all cultures shown containing pemetrexed ("+") were at a concentration of 10 nM. Unless indicated ("TReg, no IL-4"), all T REG Conditions all contained IL-2, TGF-β, and IL-4 ("TReg"). After a total of 10 days in culture, cultures were harvested and assessed by flow cytometry for: naive CD4 + The content of T cells (expressed as CD4 + Percentage of total T cells; Fig.14A ); Central memory CD4 + The content of T cells (expressed as CD4 + Percentage of total T cells; Fig. 14B ); and triple-positive central memory CD4 + The content of T cells (expressed as CD4 + Percentage of total T cells; Fig. 14C ).

[0345] like Fig.14A As shown, hybrid T REG -Step 2 redifferentiation under Th2 conditions (with or without 10 nM pemetrexed) produced high frequencies of CD4 + CD45RA + Naive T cell subsets, which are advantageous in experimental mouse models of adoptive T cell therapy.

[0346] In addition, if Fig. 14C As shown, hybrid TREG -Step 2 redifferentiation under Th2 conditions (with or without 10 nM pemetrexed) resulted in high frequencies of CD4 T cells with triple positive co-expression of the memory markers CD62L, CCR7, and CD127. + T cells. This triple positive memory phenotype is a marker of T cells with a very primitive differentiation state.

[0347] like Fig. 14C As shown, compared with pure T REG Polarization conditions, CD4 triple positive for CD62L, CCR7 and CD127 + Hybrid T cells REG -The frequency was higher under Th2 polarization conditions.

[0348] Moreover, if Fig. 14C As shown, more stringent step 1 dedifferentiation conditions (containing not only temsirolimus and vitamin D but also anti-IL-2 receptor monoclonal antibody) were used in the hybrid T REG -Th2 polarization conditions produced the highest frequency of CD4 cells that were triple positive for CD62L, CCR7, and CD127.

[0349] Figures 15A-15B Demonstrated that culture of dedifferentiated T cells under hybrid Th2 / TReg polarization conditions resulted in triple positive T central memory CD8 + T cell production. Make human CD4 + and CD8 + T cells were subjected to a 3-day dedifferentiation process and then cultured in media containing different polarizing culture conditions and the presence of pemetrexed, such as Fig.13 As described. Of the total n=24 culture conditions, only cultures with good cell yields were further evaluated; all cultures shown containing pemetrexed ("+") were at a concentration of 10 nM. Unless indicated ("TReg, no IL-4"), all T REG Conditions all contained IL-2, TGF-β, and IL-4 ("TReg"). After a total of 10 days in culture, cultures were harvested and assessed by flow cytometry for: central memory CD8 + The content of T cells (expressed as CD8 + Percentage of total T cells; Fig.15A ); and triple positive central memory CD8 + The content of T cells (expressed as CD8 + Percentage of total T cells; Fig. 15B ).

[0350] Moreover, if Fig. 15B As shown, compared with pure T REG Polarization conditions, CD8 cells that are triple positive for CD62L, CCR7, and CD127 in hybrid T REG -Th2 polarization conditions are higher. In addition, Figures 15A-15B As shown, more stringent step 1 dedifferentiation conditions (containing not only temsirolimus and vitamin D but also anti-IL-2 receptor monoclonal antibody) were used in the hybrid T REG -Th2 polarization conditions produced the highest frequency of CD8 cells that were triple positive for CD62L, CCR7, and CD127 + T cells.

[0351] Altogether, these data suggest that the use of hybrid T REG -Th2 cytokine polarization conditions (IL-2, TGF-β, and IL-4) and T cell redifferentiation with pemetrexed (10 nM) after step 1 dedifferentiation resulted in CD4 + and CD8 + T cells.

[0352] Example 14: Dedifferentiated T cells in hybrid T REG / Th2 polarization conditions produce T cells with a primitive Th2 cytokine phenotype

[0353] The cytokine secretion pattern of T cells redifferentiated under the culture conditions of step 2 after step 1 dedifferentiation was also assessed. Cytokine secretion is an indicator of T cell effector function and, as such, is generally expected to be REG The cells have reduced cytokine secretion potential, especially for key inflammatory cytokines such as IL-17, IFN-γ, and TNF-α. REG In the case of a Th2 cell population, it can be expected that such cells will also secrete a certain profile of Th2 cytokines.

[0354] Figures 16A-16C We show that culture of dedifferentiated T cells under hybrid Th2 / TReg polarization conditions results in the production of cells with a primitive Th2 cell cytokine phenotype, as indicated by high levels of IL-2 and IL-4 secretion and low levels of IL-5 secretion. + and CD8 + T cells were subjected to a 3-day dedifferentiation process and then cultured in media containing different polarizing culture conditions and the presence of pemetrexed, such as Fig.13As described. Of the total n=24 culture conditions, only cultures with good cell yields were further evaluated; all cultures shown containing pemetrexed ("+") were at a concentration of 10 nM. Unless indicated ("TReg, no IL-4"), all T REG Conditions all contained IL-2, TGF-β, and IL-4 ("TReg"). After a total of 10 days of culture, T cells were harvested and washed, and restimulated with 3 / 28 beads (3:1 ratio) for 24 hours; the resulting supernatant was harvested and cytokine content was measured by Luminex multi-analyte method. All results shown are expressed as cytokine levels in pg / ml / 1×10 6 cells / ml / 24 hours.

[0355] like Fig.16A As shown, in the case of culture with or without the addition of pemetrexed, the REG - T cells redifferentiated in Th2 cytokine polarization conditions (IL-2, TGF-β and IL-4) had the highest IL-2 secretion values. This result is consistent with the previous understanding that IL-2 secretion in T cells is a characteristic of T cells in an early differentiation state, in which T cells are redifferentiated under hybrid culture conditions.

[0356] Figures 17A-17C showed that in hybrid Th2 / T Reg Cultivation of dedifferentiated T cells under polarizing conditions resulted in the production of cells with a primitive Th2 cell cytokine phenotype, as indicated by low levels of IL-10, IL-13, and IL-17 secretion. + and CD8 + T cells were subjected to a 3-day dedifferentiation process and then cultured in media containing different polarizing culture conditions and the presence of pemetrexed, such as Fig.13 As described. Of the total n=24 culture conditions, only cultures with good cell yields were further evaluated; all cultures shown containing pemetrexed ("+") were at a concentration of 10 nM. Unless indicated ("TReg, no IL-4"), all T REG Conditions all contained IL-2, TGF-β, and IL-4 ("TReg"). After a total of 10 days of culture, T cells were harvested and washed, and restimulated with 3 / 28 beads (3:1 ratio) for 24 hours; the resulting supernatant was harvested and cytokine content was measured by Luminex multi-analyte method. All results shown are expressed as cytokine levels in pg / ml / 1×10 6 cells / ml / 24 hours.

[0357] Figures 18A-18C showed that in hybrid Th2 / T REG Cultivation of dedifferentiated T cells under polarizing conditions resulted in the production of cells with a primitive Th2 cytokine phenotype, as indicated by low levels of IFN-γ, TNF-α, and GM-CSF secretion. + and CD8 + T cells were subjected to a 3-day dedifferentiation process and then cultured in media containing different polarizing culture conditions and the presence of pemetrexed, such as Fig.13 As described. Of the total n=24 culture conditions, only cultures with good cell yields were further evaluated; all cultures shown containing pemetrexed ("+") were at a concentration of 10 nM. Unless indicated ("TReg, no IL-4"), all T REG Conditions all contained IL-2, TGF-β, and IL-4 ("TReg"). After a total of 10 days of culture, T cells were harvested and washed, and restimulated with 3 / 28 beads (3:1 ratio) for 24 hours; the resulting supernatant was harvested and cytokine content was measured by Luminex multi-analyte method. All results shown are expressed as cytokine levels in pg / ml / 1×10 6 cells / ml / 24 hours.

[0358] In addition, if Fig. 16B As shown, in the case of culture with or without the addition of pemetrexed, the REG - T cells redifferentiated in Th2 conditions (IL-2, TGF-β and IL-4) had the highest IL-4 secretion values. Since IL-4 is a key cytokine determining Th2 polarization, T cells made under hybrid conditions are actually Th2 polarized. On the other hand, consistent with their limited differentiation state, cells redifferentiated under hybrid conditions did not secrete high levels of effector Th2 cytokines (IL-5, see Fig. 16C IL-10, see Fig.17A IL-13, see Fig. 17B ) or effector Th1 / Th17 cytokines (IFN-γ, see Fig.18A ; TNF-α, see Fig.18B ; GM-CSF, see Fig.18C IL-17, see Fig. 17C ).

[0359] Importantly, the absence of IL-4 during T cell redifferentiation resulted in higher levels of the inflammatory cytokines IFN-γ, TNF-α, and GM-CSF (see Figures 18A-18C ).

[0360] Taken together, these data suggest that T cells undergo a process of dedifferentiation from step 1 to T REG Phenotypic redifferentiation should be optimally utilized in hybrid T REG -Th2 polarization conditions, as T cells derived from such conditions have a greatly reduced capacity to secrete cytokines associated with inflammatory diseases.

[0361] Example 15: Dedifferentiated T cells in hybrid T REG Culture under Th2 conditions will produce hybrid T cells with enhanced REG T cells / Th2 transcription factor profile

[0362] T cell cytokine phenotype is determined by key transcription factors. The association between transcription factors and T cell subsets is as follows: FOXP3 determines T REG TBET determines the development of Th1 cells; and GATA3 determines the development of Th2 cells.

[0363] To evaluate these transcription factors in a manufacturing process, T cells were first subjected to a step 1 dedifferentiation process and then in a hybrid T REG -Th2 culture conditions (IL-2, TGF-β and IL-4). In addition, the effect of pemetrexed and classical mTOR inhibitors were compared. In the experiment, instead of using mTOR inhibitors (rapamycin; ) using a water-soluble parent form of the drug temsirolimus

[0364] iT REG The phenotype was considered unstable; thus, the expression of hybrid T cells was assessed at delayed time points (including days 20 and 32 of culture). REG - Stability of T cells redifferentiated by Th2 culture conditions. In addition, to rigorously test phenotypic stability, T cells received high levels of co-stimulation (3:1 beads to T cells ratio) and were propagated in medium without cytokines or pharmacological agents between days 24 and 32 of culture.

[0365] Figures 19A-19D showed that hybrid Th2 / T REG Extended culture of dedifferentiated T cells under polarizing conditions resulted in the generation of CD4+ T cells expressing FOXP3 and GATA3 transcription factors. + and CD8 + T cells were subjected to a 3-day dedifferentiation process and then cultured in a hybrid Th2 / T REGPolarizing conditions (IL-2; TGF-β; IL-4) were co-stimulated (3: 1 beads to T cell ratio) and propagated in medium without or with the drug inhibitor temsirolimus (1.0 μM) or pemetrexed (10 nM). On days 14 and 24 of culture, the cultures were restimulated using 3 / 28 beads; on day 24 of culture, to assess the stability of transcription factor expression, the culture medium did not contain exogenous cytokines or pharmacological inhibitors. On days 12, 20, and 32 of culture, T cells were harvested and surface flow cytometry (CD4 marker) and intracellular staining were performed for the following transcription factors FOXP3, Tbet, and GATA3. The above data show the percentage of CD4 cells in the total culture population ( Fig.19A ); Expression T REG The percentage of CD4 cells expressing the transcription factor FOXP3 ( Fig.19B ); The percentage of CD4 cells expressing Th1 transcription factor Tbet ( Fig.19C ); and the percentage of CD4 cells expressing the Th2 transcription factor GATA3 ( Fig.19D ).

[0366] like Figures 19A-19D As shown in the details, during the culture process, over time, at T REG - T cells redifferentiated under Th2 conditions have the advantage of gradually transforming into CD4 cells ( Fig.19A ).like Fig.19B As shown, CD4 cells expressed FOXP3 at high frequencies in a stable manner from day 12 to day 32 of culture, regardless of the presence or absence of temsirolimus or pemetrexed in the culture.

[0367] like Fig.19C , contamination with the Th1 transcription factor TBET was low even in the absence of pharmacological inhibitors. However, the most consistently reduced TBET values ​​were observed under hybrid polarization conditions that also included pemetrexed. Fig.19D As shown, in the hybrid T supplemented with pemetrexed REG -The highest value of Th2-associated GATA3 expression at the end of culture was observed in T cells generated under Th2 conditions.

[0368] Figures 20A-20D showed that hybrid Th2 / T REG Extended culture of dedifferentiated T cells under polarizing conditions results in CD8 + T cell production. Make human CD4 + and CD8 + T cells were subjected to a 3-day dedifferentiation process and then cultured in a hybrid Th2 / TREG Polarizing conditions (IL-2; TGF-β; IL-4) were co-stimulated (3: 1 beads to T cell ratio) and propagated in medium without or with the drug inhibitor temsirolimus (1.0 μM) or pemetrexed (10 nM). On days 14 and 24 of culture, the cultures were restimulated using 3 / 28 beads; on day 24 of culture, to assess the stability of transcription factor expression, the culture medium did not contain exogenous cytokines or pharmacological inhibitors. On days 12, 20, and 32 of culture, T cells were harvested and surface flow cytometry (CD8 marker) and intracellular staining were performed for the following transcription factors FOXP3, Tbet, and GATA3. The above data show the percentage of CD8 cells in the total culture population ( Fig. 20A ); Expression T REG The percentage of CD8 cells expressing the transcription factor FOXP3 ( Fig. 20B ); The percentage of CD8 cells expressing Th1 transcription factor Tbet ( Fig. 20C ); and the percentage of CD8 cells expressing the Th2 transcription factor GATA3 ( Fig.20D ).

[0369] like Fig. 20A As shown in Figure 2, the content of CD8 cells decreased slightly over time during the culture process. REG Cell functions are usually attributed to CD4 cell subsets, but CD8 + T REG cells have also been well described; due to the diversity of antigen specificity, the use of + and CD8 + T cell subsets REG Thus, the described method has potential advantages, in part because it generates both CD4 and CD8 T cells. REG .

[0370] like Fig. 20B As shown in the upper right figure, CD8 + T cells were indeed enriched for FOXP3 expression, which was stable over time in culture and whose stability was independent of the presence of pharmacological inhibitors.

[0371] like Fig. 20C As shown (lower left), T REG -Redifferentiation under Th2 polarization conditions usually results in low levels of the Th1 transcription factor TBET CD8 + T cell expression; however, the most consistently lowest levels were observed in the presence of pemetrexed.

[0372] Finally, if Fig.20D As shown (lower right figure), at T REG -Redifferentiation under Th2 conditions does produce CD8 + T cells, as indicated by increased expression of the GATA3 transcription factor.

[0373] Figures 40A-40B CD4 has also been described + and CD8 + Flow cytometric analysis of GATA3 and FOXP3 in redifferentiated TREG-Th2 cells.

[0374] In summary, these transcription factor analyses suggest that in hybrid T REG -Th2 culture conditions and redifferentiation with the addition of pemetrexed are optimal because they preserve CD4 + and CD8 + T cells, while having limited TBET expression.

[0375] Example 16: Dedifferentiated T cells in hybrid T REG Culture under Th2 conditions generates T cells with an enhanced Th2 cytokine secretion profile

[0376] In addition to transcription factor measurements, we also evaluated the expression of REG -The cytokine secretion capacity of T cells redifferentiated under Th2 polarization conditions. Fig.21A -21D, at T REG -All redifferentiated cultures propagated under Th2-polarizing conditions generated T cells capable of secreting IL-4, demonstrating the inherent ability of this approach to achieve Th2 polarity even in the absence of pharmacological inhibitors.

[0377] Fig.21A -21D showed a strong affinity for hybrid Th2 / T REG Extended culture of dedifferentiated T cells under polarizing conditions resulted in the generation of T cells expressing the major Th2 cytokine phenotype: secretion of IL-4, IL-5, and IL-13. + and CD8 + T cells were subjected to a 3-day dedifferentiation process and then cultured in a hybrid Th2 / T REGPolarizing conditions (IL-2; TGF-β; IL-4) were co-stimulated (3:1 beads to T cells ratio) and propagated in medium without or with the drug inhibitor temsirolimus (1.0 μM) or pemetrexed (10 nM). On days 14 and 24 of culture, the culture was restimulated with 3 / 28 beads; on day 24 of culture, in order to assess the stability of transcription factor expression, the culture medium did not contain exogenous cytokines or pharmacological inhibitors. On days 12, 20, and 32 of culture, T cells were harvested and washed, and restimulated with 3 / 28 (ratio 3:1) for 24 hours; the resulting supernatant was harvested and cytokine content was detected by Luminex multi-analyte method. All results shown are expressed as cytokine levels in pg / ml / 1×10 6 cells / ml / 24 hours. The Th2 cytokine IL-10 was also evaluated: all values ​​were less than 20pg / ml / 1×10 6 cells / ml / 24 hours.

[0378] It is worth noting that although temsirolimus attenuated the REG -Th2-derived T cells secrete the effector Th2 cytokine IL-5 ( Fig. 21B ) and IL-13( Fig. 21C ), but the use of pemetrexed completely preserved the ability of T cells to secrete IL-5 and IL-13. Thus, these data provide further evidence that, compared with the use of conventional T REG Compared with mTOR-promoting agents (such as the mTOR inhibitor temsirolimus), the use of pemetrexed is advantageous because pemetrexed is closely related to T REG -The production of Th2 hybrid subsets is more compatible.

[0379] In addition, in T REG -All T cells redifferentiated under Th2 polarization conditions had relatively low levels of IL-2 ( Fig.22A ), IFN-γ( Fig. 22B ), IL-17 (all values ​​were less than 20 pg / ml) and TNF-α (all values ​​were less than 20 pg / ml) expressions.

[0380] Fig.22A -22D demonstrated that extended culture of dedifferentiated T cells under hybrid Th2 / TREG polarization conditions resulted in the generation of T cells expressing the major Th2 cytokine phenotype: IL-2, IFN-γ, and GM-CSF secretion. + and CD8 + T cells were subjected to a 3-day dedifferentiation process and then cultured in a hybrid Th2 / T REGPolarizing conditions (IL-2; TGF-β; IL-4) were co-stimulated (3:1 beads to T cells ratio) and propagated in medium without or with the drug inhibitor temsirolimus (1.0 μM) or pemetrexed (10 nM). On days 14 and 24 of culture, the culture was restimulated with 3 / 28 beads; on day 24 of culture, in order to assess the stability of transcription factor expression, the culture medium did not contain exogenous cytokines or pharmacological inhibitors. On days 12, 20, and 32 of culture, T cells were harvested and washed, and restimulated with 3 / 28 (ratio 3:1) for 24 hours; the resulting supernatant was harvested and cytokine content was detected by Luminex multi-analyte method. All results shown are expressed as cytokine levels in pg / ml / 1×10 6 cells / ml / 24 hours. Inflammatory cytokines IL-17 and TNF-α were also evaluated: all values ​​were less than 20 pg / ml / 1×10 6 cells / ml / 24 hours.

[0381] Notably, the cytotoxicity of the 24-histidine-containing group was significantly increased after further supplementation with pemetrexed (10 nM; Fig. 22C ) REG Higher levels of GM-CSF secretion were observed in T cells redifferentiated under Th2 hybrid culture conditions. REG - Whether the ability to enhance GM-CSF in Th2 hybrid populations is necessarily harmful or beneficial.

[0382] Taken together, these data suggest that T REG - T cell redifferentiation under Th2 polarization conditions is advantageous because it generates T cells with reduced capacity to secrete Th1 and Th17 cytokines associated with inflammatory diseases. REG - The inclusion of pemetrexed in Th2 polarizing conditions is advantageous as this will increase the capacity for Th2 cytokine production, which will further provide limited plasticity towards differentiation towards Th1 and Th17 subsets.

[0383] Example 17: Use of Selected Anti-TNF-α Agents Prior to Lymphocyte Collection by Apheresis to Beneficially Alter the Input T Cell TCR Repertoire

[0384] Fig.23A and 23BDepicted is the use of RNA-based T cell receptor sequencing to detect widespread upregulation and downregulation of T cell TCR specificity following treatment with the TNF-α inhibitor etanercept. In Figure 23, RNA was isolated from peripheral blood mononuclear cells of ALS patients before and after treatment with etanercept therapy. The RNA was subjected to TCR repertoire analysis 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): 61. Fig.23A In the figure, approximately 25% of TCR specificities were upregulated in the post-treatment samples (indicated in red); in stark contrast, approximately 25% of TCR specificities were downregulated in the post-treatment samples (indicated in blue). As indicated in the upper right panel (B), etanercept therapy produced a significant T cell clonal expansion, as several T cell clones increased in frequency from 0.01 before etanercept (close to the detection limit of the assay) to values ​​of 247 to 486 after treatment, consistent with a greater than 4-log T cell expansion. Fig. 23B As indicated, etanercept therapy produced a significant T-cell clonal contraction, as the frequencies of several T-cell clones decreased from 259 to 598 before etanercept to a value of 0.01 after treatment, consistent with a greater than 4-log contraction of T-cell clones.

[0385] Fig.23A -B showed that anti-TNF-α therapy with etanercept, which preferentially inhibits serum, cell-free forms of TNF-α (promoting Th1-type cells expressing TNFR1), was associated with extensive changes in T cell receptor up- and down-regulation. These observations suggest that pretreatment of subjects with etanercept or any other anti-TNF-α therapeutic that preferentially inhibits serum, cell-free forms of TNF-α (such as the monoclonal antibody adalimumab) can be used to shift the T cell receptor repertoire away from T cells of a Th1-type phenotype on the basis of antigen specificity, thereby enriching T cells of a Th1-type phenotype.

[0386] Example 18: Characterization of TREG-Th2 hybrid population as a cell product enriched for CD25, CD27, 2B4, BTLA and CTLA4 expression.

[0387] Fig.24The generated iTREG / Th2 hybrid population showed increased expression of CD25, CD27, 2B4, BTLA, and CTLA4 relative to control Th1 / Tc1 cultures. Fig.24 In the iTREG / Th2 hybrid population, the initial phase of T cell dedifferentiation and subsequent redifferentiation in medium containing IL-2, TGF-β and IL-4 was generated by the method detailed previously. On day 11 of iTREG / Th2 production, cells were harvested and flow cytometry was performed to evaluate CD4+ and CD8+ T cell expression of relevant molecules (i.e., CD25, CD27, 2B4, BTLA and CTLA4); three independent control conditions evaluating Th1 / Tc1 polarization were compared.

[0388] Fig.24 It was shown by flow cytometry analysis that hybrid TREG-Th2 cells made according to the described conditions have increased expression of the following cell surface molecules relative to control Th1 / Tc1 cells: CD25, CD27, 2B4, BTLA and CTLA4.

[0389] like Fig.24 As shown, the iTREG / Th2 hybrid cell product has CD4+ and CD8+ T cells expressing at least 10% higher and more preferably 50% higher levels of CD25, CD27, 2B4, BTLA and CTLA4 relative to control Th1 / Tc1 cells.

[0390] CD25, the IL-2 receptor, is critical to the ability of TREG cells to control autoimmunity, particularly CD8+ T cell driven responses. Therefore, expression of CD25 on iTREG / Th2 manufactured cell products is a desirable property.

[0391] CD27, a co-stimulatory molecule with increased expression on TREG cells, has been shown to contribute to the inhibitory function of TREG. Therefore, expression of CD27 on the cell product of iTREG / Th2 production is a desirable property.

[0392] 2B4 (CD244) has recently been shown to inhibit CD8+ T cell responses by attenuating glycolysis and cell division. Therefore, expressing 2B4 on iTREG / Th2 manufactured cell products is a desirable property.

[0393] BTLA (CD272) is a co-inhibitory receptor, and ligation of BTLA with the herpes virus entry mediator HVEM promotes the induction and suppression of effector immune responses in TREG cells. Therefore, expression of BTLA on iTREG / Th2 manufactured cell products is a desirable property.

[0394] CTLA4 is a key effector molecule of TREG cells as it has recently been shown to have the ability to limit immunity to malaria infection. Therefore, expressing CTLA4 on the cell products of iTREG / Th2 production is a desirable property.

[0395] Example 19: Characterization of TREG-Th2 hybrid population as a cell product enriched for TIGIT, TIM3, ICOS, LAIR1 and OX40 expression.

[0396] Fig.25 The generated iTREG / Th2 hybrid populations showed increased expression of TIGIT, TIM3, ICOS, LAIR1, and OX40 relative to control Th1 / Tc1 cultures. Fig.25 In the iTREG / Th2 hybrid population, the initial phase of T cell dedifferentiation and subsequent redifferentiation in medium containing IL-2, TGF-β and IL-4 was generated by the method described in detail previously. On day 11 of iTREG / Th2 production, cells were harvested and flow cytometry was performed to evaluate CD4+ and CD8+ T cell expression of relevant molecules (i.e., TIGIT, TIM3, ICOS, LAIR1 and OX40); three independent control conditions evaluating Th1 / Tc1 polarization were compared. Fig.25 It was shown by flow cytometry analysis that hybrid TREG-Th2 cells made according to the described conditions have increased expression of the following cell surface molecules relative to control Th1 / Tc1 cells: TIGIT, TIM3, ICOS, LAIR1 and OX40.

[0397] like Fig.25 As shown, the iTREG / Th2 hybrid cell product has CD4+ and CD8+ T cells expressing at least 10% higher and more preferably 50% higher levels of TIGIT, TIM3, ICOS, LAIR1 and OX40 relative to control Th1 / Tc1 cells.

[0398] TIGIT is a cell surface co-inhibitory receptor molecule associated with regulatory T cell function. Therefore, expression of TIGIT on iTREG / Th2 manufactured cell products is a desirable property.

[0399] TIM3 is a co-inhibitory receptor that mediates the inhibitory effects of TREG cells. Therefore, expressing TIM3 on iTREG / Th2 manufactured cell products is a desirable property.

[0400] ICOS is a co-stimulatory molecule that has recently been identified to help maintain immunosuppression by regulatory T cells to control immune responses in the central nervous system. Therefore, expression of ICOS on iTREG / Th2 manufactured cell products is a desirable property.

[0401] LAIR1 (CD305) is a multifaceted inhibitory molecule that can block inflammation at multiple steps, including inhibition of activated effector memory T cells. Therefore, expression of LAIR1 on iTREG / Th2 manufactured cell products is a desirable property.

[0402] OX40 is a co-stimulatory molecule. Therefore, expression of OX40 on the cell product of iTREG / Th2 production is a desirable property.

[0403] Example 20: Characterization of GATA3 and FOXP3 expression in TREG / Th2 hybrid populations

[0404] Steady-state apheresis samples were obtained and lymphocytes were enriched by Ficoll gradients, and then plated in G-Rex culture dishes and incubated in culture medium containing vitamin D (0.3 nM), temsirolimus (3.0 μM) and basiliximab (30 μg / mL). After the initial dedifferentiation interval, T cells were co-stimulated with anti-CD3 / anti-CD28 coated magnetic beads at a bead to T cell ratio of 3:1, and cytokines (IL-4 (1000 IU / mL), IL-2 (10,000 IU / mL) and TGF-β (100 ng / mL)) were added. After 6 days of culture, T cells were harvested, stained for surface markers (CD4 and CD8) and intracellular molecule expression (GATA3 and FOXP3), and evaluated by flow cytometry. Fig.26A -The results in B show the FOXP3 and GATA3 expression of CD4+ and CD8+ T cells at the beginning of culture and after culture (Th2 / TREG) as measured by flow cytometry. The percentages provided indicate the number of cells that are considered to be positive for CD4+ or CD8+ and intracellular markers (shown in boxes).

[0405] Fig.26A-The results shown in B show the results of the Th2 / TREG cell product phenotype indicated for manufacture. The T cells of type II cytokine phenotype can be characterized in part by the expression of its transcription factor GATA3, while the regulatory T cell group can be identified in part by the expression of its FoxP3 transcription factor. At the beginning of culture, very low frequency T cells express GATA3 or FoxP3. In sharp contrast, the T cell products manufactured under Th2 / TREG culture conditions express high frequency T cells, which are single positive for GATA3, single positive for FOXP3, or double positive for GATA3 and FOXP3 (not shown); Importantly, as shown, this transcription factor spectrum is expressed in manufactured CD4+ (upper figure) and CD8+ (lower figure) T cells. The control manufacturing culture not comprising IL-4 greatly reduces the frequency of GATA3 positive T cells, which shows the important role of IL-4 in the manufacture of Th2 / TREG hybrid groups (not shown).

[0406] Most of the phenotypic characterizations of T cell products manufactured according to the TREG / Th2 method detailed in the present disclosure can be determined at the end of the culture. However, it is important to note that T cell products can be cryopreserved, and in this way, the phenotypic characteristics of T cells in the thawed state reflect the actual product that will be adopted and transferred to the subject. Relative to control Th1 / Tc1 cells, TREG / Th2 cells in the thawed state can be characterized by the following: (a) by flow cytometry, the expression of CD25, CD27, 2B4, BTLA, CTLA4, TIGIT, TIM3, ICOS, LAIR1 and OX40 increases; (b) by Luminex cytokine secretion analysis, IFN-g and TNF-α are reduced, but the secretion of IL-4 is increased; and (c) the expression of T cell fate transcription factors is changed, that is, TBET is reduced and FOXP3 and GATA3 are increased.

[0407] Example 21: Characterization of CD73 and CD103 expression in TREG / Th2 hybrid populations

[0408] Steady-state apheresis samples were obtained and lymphocytes were enriched by Ficoll gradients, and then plated in G-Rex culture dishes and incubated in culture medium containing vitamin D (0.3 nM), temsirolimus (3.0 μM) and basiliximab (30 μg / mL). After the initial dedifferentiation interval, T cells were co-stimulated with anti-CD3 / anti-CD28 coated magnetic beads at a bead to T cell ratio of 3:1, and cytokines (IL-4 (1000 IU / mL), IL-2 (10,000 IU / mL) and TGF-β (100 ng / mL)) were added. After 6 days of culture, T cells were harvested, stained for surface markers (CD4 and CD8) and ectonucleotidase molecules (CD73) or integrin molecules (CD103), and evaluated by flow cytometry. Fig.27A -The results in B show the CD73 and CD103 expression of CD4+ and CD8+ T cells at the beginning of culture and after culture (Th2 / TREG) as measured by flow cytometry. The percentages provided indicate the number of cells that are considered to be positive for CD4+ or CD8+ and ectonucleotidase molecules or integrin molecules (shown in boxes), respectively.

[0409] Regulatory T cell populations can suppress pathogenic effector T cell populations through several established mechanisms, including through the expression of CD39 and CD73 ectonucleotidase molecules, which serve to hydrolyze proinflammatory ATP into immunosuppressive adenosine substrates. Indeed, TREG cells expressing CD39 have enhanced suppressive function and are associated with the regression of inflammatory bowel disease. Furthermore, the suppressive function of human TREG cells is mediated in part by CD73. As follows Fig.27A As shown, the expression of TREG-related effector molecule CD73 of T cells produced under Th2 / TREG culture conditions can be increased; CD39 is also highly expressed on T cells produced by TREG / Th2 (not shown). In addition to CD39 / CD73 ectonucleases, TREG cell function is also associated with the expression of CD103, an integrin that determines the localization of epithelial lymphocytes. In fact, CD103 and IL-2 receptor signaling can synergistically maintain the immune tolerance of the intestinal mucosa; in addition, TREG cells expressing CD103 are essential for improving experimental chronic GVHD. As follows Fig.27B As shown, the expression of the TREG-related effector molecule CD103 of T cells produced under Th2 / TREG culture conditions can be increased.

[0410] Example 22: Characterization of CD150 and CD27 / CD95 expression of TREG / Th2 hybrid population

[0411] Steady-state apheresis samples were enriched for lymphocytes by Ficoll gradient, plated in G-Rex dishes, and incubated in medium containing vitamin D (0.3 nM), temsirolimus (3.0 μM), and basiliximab (30 μg / mL). After this initial dedifferentiation interval, T cells were co-stimulated with anti-CD3 / anti-CD28-coated magnetic beads at a 3:1 bead:T cell ratio, and cytokines (IL-4 (1000 IU / mL), IL-2 (10,000 IU / mL), and TGF-β (100 ng / mL)) were added. After 6 days of culture, T cells were harvested, stained for surface markers, and subjected to multicolor flow cytometric analysis for CD4, CD8, CD150, CD27, CD95, CD45RA, CD62L, and CCR7. Results are shown in Fig.28A - shown in B.

[0412] T cells cultured under TREG (RAPA-501) conditions were compared to culture input T cells ("Day 0") and also compared to control culture T cells propagated in the absence of mTOR inhibitor ("Control"). Fig.28A As shown, the CD4+ and CD8+ T cell subsets contained in the RAPA-501 cell product have greatly increased expression of the stem cell marker CD150 relative to the culture input T cells and the control culture T cells. Fig.28B As shown, the RAPA-501 cell product is also enriched for the T stem cell memory (TSCM) phenotype relative to the culture input cells; this population is absent in the control cultures because the T cells generated under these conditions are effector memory CD45RO+ (not shown). The left panel (culture input T cells) and right panel (RAPA-501 cells) show the expression of TSCM markers CD95 and CD27 after gating on TSCM markers CD45RA, CD62L and CCR7; similar differences in the expression of these TSCM markers were not observed for CD8+ T cells (not shown).

[0413] In experimental models, the efficacy of adoptive T cell therapy depends on the successful engraftment and in vivo persistence of T cell populations. Importantly, the differentiation state of T cells helps dictate in vivo persistence, with less differentiated cells having greater persistence. In initial studies, murine rapamycin-resistant T cells expressing a T central memory (TCM) phenotype had enhanced in vivo engraftment potential relative to control T cells; in addition, human rapamycin-resistant T cells also had increased engraftment in a human-to-mouse model of xenogeneic graft-versus-host disease. Other researchers have determined that T cells with reduced differentiation have greater in vivo persistence and mediate increased in vivo effects relative to the T effector memory (TEM) population, including TCM subsets, naive T cell subsets, and more recently, T stem cell memory (TSCM) subsets. This relationship between T cell differentiation state and in vivo T cell function is actionable with respect to TREG cells, such as: (1) compared with TREG cells with TEM phenotype, TREG cells with TCM phenotype are more effective in reducing experimental GVHD; (2) TREG cells expressing the stem cell marker CD150 are very effective in preventing stem cell transplant rejection. Fig.28A As shown, T cells produced under Th2 / TREG culture conditions are enriched for cells with a reduced differentiation state consistent with a T stem cell subset, including expression of the CD150 marker.

[0414] Example 23: Characterization of cytokine secretion by TREG / Th2 hybrid populations

[0415] Steady-state apheresis samples were enriched for lymphocytes by Ficoll gradient, plated in G-Rex dishes, and incubated in culture medium containing vitamin D (0.3 nM), temsirolimus (3.0 μM) and basiliximab (30 μg / mL). After the initial dedifferentiation interval, T cells were co-stimulated with anti-CD3 / anti-CD28 coated magnetic beads at a bead:T cell ratio of 3:1, and cytokines (IL-4 (1000 IU / mL), IL-2 (10,000 IU / mL) and TGF-β (100 ng / mL)) were added. This culture was referred to as condition "A". Condition "B" is the same culture condition, but IL-4 is not added. Condition "C" reflects the standard TREG culture conditions of rapamycin (1 μM), IL-2 (100 IU / mL) and TGF-β (10 ng / mL). Condition "D" reflects a Th1 type control culture made in the presence of IFN-α in the absence of an mTOR inhibitor. At the end of the culture, T cells were harvested, stimulated with anti-CD3 / anti-CD28 beads, and the resulting supernatants were tested for cytokine content by Luminex assay.

[0416] It may be important to evaluate cytokine secretion by the manufactured Th2 / TREG cells. First, it is critical that the cell product can secrete IL-4, which serves as a driving cytokine for subsequent Th2 differentiation. Second, it is desirable that the adoptively transferred T cell population be able to secrete IL-2, as this ability indicates progenitor cell function that allows the T cells to expand more easily in vivo without the need for exogenous IL-2. Finally, it is important that the Th2 / TREG cell population has reduced secretion of the Th1-type or Th17-type cytokines IFN-α, TNF-α, IL-17, and GM-CSF. Fig.29 As shown, the manufactured Th2 / TREG cell products secreted IL-4 and IL-2, with minimal secretion of Th1 or Th17 type cytokines.

[0417] Example 24: Characterization of Th1 / Tc1 inhibition by TREG / Th2 hybrid population

[0418] Steady-state apheresis samples were enriched for lymphocytes by Ficoll gradient, plated in G-Rex culture dishes, and incubated in a culture medium containing vitamin D (0.3 nM), temsirolimus (3.0 μM) and basiliximab (30 μg / mL). After the initial dedifferentiation interval, anti-CD3 / anti-CD28 coated magnetic beads were used to co-stimulate T cells at a bead:T cell ratio of 3:1, and cytokines (IL-4 (1000 IU / mL), IL-2 (10,000 IU / mL) and TGF-β (100 ng / mL)) were added to make Th2 / TREG cells in vitro. At the same time, T cells were cultured in the presence of type I polarization cytokine IFN-α to produce effector Th1 / Tc1 cells; Th1 / Tc1 cultures were produced from the same donor (autologous; "AUTO") or unrelated donor (allogeneic; "ALLO") as RAPA-501 cell cultures. After in vitro culture, Th1 / Tc1 effector T cells were plated in the bottom chamber of the transwell plate and co-stimulated with anti-CD3 / anti-CD28 coated beads at a 3:1 bead to T cell ratio. After 24 hours of Th1 / Tc1 cell co-stimulation, RAPA-501 cells were added to the top chamber of the transwell plate at a 1:1 Th1 / Tc1 to RAPA 501 ratio. (A) RAPA-501 regulation of cytokine content. At the 24th hour of culture (before RAPA-501 cells were added to the upper chamber) and the 48th hour (with or without RAPA-501 cells), the culture supernatant was harvested and the cytokine content was detected by Luminex assay. The results of IL-2, IFN-γ, GM-CSF and TNF-α content were expressed in pg / ml / 24 hours / 1×106 cells / ml. (B) RAPA-501 cell modulation of Th1 / Tc1 cell expression of PD1 occurs in an antigen-independent manner. Autologous or allogeneic Th1 / Tc1 cells were harvested at 48 hours after addition or absence of RAPA-501 cells at the 24 hour time point. Th1 / Tc1 cells were then subjected to flow cytometry to assess PD1 expression.

[0419] During the development of the RAPA-501 cell product, experiments were performed to characterize the molecular mechanisms of the observed T cell suppression. One method used to evaluate potential mechanisms is a transwell assay in which effector T cells and RAPA-501 cells are separated by filters that prevent cell-to-cell contact but allow soluble small mediators such as cytokines for cell communication. Fig. 30A-B shows that RAPA-501 cells regulate effector T cells in a contact-independent manner (experiments performed in transwell dishes). RAPA-501 cells act in a T cell receptor-independent manner to inhibit the cytokine secretion capacity of effector T cells; that is, because no costimulatory beads were added to the transwell chamber containing RAPA-501 cells, RAPA-501 cells do not require costimulation to regulate the levels of inflammatory cytokines, including IL-2, IFN-γ, GM-CSF, and TNF-α ( Fig. 30A ). The ability of TREG cells to consume IL-2 is a commonly described phenomenon, although previous studies have determined that IL-2 consumption requires cell-to-cell contact. Thus, RAPA-501 cells appear to have a somewhat unique ability to modulate the levels of multiple inflammatory cytokines in a contact-independent manner. These results suggest that RAPA-501 cells are suitable candidate cells for neutralizing cytokines. Secondly, it was found that RAPA-501 cells modulate additional aspects of effector T cell biology in a contact-independent manner (using a transwell assay), namely, promoting the expression of programmed death 1 (PD-1) checkpoint molecules on effector T cells. Importantly, as Fig. 30B As shown, RAPA-501 cells upregulated PD1 expression on autologous and allogeneic Th1 / Tc1 cells, further elucidating that one mechanism of RAPA-501 cell suppressive function occurs in a TCR-independent manner through soluble mediators.

[0420] Example 25: Characterization of the inhibition of cytokine secretion by CNS microglia by TREG / Th2 hybrid population

[0421] Human microglia (HMC3 cell line) were first activated with IFN-γ (10 ng / ml; 24 hours), and then activated with LPS (10 ng / ml; 3 hours); RAPA-501 cells prepared as described above were not added to the upper chamber (left figure) or added to the upper chamber (right figure) (RAPA-501 to HMC3 ratio of 1:40), and then the treated HMC3 cells were plated into the lower chamber of the transwell. RAPA-501 cells were produced using the method described in the patent application to produce hybrid Th2 / TREG phenotype T cells. After 24 hours, the cell-free supernatant was harvested and the content of IL-6, IFN-γ and IP-10 was evaluated by Luminex assay (cytokine secretion was measured in pg / ml / 1×106 cells / ml / 24 hours). The results are shown in Figure 31.

[0422] Microglia are CNS resident antigen presenting cells that can develop into pro-inflammatory factors in ALS. To our knowledge, the ability of manufactured human Th2 / TREG cells to suppress human microglial inflammation has not been previously reported. To address this issue, the human microglial cell line HMC3 was induced into a pro-inflammatory state by sequentially culturing in IFN-γ and LPS endotoxin. Figures 31A-31B As shown, the addition of RAPA-501Th2 / TREG cell products to proinflammatory microglia reduced the culture supernatant content of proinflammatory cytokines IL-6, IP-10, and IFN-γ. In this experiment, the observed immunosuppression occurred at a very low 1:40 ratio of TREG to inflammatory microglia in transwell dishes, indicating that RAPA-501 cells can reduce CNS inflammation in a contact-independent manner (as indicated by the transwell design) and with high potency (as indicated by the 1:40 ratio of TREG to microglia).

[0423] Example 26: ALS iT using a host conditioning platform of pentostatin, cyclophosphamide, and lamivudine REG Cell therapy.

[0424] Fig.32 The PC regimen and overall treatment approach are described in detail. The PC regimen will be administered in 2-week cycles, with a stepwise increase in the dose of pentostatin or cyclophosphamide in cycles 1 to 4, as indicated (total duration of the PC regimen is 8 weeks). Pentostatin will be administered on day 1 or day 1 and 4 of a 14-day cycle; cyclophosphamide (Cy) will be administered on days 1, 2, and 3 or day 1, 2, 3, 4, and 5 of a 14-day cycle. For cycle 4, if ALC is < 1250 cells per microliter, the Cy dose will be increased to 200 mg per day. After achieving immunodepletion and immunosuppression by administering the PC regimen, the first iT REG Cell infusion will be performed in week 8 of therapy. Lamivudine, an inflammasome inhibitor, will be administered continuously at a dose of 150 mg BID from week 8 to week 26 of the regimen.

[0425] Fig.33 Provides information about iT REG Additional details of cell manufacturing, which depicts lymphocyte collection by apheresis before and after PC treatment. Lymphocytes from ALS patients will be collected by steady-state apheresis (collection of 10 to 15 liters), which will be performed just before or after the PC regimen. Collection before the PC regimen is important for iT REG Production after a PC regimen may be more advantageous because the number of T cells found will be increased and will not be immunosuppressed; in contrast, collection after a PC regimen may be advantageous because of the risk of contamination of iT cells.REG The inflammatory Th1 / Tc1 cells in culture were depleted in vivo before production. REG Afterwards, the product will be cryopreserved at therapeutic doses to allow iT REG Repeated dosing of cells as indicated by infusions #2, #3, and #4.

[0426] Fig.34 Further details on iT REG The strategy of multiple cell infusions, which depicts the iT REG Sequencing of PC protocols before each administration in repeated administration of cells. REG The PC regimen is administered prior to infusion to (1) deplete and suppress inflammatory Th1 / Tc1 cells that contribute to disease pathogenesis; and (2) elevate levels of homeostatic cytokines (such as IL-7 and IL-15) that will allow the adoptively transferred iT cells to REG The PC regimen will consist of 2 mg / m on days 1 and 4. 2 dose of pentostatin and a fixed dose of cyclophosphamide 100 mg per day on days 1 to 5. After a two-day pause, iT REG cells (Day 8 of the protocol). From Week 8 onwards, the inflammasome inhibitor lamivudine will be continuously administered to limit iT REG Inflammatory drivers during cell therapy.

[0427] Fig.35 Provides information about using iT REG More details of monitoring of cell-treated patients, showing that ALS monitoring will be performed approximately monthly, both via the patient-reported ALSFRS-R and clinician-reported Appel scores, as indicated. As indicated, immunological laboratories for monitoring the inflammatory state of ALS patients will be assessed approximately monthly.

[0428] Example 27: Use of Selected Anti-TNF-α Agents Prior to Lymphocyte Collection by Apheresis to Beneficially Alter the Input T Cell TCR Repertoire

[0429] Figures 36A-36B showed that anti-TNF-α therapy with etanercept, which preferentially inhibits serum, cell-free forms of TNF-α that promote Th1-type cells expressing TNFR1, was associated with widespread changes in both up- and down-regulation of T cell receptors. Figures 36A-36B Describe the use of RNA-based T cell receptor sequencing to detect widespread upregulation and downregulation of T cell TCR specificity after treatment with the TNF-α inhibitor etanercept. Figures 36A-36BIn the present study, RNA was isolated from peripheral blood mononuclear cells of ALS patients before and after treatment with etanercept therapy. The RNA was subjected to TCR repertoire analysis 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): 61. Fig.36A As shown, it was demonstrated that approximately 25% of the TCR specificity was upregulated in the post-treatment samples (as indicated by red); in sharp contrast, approximately 25% of the TCR specificity was downregulated in the post-treatment samples (as indicated by blue). Fig.36B As shown, etanercept therapy produced significant T-cell clonal expansion, as the frequencies of several T-cell clones increased from 0.01 before etanercept (close to the detection limit of the assay) to values ​​of 247 to 486 after treatment, consistent with a greater than 4-log T-cell expansion. Fig.36B As shown in Figure 2, etanercept therapy produced a significant T cell clonal contraction, as several T cell clones decreased in frequency from 259 to 598 before etanercept to a value of 0.01 after treatment, consistent with a greater than 4-log contraction of T cell clones. These observations suggest that pretreatment of subjects with etanercept or any other anti-TNF-α therapeutic that preferentially inhibits serum, cell-free forms of TNF-α (e.g., the monoclonal antibody adalimumab) can be used to shift the T cell receptor repertoire away from T cells of a Th1 phenotype on the basis of antigen specificity, thereby enriching for T cells that are more likely to be resistant to TNF-α. REG Phenotype of T cells.

[0430] Example 28: T cells as a product enriched for the expression of CD25, CD27, 2B4, BTLA and CTLA4 REG - Characterization of Th2 hybrid population.

[0431] Fig.37 Flow cytometry analysis showed that hybrid T cells produced under the described conditions REG -Th2 cells have increased expression of the following cell surface molecules relative to control Th1 / Tc1 cells: CD25, CD27, 2B4, BTLA and CTLA4. Fig.37 In, iT REG The hybrid T / Th2 population was generated by the methods described in detail previously, using an initial phase of T cell dedifferentiation followed by hybrid T cell differentiation in the presence of IL-2, TGF-β, and IL-4.REG / Th2 medium; cells were harvested and flow cytometry was performed to assess the expression of relevant molecules (i.e., CD25, CD27, 2B4, BTLA, and CTLA4) + and CD8 + T cell expression; three independent control conditions assessing Th1 / Tc1 polarization were compared.

[0432] like Fig.37 As shown, compared with the control Th1 / Tc1 cells, iT REG The Th2 / Th2 hybrid cell product has CD4+ expressing levels of CD25, CD27, 2B4, BTLA and CTLA4 that are at least 10% higher and more preferably 50% higher. + and CD8 + T cells.

[0433] CD25, the IL-2 receptor, is important for T REG Cells control autoimmunity (especially CD8 + The ability of iT cells to drive responses is crucial. REG Expression of CD25 on cell products of Th2 / Th2 production is a desirable property.

[0434] CD27 (a REG Increased expression of co-stimulatory molecules on T cells has been shown to contribute to REG Therefore, in iT REG Expression of CD27 on cell products produced by Th2 / Th2 is a desirable property.

[0435] 2B4 (CD244) has recently been shown to inhibit CD8 by attenuating glycolysis and cell division. + T cell response. REG Expression of 2B4 on cell products produced by Th2 / Th2 is a desirable property.

[0436] BTLA (CD272) is a co-inhibitory receptor, and ligation of BTLA with the herpes virus entry mediator HVEM promotes T REG Therefore, in iT REG Expression of BTLA on cell products produced by Th2 / Th2 is a desirable property.

[0437] CTLA4 is T REG It is a key effector molecule of the mitochondrial cell, as it has recently been shown to have the ability to limit immunity to malarial infection. REG Expression of CTLA4 on cell products produced by Th2 / Th2 is a desirable property.

[0438] Example 29: T cells as products enriched for TIGIT, TIM3, ICOS, LAIR1 and OX40 expression REG - Characterization of Th2 hybrid population.

[0439] Fig.38 It was shown by flow cytometry analysis that hybrid TREG-Th2 cells made according to the described conditions had increased expression of the following cell surface molecules relative to control Th1 / Tc1 cells: TIGIT, TIM3, ICOS, LAIR1 and OX40. Fig.38 In, iT REG The Th2 / Th2 hybrid population was generated by the methods detailed previously, using an initial phase of T cell dedifferentiation followed by redifferentiation in medium containing IL-2, TGF-β, and IL-4. REG On day 11 of Th2 production, cells were harvested and flow cytometry was performed to assess the expression of CD4 TIGIT, TIM3, ICOS, LAIR1, and OX40. + and CD8 + T cell expression; three independent control conditions assessing Th1 / Tc1 polarization were compared.

[0440] like Fig.38 As shown, compared with the control Th1 / Tc1 cells, iT REG The Th2 / Th2 hybrid cell product has CD4+ and CD8+ T cells expressing at least 10% higher and more preferably 50% higher levels of TIGIT, TIM3, ICOS, LAIR1 and OX40.

[0441] TIGIT is a cell surface co-inhibitory receptor molecule that is associated with regulatory T cell function and contributes to the immunosuppressive environment in B-cell non-Hodgkin lymphoma. REG Expression of TIGIT on Th2 / Th2-producing cell products is a desirable property.

[0442] TIM3 is a co-inhibitory receptor that mediates T REG The inhibitory effect of iT cells includes, for example, the inhibition of T cells infiltrating head and neck squamous cell carcinoma. REG Expression of TIM3 on cell products produced by Th2 / Th2 is a desirable property.

[0443] ICOS is a co-stimulatory molecule that has recently been identified to contribute to the maintenance of immunosuppression by regulatory T cells to control immune responses in the central nervous system. REG Expression of ICOS on Th2 / Th2-producing cell products is a desirable property.

[0444] LAIR1 (CD305) is a multifaceted inhibitory molecule that blocks inflammation at multiple steps, including inhibiting activated effector memory T cells. REG Expression of LAIR1 on cell products produced by Th2 / Th2 is a desirable property.

[0445] OX40 is a co-stimulatory molecule. REG Expression of OX40 on Th2 / Th2-producing cell products is a desirable property.

[0446] At the end of the culture, the T REG Most phenotypic characterizations of T cell products made using the Th1 / Tc1 approach are limited to the phenotypic characterization of T cell products made using the Th1 / Tc2 approach. However, it is important to note that T cell products can be cryopreserved and, as such, the phenotypic characteristics of T cells in the post-thaw state reflect the actual product that will be adoptively transferred into a subject. REG Th2 / Th2 cells can be characterized by: (a) increased expression of CD25, CD27, 2B4, BTLA, CTLA4, TIGIT, TIM3, ICOS, LAIR1, and OX40 by flow cytometry; (b) decreased IFN-g and TNF-α, but increased secretion of IL-4 by Luminex cytokine secretion analysis; and (c) altered expression of T cell fate transcription factors, i.e., decreased TBET and increased FOXP3 and GATA3.

[0447] To generate iT REG Apheresis

[0448] Prior to treatment with the pentostatin / cyclophosphamide regimen, subjects will undergo a lymphocyte apheresis procedure. The purpose of collecting these peripheral lymphocytes is to create iT cells for adoptive T cell therapy. REG cell.

[0449] The apheresis will consist of a 10 to 15 liter collection performed on a CS-3000 or equivalent machine. The apheresis product will be sent to the protocol sponsor, Rapa Therapeutics, and iT REG The cells will be produced by in vitro culture using specific culture conditions.

[0450] Purpose

[0451] Main purpose. To determine the iT in the context of PC solutions REG Safety of cell infusion and maintenance lamivudine therapy in inflammatory subgroups of ALS patients.

[0452] Secondary purpose. Determine iTREG Therapy can suppress inflammatory markers in ALS patients. REG Effects of therapy on patient-reported and clinician-reported ALS scores.

[0453] Eligibility criteria.

[0454] Subjects diagnosed with laboratory-supported suspected, probable, or definite sporadic or familial ALS according to the World Federation of Neurology El Escorial Criteria. Age >18 years and less than or equal to 75 years. Karnofsky performance status of 70% or greater. Ejection fraction obtained by MUGA or 2-D echocardiography within tissue normal limits. Serum creatinine less than or equal to 2.0 mg / dl. AST and ALT less than or equal to 3 times the upper limit of normal. Bilirubin less than or equal to 1.5 (unless due to Gilbert's disease). Corrected DLCO greater than or equal to 50% on pulmonary function testing.

[0455] To evaluate the secondary study endpoints, patients must have evidence of inflammatory markers in peripheral blood cell populations after evaluating at least two independent blood samples during the screening interval. Assays for evaluating the inflammatory state of potential patients will include: flow cytometry; cytokine secretion analysis; and cell signaling events analyzed by Western blot. Other tests may include tests of T cell receptor lineages or in vitro sensitization tests for potential autoantigens (such as motor neuron proteins or ALS-related protein aggregates). Cytokine secretion is assessed in the absence of stimulation (autonomous cytokine secretion) and various forms of stimulation, including but not limited to: anti-CD3 / anti-CD28 co-stimulation; LPS endotoxin exposure; CD40 ligand exposure; adenosine A2a and A3 receptor agonism and antagonism; T cell (PD1, TIM-3) and monocyte (CD47, CD200) checkpoint inhibition; T cell receptor clonality is assessed by RNA sequencing technology. The decision as to whether a potential subject has sufficient inflammation to warrant inclusion in the study will be based on a matrix analysis of all of these tests and will be made by the study PI or Principal Investigator in consultation with the Medical Director of the Rapa Therapeutics Laboratory, which performs the immunoassays.

[0456] Exclusion criteria included patients actively taking riluzole Edaravone therapeutic agents (unless on a stable dose for more than one month). The following will also represent exclusion criteria: receipt of any investigational intervention within 30 days of the protocol; spirometry <60% of expected values; active uncontrolled infection; hypertension not adequately controlled with 3 or fewer medications; history of cerebrovascular accident within 6 months of enrollment; history of pulmonary embolism within 6 months of enrollment; or clinically significant cardiac pathology (defined as myocardial infarction within 6 months prior to enrollment; grade III or IV heart failure according to NYHY; uncontrolled angina; severe uncontrolled ventricular arrhythmias; ECG evidence of acute ischemia or active conduction system abnormalities). Patients with a history of coronary artery bypass grafting or angioplasty will receive a cardiology evaluation and will be considered on a case-by-case basis. Patients seropositive for HIV, hepatitis B, or hepatitis C will be excluded. Patients with known or discovered pregnancy and patients of childbearing age who do not wish to use contraception will be excluded. Patients may be excluded at the PI's discretion or if it is determined that allowing participation would constitute an unacceptable medical or psychiatric risk.

[0457] Treat ALS patients with PC regimen.

[0458] The purpose of the 8-week PC regimen was to cause partial depletion and inhibition of Th1 / Tc1 cells that contribute to the pathogenesis of ALS disease. In addition, the PC regimen was designed to acutely produce T cell homeostatic cytokines (particularly IL-7 and IL-15) by infusing Hreg cells at week 8 and immediately after the subsequent PC regimen.

[0459] The PC regimen will be administered in 14-day cycles; however, a delay of up to two weeks between cycles will be allowed if dysfunction occurs or additional time is required to evaluate and / or treat any adverse events. For cycle #1, pentostatin (1 mg / m IV on day 1) 2 Cycle #2 (which may be used as long as no dose-limiting toxicity occurs) will include an increased dose of pentostatin (2 mg / m2 IV on day 1) in combination with the same dose of cyclophosphamide (100 mg / day orally on days 1, 2, and 3). 2 Cycles #3 and #4 (which may be used as long as dose-limiting toxicity does not occur and the absolute lymphocyte count is greater than 750 cells per microliter) will consist of 2 doses of pentostatin (2 mg / m2 IV on days 1 and 4) combined with 5 days of cyclophosphamide (100 mg orally daily on days 1, 2, 3, 4, and 5) 2 ).

[0460] If the ALC count is 750 cells per microliter or less before cycle #3 or #4, no further cycles will be administered and the patient will continue on lamivudine for maintenance therapy. If the absolute lymphocyte count is greater than 1250 cells per microliter before cycle #4, the dose of cyclophosphamide will be doubled (200 mg orally daily on days 1, 2, 3, 4, and 5).

[0461] Details related to pentostatin administration: (a) Preparation: The pharmacy will reconstitute the pentostatin concentration to 2 mg / ml according to the vial instructions. Then the appropriate patient-specific dose will be added to 0.9% sodium chloride to make a total volume of 50 mL; (b) Dose and administration: The pentostatin dosing will be adjusted for renal insufficiency (see below); each dose of pentostatin will be administered intravenously over 30 - 60 minutes; (c) Prescription and antiemetic therapy: Before the infusion, 1 liter of 0.9% sodium chloride will be infused over 30 - 60 minutes. Pentostatin can cause vomiting. The antiemetic protocol guidelines are as follows (the PI may decide whether to allow changes at their discretion): (1) Intravenous infusion of 12 mg of dexamethasone 60 minutes before each dose of pentostatin; (2) Dexamethasone may be taken orally for the first five days of each cycle if needed to control vomiting; (3) Intravenous infusion of 8 mg of ondansetron 60 minutes before each dose of pentostatin; (4) For the remainder of the treatment, ondansetron may be taken orally as prescribed every 12 hours at a dose of 8 mg (tablets) from day 1 to day 14; (5) For patients with uncontrolled nausea and vomiting, aprepitant may be added to the antiemetic protocol as needed.

[0462] Details related to pentostatin dose reduction: Serum creatinine levels will be obtained before calculating each scheduled dose of pentostatin and CrCl. CrCl can be obtained from urine over 24 hours or calculated using the Cockcroft - Gault formula. If the creatinine level increases during pentostatin and cyclophosphamide treatment in the subject, subsequent dosing will be modified as follows: When CrCl > 60 (mL / min / 1.73 m2): Administer 100% of the expected pentostatin dose (for cycles #1, #2, 1 mg / m 2 of pentostatin; for cycles #3, #4, the dose is 2 mg / m 2 ); When 30 < CrCl < 60: Administer 50% of the expected pentostatin dose (for cycles #1, #2, 0.5 mg / m 2 of pentostatin; for cycles #3, #4, 1 mg / m 2 of pentostatin); When CrCl < 30: Continue pentostatin use.

[0463] Since pentostatin has rarely been associated with neurologic toxicity (seizures, coma), particular attention should be paid to CNS evaluation. If the PC regimen is associated with any new neurological toxicity of grade 2 or greater severity or worsening of any pre-existing neurological toxicity, the institutional PI should be contacted to discuss the need for approval of further pentostatin therapy and further protocol treatment.

[0464] Specific Aspects of Cyclophosphamide Administration: Hydration. Because cyclophosphamide can cause cystitis, it is important for patients to stay well hydrated. Patients should drink at least 2 to 4 liters of fluid per day to keep the color of their urine clear. It is also particularly important to empty the bladder before going to bed. Oral cyclophosphamide will be administered at a fixed dose of 100 mg per day on Days 1, 2, and 3 (for Cycle #1 and Cycle #2) or Days 1, 2, 3, 4, and 5 (Cycle #3 and Cycle #4). However, for patients who do not have a significant decrease in ALC prior to Cycle #4 (defined as ALC greater than 1250 cells per microliter), the dose of cyclophosphamide for Cycle #4 will be increased to 200 mg per day on Days 1, 2, 3, 4, and 5. If patients cannot tolerate oral therapy, intravenous infusions of cyclophosphamide are permitted; the intravenous infusion dose will be the same as the intended oral dose. For intravenous infusions, cyclophosphamide will be reconstituted to a concentration of 20 mg / ml by the HUMC pharmacy according to the bottle instructions. The appropriate dose (100 mg or 200 mg) is then diluted in 100 ml of D5W or 0.9% sodium chloride and infused intravenously over 30 minutes.

[0465] It is not expected that a PC cycle will result in a large decrease in the absolute neutrophil count. However, if the ANC is below certain values ​​in the assay before the next cycle, the dose of cyclophosphamide will be adjusted as follows: (1) for ANC values ​​of 1000 cells per microliter or more, 100% of the expected dose will be administered; (2) for ANC values ​​between 500 and 999 cells per microliter, 50% of the expected dose will be administered; and (3) for ANC values ​​less than 500 cells per microliter, cyclophosphamide will not be administered. In addition, the PI may consider the decision to start G-CSF therapy in cases where the ANC value is less than 500 cells per microliter.

[0466] The quantitative goal of the 8-week PC regimen is to reduce the ALC value to approximately 750 cells per microliter. It is hypothesized that this degree of depletion and suppression of T cells that contribute to disease pathogenesis will allow iT REG The cells were successfully engrafted and biologically active to control the neuroinflammatory process. However, a more stringent reduction of host Th1 / Tc1 cells may be required to allow iT reg cells exert their full inhibitory effect; in this case, the PC regimen can be intensified or prolonged to REGPrior to cell therapy, lower ALC values ​​were targeted, such as 500, 250, or 0 ALC per microliter. REG Cell therapies may be so potent that even an ALC value of 750 cells per microliter may be considered too stringent. In these cases, the intensity of the PC regimen may be reduced or the duration of treatment shortened to target higher ALC values, such as 1000, 1250, or 1500 cells per microliter.

[0467] Practice related to lamivudine maintenance therapy.

[0468] After completion of the PC regimen, patients will continue to receive maintenance therapy with lamivudine, which will continue until the end of the study, which is month 6 of the regimen. Lamivudine (oral tablets) will be administered at a dose of 150 mg twice daily. In the event that the estimated creatinine clearance decreases below 50 ml / min, the dose of lamivudine will be reduced to 150 mg orally once daily. Lamivudine will be discontinued when the estimated creatinine clearance is less than 30 ml / min.

[0469] As previously mentioned, the established target of lamivudine is to downregulate the NLRP3 inflammasome, which represents the proximal event in the pathogenesis of ALS. Therefore, it is envisioned that other inflammasome inhibitors would be suitable or may be more suitable for use in our regimen platform; for example, inflammasome inhibitors with potentially improved risk:benefit ratios have been developed:

[0470] It is important to note that the predicted effect of lamivudine on iT REG Cell therapies have antagonistic effects because their mechanisms of action are actually complementary. This complementarity is consistent with targeting T REG This is in stark contrast to other proposed interventions for cell therapy, such as rapamycin, which inhibits a variety of T cell responses, and IL-2, which promotes T cell responses in vivo. REG have a narrow therapeutic window in terms of expansion and can elevate inflammatory T cell populations).

[0471] Supportive care treatment.

[0472] If neutropenia develops, patients do not need to be on systemic antibiotic prophylaxis. The decision to initiate antibiotics will depend on the regimen PI.

[0473] All patients will be assigned to oral acyclovir (or its prodrug valacyclovir) for antiviral prophylaxis against HSV or VZV at the end of the study and when protocol treatment is initiated.

[0474] All patients will be placed on oral antifungal prophylaxis (first-line antimicrobial: fluconazole) at the end-of-study visit and when protocol therapy is initiated. Substitutions will be permitted with approval from the protocol PI.

[0475] All patients will begin Pneumocystis jiroveci prophylaxis upon study entry. All patients will be scheduled for oral sulfamethoxazole (160 mg of trimethoprim / 800 mg of sulfamethoxazole): one tablet orally on Monday, Wednesday, and Friday; substitutions or alternative regimens will be permitted with approval of the protocol PI.

[0476] Using iT REG Cell therapy for ALS patients: manufacturing and phenotype of cell products.

[0477] As detailed above, iT REG The cell product will be made from autologous T cells that can be collected by apheresis either before the PC regimen or at the end of the 8-week PC regimen prior to study entry. Each apheresis collection has inherent advantages: the initial collection will have a higher T cell yield, while the collection after the PC regimen will include a relatively depleted T cell population of Th1 / Tc1 cells.

[0478] Because it reg The cells are based on the change of effector T cells from inflammatory phenotype to anti-inflammatory T REG The principle of phenotypic transformation is adopted, so there is no need for expensive and laborious natural T REG Purification steps that require the use of monoclonal antibodies / column selection methods or flow cytometry to obtain CD4 + 、CD25 + The relatively rare nTREG population characterized by low expression of CD127 is also characterized by low expression of CD8 + T REG Cells can mediate immunosuppression and play a role in iT REG Cell therapy may be beneficial in terms of providing increased diversity, thus eliminating the need for CD8 + T cells from iT REG Elimination of cell populations.

[0479] It has been demonstrated that T cells can be defined as a central memory type of limited differentiation state based on the expression of cell surface markers such as CD62L and CCR7. REG cells, with increased in vivo regulatory functions. On the other hand, it is well known that after T cells acquire a more differentiated effector memory state, molecules that mediate inhibitory functions are upregulated, such as: IL-10; CTLA-4; ectonucleotidase molecules CD39 and CD73; cytolytic molecules such as perforin and fas ligand. These data suggest that infusion of iT cells containing both central memory and effector memory subsets may be beneficial. REG Cell products will be beneficial, and therefore, the iT REG The cell product will have signatures from both subpopulations.

[0480] In addition, iT REG The cells must express FoxP3, a transcription factor that determines the differentiation program of regulatory T cells. Furthermore, since FoxP3 expression and the resulting regulatory function have been shown to degenerate over time, iT REG The cell product must have stable FoxP3 expression over a long period of time in culture.

[0481] Furthermore, it has been demonstrated that FoxP3 alone in humans is insufficient to identify regulatory T cell phenotypes, as it can be transiently expressed by bona fide inflammatory T cell subsets. Therefore, it would be desirable to generate iT cells that express FoxP3 but relatively lack co-expression of molecules associated with inflammatory T cell subsets. REG Cell products are crucial, such as the Th1 / Tc1-type transcription factor TBET or the Th1-type cytokines IL-2 or IFN-γ.

[0482] Finally, it is important that iT REG The plasticity of cells to differentiate from a regulatory phenotype to an inflammatory phenotype is reduced. That is, it has been well documented that the suppressive phenotype of Treg cells may be relatively unstable, which may lead to the transformation into inflammatory T cell subsets that actually contribute to mediating neurodegenerative diseases. REG The cell product must stably express FoxP3 and also show a reduced ability to switch to the Th1 / Tc1 subset. REG As an additional preventive measure for cell dedifferentiation plasticity towards Th1-type subsets, IL-4 was purposefully added to iT cells. REG The cells are produced so that any such differentiation will be towards the Th2 lineage, which is important for T REG Cell maintenance and T REG The function of cytosuppressors appears to be important; they have been described as REG The default pathway for regulatory T cells; it may mediate anti-inflammatory effects in the setting of ALS. Although there is evidence that the Th2-like state of regulatory T cells has a potential beneficial effect, the use of REG Manufacturing methods for cell therapies have not yet included the purposeful addition of exogenous IL-4 during culture (as demonstrated in the recent Treg manufacturing example).

[0483] At the end of manufacturing, iT REG The cell product was divided into at least four equal portions of individual therapeutic cell doses and cryopreserved (1×10 per kg). 6 Up to 5×10 6 cells).

[0484] Using iT REG Cell therapy for ALS patients: T REGCombination of cell populations.

[0485] iT REG The infusion dose of the cell population was 1×10 per kg of recipient body weight. 6 to 5×106 cells. This is a relatively low T cell number compared with previous studies. REG The dosage of cell therapy is determined by several factors: PC regimen will be iT REG Implantation of cells provides sufficient immune space; iT REG Cells will express a memory profile associated with cell persistence after adoptive transfer; iT REG The cell product is divided into at least four equal portions into clinically relevant therapeutic doses, thereby permitting multiple treatment cycles.

[0486] As mentioned earlier, iT REG The cell product will contain multiple memory differentiation states (central memory [CM] plus effector memory [EM]), which can respectively control neuroinflammation long-term or transiently. It will be possible to control the ratio of these central memory populations to effector memory populations to obtain the most ideal results according to the clinical situation; that is, the ratio of CM cells:EM cells to T cells based on clinical parameters. REG The distribution can be 1:1, 3:1, 10:1, 1:3 or 1:10.

[0487] It will be possible to control CD4 in a similar manner, depending on the clinical situation. + iTREG cells:CD8 + iT REG ratio of cells to achieve improved therapeutic effects.

[0488] Finally, because of iT REG Cells and nT REG Since cells express different T cell receptor lineages and can complement each other in mediating immunosuppression, it is conceivable that co-administration of nT REG Cells were obtained using iT REG The best cell therapy.

[0489] Using iT REG Cell therapy for ALS patients: combination with pharmacological agents

[0490] It is possible that when used in combination with a platform that includes the immunomodulatory effects of a PC regimen and the inflammasome inhibitory effects of lamivudine, iT REG Therapy may be enough to control neuroinflammation.

[0491] However, it is conceivable that iT can be optimized by changing the platform REGCell therapy. For example, and not limitation, treatment can be optimized by changing the intensity of the PC regimen; replacing cyclophosphamide with another agent that works synergistically with pentostatin; or adding a third component to the PC regimen, such as low-dose IL-2 therapy after anti-TNF therapy, which is inferred to predictably increase T cells in vivo. REG Cells. By way of example and not limitation, low-dose IL-2 therapy as described in Pham MN, von Herrath MG, Vela JL. "Antigen-Specific Regulatory T Cells and Low Dose of IL-2 in Treatment of Type 1 Diabetes". Frontiers in Immunology. 2015; 6: 651.

[0492] Furthermore, it is foreseen that lamivudine may be replaced by more potent or specific inflammasome inhibitors similar to recently synthesized molecules.

[0493] Ultimately, more proximal events drive the inflammation that drives ALS, such as the accumulation of misfolded RNA elements and insufficient autophagy. In this context, it is reasonable to use drugs that promote autophagy to treat ALS, especially rapamycin. However, clinical trials of rapamycin for the treatment of ALS have just begun (ClinicalTrials.gov Identifier: NCT03359538); in addition, the program is evaluating continuous treatment with rapamycin (which may cause severe toxicity), fixed doses of rapamycin (which may result in a large degree of drug variability between patients), and relatively small doses of rapamycin (which will not ensure the high drug levels necessary to effectively inhibit the mTOR pathway and therefore promote autophagy). To circumvent these limitations, iT was used to treat ALS using the following parameters: REG Combining cell therapy with rapamycin to promote autophagy: Using intermittent rapamycin therapy to limit drug toxicity and restrict the effects of rapamycin on iT REGThe invention relates to the inhibition potential of cells (for example, but not limitation, one week of mTOR inhibition treatment, followed by three weeks of mTOR treatment followed by recovery); using variable doses of rapamycin (including a loading dose of rapamycin), combined with serum monitoring of rapamycin levels to ensure uniform drug levels, to more consistently inhibit the mTOR pathway; and using high-dose rapamycin treatment to achieve rapamycin serum levels of 30 ng / ml, which is better than the typical target value of about 5 to 12 ng / ml. See Mossoba ME, Halverson DC, Kurlander R et al. "High-Dose Sirolimus And Immune Selective Pentostatin Plus Cyclophosphamide Conditioning Yields Stable Mixed Chimerism and Insufficient Graft-Versus-Tumor Responses". Clinical Cancer Research 2015; 21(19): 4312-4320.

[0494] Furthermore, rapamycin treatment may not be sufficient to promote autophagy in neurodegenerative cells due to the drug's inability to adequately penetrate the CNS; to date, even intravenous treatment with the rapamycin analog temsirolimus has not resulted in significant drug levels in the CSF. To overcome this limitation, we envision that we would administer temsirolimus via an indwelling Ommaya reservoir (in a manner similar to treatments for lysosomal storage disorders) to achieve steady CSF drug levels of the mTOR inhibitor, thereby optimally promoting autophagy in the setting of neurodegenerative disease.

[0495] Using iT REG Cell therapy for ALS patients: immune monitoring.

[0496] iT at ALS REG In the context of cell therapy, it will be important to quantify the success of cell therapy in terms of its ability to modulate disease-associated neuroinflammatory pathways. That said, monitoring the clinical course of neurodegenerative diseases is not sufficient, as there is a large degree of variability in disease progression across patient groups: multiple infusions of iT cells may be required to achieve optimal ability to treat neuroinflammation. REG cells, which include combined use with multiple pharmacological agents; therefore, the use of immune biomarkers to help guide treatment decisions will be critical.

[0497] About iTREG Treatment decisions for cell infusion and repeated administration of relevant pharmacological agents will be based on specialized testing of cultured peripheral blood mononuclear cells. These tests address several key issues related to inflammation monitoring, including: spontaneous cytokine measurement; cooperation between T cells and monocytes in cytokine measurement; the role of recombinant human CD40 ligand, T cell checkpoint inhibitor pathways, and monocyte checkpoint pathways in revealing cytokine secretion; assessment of inflammasome activation by multiple techniques, such as quantification of proteins by Western blotting; evaluation of adenosine receptor biology on peripheral T cells as an indicator of inflammatory events; assessment of co-expression of FoxP3 transcription factor with Th1-associated molecules TBET, IL-2, or IFN-γ using flow cytometry; characterization of T cell receptor repertoires by RNA sequencing; and detection of antigen-specific T cell responses against potential nervous system autoantigens, such as protein aggregates generated during disease pathogenesis.

[0498] Program evaluation.

[0499] Clinical evaluations by a physician or mid-level healthcare provider will occur on day 1 of each cycle of the PC regimen, which is scheduled for 14 days. Patients will also be seen by their local healthcare provider once more during the 14-day PC regimen (ideally around day 8 of the cycle). At these visits, a CBC with differential and a complete metabolic panel (a complete metabolic panel typically includes approximately 14 tests, including electrolytes, creatinine, hepatic transaminases, and bilirubin; the specific panel used is not specified by the protocol) will be obtained, and laboratory results will be sent to the protocol investigators.

[0500] Upon completion of the interval covering the PC regimen (approximately Month 2), patients will be seen monthly at Months 3, 4, 5, and 6; the Month 6 visit will represent the end-of-study visit. Tests to be performed at the time of these clinical evaluations include: (1) interim history and physical examination; (2) complete blood count (CBC) with differential and platelet count; (3) complete metabolic panel; and (4) immune subset enumeration (TBNK panel).

[0501] To monitor the immune parameters of the study, peripheral blood samples will be sent to Rapa Therapeutics for centralized monitoring and more in-depth analysis. Blood samples will consist of 30ml in green top heparinized tubes (for cell analysis) and 5ml in red top tubes (for serum analysis) sent to Rapa Therapeutics.

[0502] Using the same samples sent to Rapa Therapeutics, the effects of the PC regimen and lamivudine maintenance therapy will be studied on pro- or anti-inflammatory cytokines or cell subsets as measured by RNA expression, supernatant / Luminex analysis, flow cytometry, and cellular signaling events as measured by phosphorylation analysis by western blot.

[0503] Potential biomarkers for ALS in serum will be assessed. By way of example and not limitation, biomarkers such as those described in: Beach TG. "A Review of Biomarkers for Neurodegenerative Disease: Will They Swing Us Across the Valley?" Neurology and Therapy. 2017; 6(Suppl 1): 5-13.

[0504] The patient's TCR repertoire will be characterized and whether therapeutic interventions affect this repertoire will be assessed.

[0505] In vitro studies will fall under the general category of “immune characterization studies.” They will focus on isolating different cell subsets by multiparameter FACS analysis or on separation by magnetic beads with subsequent characterization. Specifically, peripheral blood mononuclear cells (PBMCs) will be analyzed by flow cytometry for the expression of markers indicative of hematopoietic lineages, immune functional subsets, cytokine production, and activation status. Cell subsets will be analyzed for T cell receptor repertoire diversity. Cells can be activated in vitro with many different stimuli, including specific antigens and mitogens, which are known to activate different pathways of T lymphocyte or monocyte function. Analysis may include T cell proliferation, cytokine production, and gene expression. During the course of the study, as technology and knowledge in the field develop, the specific assays used for the ongoing data analysis may be modified, deleted, or replaced without changing the purpose of the study.

[0506] Response standards.

[0507] Patient-reported ALSFS-R scores and clinician-reported Appel scores will be measured at various time points as previously indicated.

[0508] Toxicity Standard

[0509] Toxicity will be graded according to the NCI Common Terminology Criteria for Adverse Events (CTCAE) (available at http: / / ctep.info.nih.gov). A copy of CTCAE version 4.0 may be downloaded from the CTEP homepage. A copy of CTCAE version 4.0 should be provided to all therapeutic areas and personnel involved in the study.

[0510] Any grade 4 or 5 toxicity (CTCAE version 4.0) attributed possibly or definitely to study drug (pentostatin, cyclophosphamide, lamivudine) will be considered a dose-limiting toxicity (DLT). The following toxicities will not be considered DLTs: biochemical grade 4 toxicity (except renal and liver values); grade 4 vomiting; grade 4 fever; grade 4 toxicity related to infection that resolves within 7 days.

[0511] Statistical considerations.

[0512] The study design cites the standard 3+3 approach to evaluate the safety of pentostatin and cyclophosphamide therapy and maintenance lamivudine therapy (platform). In the first three patients, if no patient develops DLT through pentostatin / cyclophosphamide therapy, it will be determined that the regimen is safe for expanding the group to a total of n=10 patients. On the other hand, if one-third of the first patients develop DLT, the accrued number will increase to a total of n=6 patients. In this case, if no more than 1 of the first 6 patients develops DLT, the number of n=10 in the group can continue to increase.

[0513] Once the platform is successfully developed, the iT REG Initially, a pilot study will be performed to evaluate the safety and potential efficacy of multiple infusions of iT REG The cells' ability to inhibit disease-associated neuroinflammatory pathways.

[0514] Once the ability to effectively modulate biomarkers associated with neurodegeneration is documented, a Phase II clinical trial (using either historical control data or a randomized cohort design) will be performed to evaluate the efficacy of iT REG Whether cell therapy can improve clinical outcomes in patients with ALS.

[0515] Risk / Benefit Analysis.

[0516] The estimated survival of patients enrolled in this study is approximately 2 to 4 years from the start of the study.

[0517] The first component of the protocol consists of four cycles of immunodepleting and immunosuppressive therapy (PC regimen), which includes pentostatin and cyclophosphamide. It is hypothesized that PC regimen will eliminate and suppress pathogenic immune cells that contribute to ALS progression; therefore, patients may benefit from this effect to improve quality of life or ultimately reduce disease progression. However, unexpected toxicities may occur with PC regimen with respect to the central nervous system. Although dosing is being modified to help ensure the relative safety of PC regimen in this new ALS patient population, PC regimen may have paradoxical effects and actually increase the rate of ALS progression or cause other neurotoxicity. Pentostatin can also be toxic to other organs, such as the heart or kidneys, in rare cases. Although this effect is part of the treatment rationale, it is expected that the most common toxic effect of PC regimen will be lymphocyte depletion. On the other hand, PC regimen may eliminate bone marrow cells, thereby increasing the likelihood of bacterial or fungal infections. PC regimen is expected to be associated with T cell immunosuppression, and therefore, opportunistic viral infections may occur.

[0518] The second regimen component includes maintenance therapy with the antiviral drug lamivudine. If the drug reduces inflammation in the central nervous system, as hypothesized, patients may benefit from this treatment. Aside from major gastrointestinal side effects and pancreatitis, lamivudine is generally a very well tolerated drug.

[0519] The third program component includes iT REG Patients may benefit from this therapy (cell therapy to control inflammation occurs directly in the microenvironment that triggers inflammation) because cell therapy works through multiple molecular mechanisms of action that cannot be easily replicated by drug therapy and because the effects of cell therapy can be long-lasting due to memory cell effects.

[0520] Alternative design solutions

[0521] Fig.39 An alternative protocol design is provided. Lymphocytes will be collected by steady-state apheresis; the apheresis product will be shipped to Rapa Therapeutics (Rockville, MD). After manufacturing RAPA-501 cells, n=4 doses of RAPA-501 cells will be cryopreserved in single-use infusion bags at the clinically indicated cell dose. The treatment interval will be 6 months, followed by a 6-month observation interval. Cohort #1 will receive RAPA-501 cells and will be infused with 4×10 6Cohort #1 represents the safety cohort and will follow a standard 3+3 design; if dose-limiting toxicity (DLT) occurs in 0 / 3 or no more than 1 / 6 patients, they will enter cohort #2. Cohort #2 will receive four cycles of RAPA-501 cells identical to the study protocol discussed previously, except that the T cell dose will be increased to 120×10 per infusion. 6 Cohort #3 will evaluate the maximum dose of RAPA-501 cells that can be safely administered as a single agent (40×10 cells per infusion, as per Cohort #1 or Cohort #2, respectively). 6 cells or 120×10 6 cells), and host conditioning with a PC regimen prior to each of the four RAPA-501 cell infusions. The PC regimen will include pentostatin (2 mg / m on days 1 and 4) 2 ), cyclophosphamide (100 mg per day, days 1 to 5), no treatment on days 6 and 7, and RAPA-501 cell infusion on day 8.

[0522] Dedifferentiation Example:

[0523] 1. A method for dedifferentiation of T cells, the method comprising:

[0524] inoculating a culture input cell population comprising T cells from a subject at a cell density in a medium comprising vitamin D, temsirolimus, and an IL-2 signaling inhibitor;

[0525] adding anti-CD3 / anti-CD28 coated magnetic beads to the T cells and culture medium at a beads:T cell ratio of 1:1 to 1:12;

[0526] The culture input cell population and culture medium are incubated for a period of time to produce dedifferentiated T cells.

[0527] 2. The method according to embodiment 1 further comprises: harvesting the dedifferentiated T cells.

[0528] 3. The method according to embodiment 2, further comprising, after harvesting the dedifferentiated T cells:

[0529] packaging at least a portion of the dedifferentiated T cells in a package; and

[0530] Freezing said package containing said portion of said dedifferentiated T cells.

[0531] 4. The method of any one of embodiments 1 to 3, further comprising, before inoculating the culture input cell population into the culture medium:

[0532] The culture input cell population is harvested from the subject.

[0533] 5. The method of any one of embodiments 1 to 4, wherein the culture medium does not contain IL-2 and no IL-2 is added to the culture medium.

[0534] 6. The method of any one of embodiments 1 to 5, wherein the cell density is at least 1.5 x 106 T cells per mL.

[0535] 7. The method of any one of embodiments 1 to 6, wherein said temsirolimus is present in said culture medium at a concentration of about 0.3 μM to about 1 μM.

[0536] 8. The method of any one of embodiments 1 to 6, wherein said temsirolimus is present in said culture medium at a concentration of about 1 μM.

[0537] 9. The method of any one of embodiments 1 to 8, wherein the IL-2 signaling inhibitor is an anti-IL-2 receptor antibody or a fragment thereof.

[0538] 10. The method of embodiment 9, wherein the IL-2 signaling inhibitor is basiliximab or daclizumab.

[0539] 11. The method of any one of embodiments 1 to 10, wherein the IL-2 signaling inhibitor is present in the culture medium at a concentration of 5 μg / mL to 50 μg / mL.

[0540] 12. The method of any one of embodiments 1 to 11, wherein the period of time is about 3 days.

[0541] 13. The method of any one of embodiments 1 to 12, wherein the bead:T cell ratio is 1:3.

[0542] 14. The method of any one of embodiments 1 to 13, wherein the culture medium further comprises 5% human serum.

[0543] 15. The method of any one of embodiments 1 to 14, wherein the culture medium comprises X-Vivo 20 medium.

[0544] 16. The method of any one of embodiments 1 to 15, wherein said vitamin D is present in said culture medium at about 0.03 nM to about 1 nM.

[0545] 17. The method of any one of embodiments 1 to 15, wherein said vitamin D is present in said culture medium at about 0.1 nM.

[0546] 18. The method of any one of embodiments 1 to 11 and 13 to 17, further comprising:

[0547] measuring the expression level of RAPTOR or RICTOR in the culture input cell population,

[0548] wherein the time period lasts until the expression level of RAPTOR or RICTOR in the culture input cell population is reduced by at least 50%, and more preferably by 90%, relative to a control T cell population, wherein the control T cell population is produced under the same conditions as the culture input cell population in the absence of temsirolimus, an IL-2 signaling inhibitor and vitamin D.

[0549] 19. The method of any one of embodiments 1 to 11 and 13 to 17, further comprising:

[0550] measuring the expression levels of RAPTOR or RICTOR and a housekeeping protein in the culture input cell population,

[0551] wherein the time period lasts until the expression level of RAPTOR or RICTOR in the culture input cell population is reduced by at least 50% relative to a control T cell population, respectively, after normalization to the expression level of a housekeeping protein, wherein the control T cell population is produced under the same conditions as the culture input cell population in the absence of temsirolimus, an IL-2 signaling inhibitor and vitamin D.

[0552] 20. The method of embodiment 19, wherein the housekeeping protein is actin or GAPDH.

[0553] 21. The method according to any one of embodiments 18 to 20, wherein said step of measuring said expression level is performed by Western blot analysis.

[0554] 22. The method of any one of embodiments 1 to 11 and 13 to 17, further comprising:

[0555] measuring the expression level of RAPTOR or RICTOR in the culture input cell population,

[0556] wherein the time period lasts until the expression level of RAPTOR or RICTOR in the culture input cell population is reduced by at least 50%, and more preferably by 90%, relative to a control T cell population, wherein the control T cell population is produced under the same conditions as the culture input cell population in the absence of temsirolimus, an IL-2 signaling inhibitor and vitamin D.

[0557] 23. A dedifferentiated T cell produced by the method according to any one of embodiments 1 to 22.

[0558] 24. A composition comprising a dedifferentiated T cell population,

[0559] wherein at least a portion of the dedifferentiated T cell population expresses less than 50% RAPTOR or RICTOR compared to a control T cell population, wherein the control T cell population is produced under the same conditions as the culture input cell population in the absence of temsirolimus, an IL-2 signaling inhibitor, and vitamin D.

[0560] 25. A method for dedifferentiation of T cells, the method comprising:

[0561] inoculating a culture input cell population comprising T cells from a subject at a cell density in a medium comprising vitamin D and temsirolimus;

[0562] adding anti-CD3 / anti-CD28 coated magnetic beads to the T cells and culture medium at a beads:T cell ratio of 1:1 or less to stimulate the T cells;

[0563] The culture input cell population and culture medium are incubated for a period of time to produce dedifferentiated T cells.

[0564] 26. The method of embodiment 25, further comprising:

[0565] The dedifferentiated T cells are harvested.

[0566] 27. The method according to embodiment 26, further comprising after harvesting the dedifferentiated T cells:

[0567] packaging at least a portion of the dedifferentiated T cells in a package; and

[0568] Freezing said package containing said portion of said dedifferentiated T cells.

[0569] 28. The method of any one of embodiments 25 to 27, further comprising, prior to inoculating the culture input cell population into the culture medium:

[0570] The culture input cell population is harvested from the subject.

[0571] 29. The method of any one of embodiments 25 to 28, wherein the culture medium does not contain IL-2 and no IL-2 is added to the culture medium.

[0572] 30. The method of any one of embodiments 25 to 29, wherein the cell density is 1.5 x 106 T cells per mL.

[0573] 31. The method of any one of embodiments 25 to 30, wherein said temsirolimus is present in said culture medium at a concentration of about 0.3 μM to about 1 μM.

[0574] 32. The method of any one of embodiments 25 to 30, wherein said temsirolimus is present in said culture medium at a concentration of about 1 μM.

[0575] 33. The method of any one of embodiments 25 to 32, wherein the period of time is about 3 days.

[0576] 34. The method of any one of embodiments 25 to 33, wherein the bead:T cell ratio is 1:3.

[0577] 35. The method of any one of embodiments 25 to 34, wherein said culture medium further comprises 5% human serum.

[0578] 36. The method of any one of embodiments 25 to 35, wherein said culture medium comprises X-Vivo 20 medium.

[0579] 37. The method of any one of embodiments 25 to 36, wherein said vitamin D is present in said culture medium at about 0.03 nM to about 1 nM.

[0580] 38. The method of any one of embodiments 25-37, wherein said vitamin D is present in said culture medium at about 0.1 nM.

[0581] 39. The method of any one of embodiments 25-32 and 34-38, further comprising:

[0582] measuring the expression level of RAPTOR or RICTOR in the culture input cell population,

[0583] wherein the time period lasts until the expression level of RAPTOR or RICTOR in the culture input cell population is reduced by at least 50% relative to a control T cell population, wherein the control T cell population is produced under the same conditions as the culture input cell population in the absence of temsirolimus, an IL-2 signaling inhibitor and vitamin D.

[0584] 40. The method of any one of embodiments 25-32 and 34-38, further comprising:

[0585] measuring the expression levels of RAPTOR, RICTOR and housekeeping proteins in the culture input cell population,

[0586] wherein the time period lasts until the expression level of RAPTOR or RICTOR in the culture input cell population is reduced by 50%, or more preferably by 90%, relative to a control T cell population after normalization for housekeeping protein expression, wherein the control T cell population is produced under the same conditions as the culture input cell population in the absence of temsirolimus, an IL-2 signaling inhibitor and vitamin D.

[0587] 41. The method of embodiment 40, wherein the housekeeping protein is actin or GAPDH.

[0588] 42. The method of any one of embodiments 39 to 41, wherein said step of measuring said expression level is performed by Western blot analysis.

[0589] 43. The method of any one of embodiments 25-32 and 34-38, further comprising:

[0590] measuring the expression level of RAPTOR or RICTOR in the culture input cell population,

[0591] wherein the time period lasts until the expression level of RAPTOR or RICTOR in the culture input cell population is reduced by at least 50%, and more preferably by 90%, relative to a control T cell population, wherein the control T cell population is produced under the same conditions as the culture input cell population in the absence of temsirolimus, an IL-2 signaling inhibitor and vitamin D.

[0592] 44. A dedifferentiated T cell produced by the method according to any one of embodiments 25 to 43.

[0593] 45. A dedifferentiated T cell population characterized in that, relative to a control T cell population cultured in the absence of the culture additives specified in these methods, the RNA expression of the following T cell differentiation molecules is reduced by at least 10%, and more preferably by 50%, respectively: cytolytic molecules, including but not limited to granzyme B; and cytokine molecules, including but not limited to IFN-γ.

[0594] 46. ​​A dedifferentiated T cell population, characterized in that, relative to a control T cell population cultured in the absence of the culture additives specified in these methods, the RNA expression of the following T cell differentiation molecules is increased by at least 10%, and more preferably by 50%: transcription factors associated with induced pluripotent stem cells, including but not limited to Nanog, KLF4 and KLF10; molecules associated with naive T cells, including but not limited to the IL-7 receptor CD127.

[0595] 47. A dedifferentiated T cell population characterized in that, relative to a control T cell population cultured in the absence of the culture additives specified in these methods, the RNA expression of the following T cell differentiation molecules is reduced by at least 10%, and more preferably by 50%: transcription factors associated with Th1 effector T cells, including but not limited to T-Bet and STAT1; however, the manufactured T cells will concomitantly have equivalent expression of transcription factors associated with cell survival (including but not limited to HIF-1-α).

[0596] 48. A dedifferentiated T cell population, characterized in that the expression of molecular markers of autophagy is increased by at least 10%, and more preferably by 50%, relative to a control T cell population cultured in the absence of the culture additives specified in these methods, including but not limited to: an increase in the protein level of the autophagy-related molecule p62 as analyzed by Western blot.

[0597] 49. A method according to any one of embodiments 1 to 22, wherein the step of adding anti-CD3 / anti-CD28 coated magnetic beads to the T cells and culture medium at a beads:T cell ratio of 1:1 to 1:12 is not performed.

[0598] 50. The method of any one of embodiments 25 to 43, wherein the step of adding anti-CD3 / anti-CD28 coated magnetic beads to the T cells and culture medium at a beads:T cell ratio of 1:1 or less to stimulate the T cells is not performed;

[0599] 51. A dedifferentiated T cell population characterized by one or more of the following properties:

[0600] The mRNA expression of one or more of granzyme B, IL-10 and IFN-γ is reduced by at least 10%, and more preferably by 50%, relative to a control T cell population incubated under the same conditions in the absence of temsirolimus, vitamin D and an IL-2 signaling inhibitor;

[0601] mRNA expression of one or more of Nanog, KLF4, KLF10 and CD127 is increased by at least 10%, and more preferably by 50%, relative to a control T cell population incubated under the same conditions in the absence of temsirolimus, vitamin D and an IL-2 signaling inhibitor;

[0602] mRNA expression of one or more of T-Bet and STAT1 is reduced by at least 10%, and more preferably by 50%, relative to a control T cell population incubated under the same conditions in the absence of temsirolimus, vitamin D, and an IL-2 signaling inhibitor;

[0603] HIF-1-α expression was within approximately 20% of control T cell populations incubated under the same conditions without temsirolimus, vitamin D, and IL-2 signaling inhibitors;

[0604] p62 expression is increased by at least 10%, and more preferably by 50%, relative to a control T cell population incubated under identical conditions in the absence of temsirolimus, vitamin D, and an inhibitor of IL-2 signaling;

[0605] The expression level of RAPTOR or RICTOR is reduced by at least 50%, and more preferably by 90%, relative to a control T cell population made under the same conditions as the culture input cell population in the absence of temsirolimus, an IL-2 signaling inhibitor, and vitamin D;

[0606] The 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 population made under the same conditions as the culture input cell population in the absence of temsirolimus, an IL-2 signaling inhibitor, and vitamin D; and

[0607] Its combination.

[0608] 52. A dedifferentiated T cell characterized by one or more of the following properties:

[0609] The mRNA expression of one or more of granzyme B, IL-10 and IFN-γ is reduced by at least 10%, and more preferably by 50%, relative to control T cells incubated under the same conditions in the absence of temsirolimus, vitamin D and an IL-2 signaling inhibitor;

[0610] mRNA expression of one or more of Nanog, KLF4, KLF10 and CD127 is increased by at least 10%, and more preferably by 50%, relative to control T cells incubated under the same conditions in the absence of temsirolimus, vitamin D and an IL-2 signaling inhibitor;

[0611] mRNA expression of one or more of T-Bet and STAT1 is reduced by at least 10%, and more preferably by 50%, relative to control T cells incubated under the same conditions in the absence of temsirolimus, vitamin D, and an IL-2 signaling inhibitor;

[0612] HIF-1-α expression was within approximately 20% of control T cells incubated under the same conditions without temsirolimus, vitamin D, and IL-2 signaling inhibitors;

[0613] p62 expression is increased by at least 10%, and more preferably by 50%, relative to a control T cell population incubated under identical conditions in the absence of temsirolimus, vitamin D, and an inhibitor of IL-2 signaling;

[0614] The expression level of RAPTOR or RICTOR is reduced by at least 50%, and more preferably by 90%, relative to a control T cell produced under the same conditions as the culture input cell population in the absence of temsirolimus, an IL-2 signaling inhibitor, and vitamin D;

[0615] The expression level of RAPTOR or RICTOR normalized by a housekeeping protein is reduced by at least 50%, and more preferably by 90%, relative to control T cells made under the same conditions as the culture input cell population in the absence of temsirolimus, an IL-2 signaling inhibitor, and vitamin D; and

[0616] Its combination.

[0617] Redifferentiation Example:

[0618] 1. A method for differentiating dedifferentiated T cells into TREG / Th2 cells, the method comprising: culturing the dedifferentiated T cells in a culture medium comprising IL-2, IL-4 and TGF-β;

[0619] Anti-CD3 / anti-CD28 coated magnetic beads were added at a ratio of 3:1 (beads:T cell ratio); the dedifferentiated T cells were incubated for a certain period of time to generate TREG / Th2 cells.

[0620] 2. The method according to embodiment 1, wherein the culture medium further comprises pemetrexed.

[0621] 3. The method of any one of embodiments 1 to 2, wherein the IL-2 is present in the culture medium at a concentration of about 100 IU / mL.

[0622] 4. The method of any one of embodiments 1 to 3, wherein the IL-4 is present in the culture medium at a concentration of about 1000 IU / mL.

[0623] 5. The method of any one of embodiments 1 to 4, wherein said TGF-β is present in said culture medium at a concentration of about 10 ng / mL.

[0624] 6. The method of embodiment 2, wherein the pemetrexed is present in the culture medium at a concentration of at most 100 nM.

[0625] 7. The method of embodiment 2, wherein the pemetrexed is present in the culture medium at a concentration of about 10 nM.

[0626] 8. The method of any one of embodiments 6 to 7, wherein said IL-7 is present in said culture medium at a concentration of about 100 IU / mL.

[0627] 9. The method of any one of embodiments 6 to 8, wherein the IL-4 is present in the culture medium at a concentration of about 1000 IU / mL.

[0628] 10. The method of any one of embodiments 6 to 9, wherein said TGF-β is present in said culture medium at a concentration of about 10 ng / mL.

[0629] 11. A method for differentiating dedifferentiated T cells into TREG / Th2 cells, the method comprising:

[0630] Culturing dedifferentiated T cells in a medium comprising IL-2, IL-4, and TGF-β, wherein the dedifferentiated T cells express RAPTOR and RICTOR at a level that is reduced by at least 10% relative to control T cells;

[0631] Anti-CD3 / anti-CD28-coated magnetic beads were added at a 3:1 ratio (bead:T cell ratio);

[0632] The dedifferentiated T cells are incubated for a certain period of time to generate TREG / Th2 cells.

[0633] 12. The method of embodiment 11, wherein the culture medium further comprises pemetrexed.

[0634] 13. The method of any one of embodiments 11 to 12, wherein said IL-12 is present in said culture medium at a concentration of about 100 IU / mL.

[0635] 14. The method of any one of embodiments 11 to 13, wherein said IL-4 is present in said culture medium at a concentration of about 1000 IU / mL.

[0636] 15. The method of any one of embodiments 11 to 14, wherein said TGF-β is present in said culture medium at a concentration of about 10 ng / mL.

[0637] 16. The method of embodiment 12, wherein said pemetrexed is present in said culture medium at a concentration of at most 100 nM.

[0638] 17. The method of embodiment 12, wherein said pemetrexed is present in said culture medium at a concentration of about 10 nM.

[0639] 18. The method of any one of embodiments 16-17, wherein said IL-17 is present in said culture medium at a concentration of about 100 IU / mL.

[0640] 19. The method of any one of embodiments 16 to 18, wherein said IL-4 is present in said culture medium at a concentration of about 1000 IU / mL.

[0641] 20. The method of any one of embodiments 16 to 19, wherein said TGF-β is present in said culture medium at a concentration of about 10 ng / mL.

[0642] 21. The method of any one of embodiments 1 to 20, wherein the culture medium is X-Vivo 20 supplemented with 5% human AB serum.

[0643] 22. The method of any one of embodiments 1 to 21, wherein the time period is between 3 days and 40 days.

[0644] 23. A TREG / Th2 cell produced by the method according to any one of embodiments 1 to 22.

[0645] 24. A method for differentiating dedifferentiated T cells into Treg cells, the method comprising:

[0646] culturing dedifferentiated T cells having reduced expression of RAPTOR and RICTOR relative to a control T cell population in a medium comprising IL-2 and TGF-β;

[0647] Anti-CD3 / anti-CD28-coated magnetic beads were added at a 3:1 ratio (bead:T cell ratio);

[0648] The dedifferentiated T cells are cultured for a certain period of time to generate TREG cells.

[0649] 25. The method of embodiment 24, wherein the culture medium further comprises pemetrexed.

[0650] 26. The method of any one of embodiments 24-25, wherein said IL-2 is present in said culture medium at a concentration of about 100 IU / mL.

[0651] 27. The method of any one of embodiments 24 to 26, wherein said TGF-β is present in said culture medium at a concentration of about 10 ng / mL.

[0652] 28. The method of embodiment 25, wherein said pemetrexed is present in said culture medium at a concentration of at most 100 nM.

[0653] 29. The method of embodiment 25, wherein said pemetrexed is present in said culture medium at a concentration of about 10 nM.

[0654] 30. The method of any one of embodiments 28-29, wherein said IL-2 is present in said culture medium at a concentration of about 100 IU / mL.

[0655] 31. The method of any one of embodiments 28 to 30, wherein said TGF-β is present in said culture medium at a concentration of about 10 ng / mL.

[0656] 32. A method for differentiating dedifferentiated T cells into Treg cells, the method comprising:

[0657] Culturing dedifferentiated T cells in a medium comprising IL-2 and TGF-β, wherein the dedifferentiated T cells express RAPTOR and RICTOR at a level that is reduced by at least 10% relative to control T cells;

[0658] Anti-CD3 / anti-CD28-coated magnetic beads were added at a 3:1 ratio (bead:T cell ratio);

[0659] The dedifferentiated T cells are cultured for a certain period of time to generate TREG cells.

[0660] 33. The method of embodiment 32, wherein said culture medium further comprises pemetrexed.

[0661] 34. The method of any one of embodiments 32-33, wherein said IL-2 is present in said culture medium at a concentration of about 100 IU / mL.

[0662] 35. The method of any one of embodiments 32 to 34, wherein said TGF-β is present in said culture medium at a concentration of about 10 ng / mL.

[0663] 36. The method of embodiment 33, wherein said pemetrexed is present in said culture medium at a concentration of at most 100 nM.

[0664] 37. The method of embodiment 33, wherein said pemetrexed is present in said culture medium at a concentration of about 10 nM.

[0665] 38. The method of any one of embodiments 36 to 37, wherein said IL-2 is present in said culture medium at a concentration of about 100 IU / mL.

[0666] 39. The method of any one of embodiments 36 to 38, wherein said TGF-β is present in said culture medium at a concentration of about 10 ng / mL.

[0667] 40. The method of any one of embodiments 24 to 39, wherein the culture medium is X-Vivo 20 medium supplemented with 5% AB serum.

[0668] 41. The method of any one of embodiments 24-40, wherein the time period is between 3 days and 40 days.

[0669] 42. The method of any one of embodiments 1 to 41, wherein the dedifferentiated T cells have reduced RAPTOR and RICTOR expression relative to a control T cell population.

[0670] 43. A TREG cell produced by the method according to any one of embodiments 1 to 22 and 24 to 42.

[0671] 44. A TREG cell or hybrid TREG / Th2 cell produced by the method of any one of embodiments 1 to 22 and 24 to 42, wherein lymphocytes collected by apheresis for subsequent T cell culture are obtained at steady state or after treatment of the subject with an anti-TNF-α therapeutic agent that is substantially selective for neutralizing serum, cell-free forms of TNF-α, most notably the recombinant receptor molecule etanercept or the monoclonal antibody adalimumab.

[0672] 45. A TREG cell or hybrid TREG / Th2 cell produced by the method of any one of embodiments 1 to 22 and 24 to 42, wherein the TREG cell or hybrid TREG / Th2 cell or a population thereof has increased expression of at least one of the following molecules relative to control Th1 / Tc1 cells by flow cytometry: CD25, CD27, 2B4, BTLA, CTLA4, TIGIT, TIM3, ICOS, LAIR1, OX40 and combinations thereof.

[0673] 46. ​​A TREG cell or hybrid TREG / Th2 cell produced by the method of any one of embodiments 1 to 22 and 24 to 42, wherein the TREG cell or hybrid TREG / Th2 cell or a population thereof has reduced secretion of inflammatory cytokines comprising IFN-γ and TNF-α relative to control Th1 / Tc1 cells.

[0674] 47. A TREG cell or hybrid TREG / Th2 cell produced by the method of any one of embodiments 1 to 22 and 24 to 42, wherein the TREG cell or hybrid TREG / Th2 cell or a population thereof has altered expression of T cell fate transcription factors relative to control Th1 / Tc1 cells, most notably, a decrease in TBET and an increase in FOXP3.

[0675] 48. A TREG cell or a hybrid TREG / Th2 cell produced by the method of any one of embodiments 1 to 22 and 24 to 42, wherein the TREG cell or hybrid TREG / Th2 cell or a population thereof has additional phenotypic traits relative to control Th1 / Tc1 cells, the additional phenotypic traits comprising: increased secretion of the Th2 cytokine IL-4; and increased expression of the Th2 transcription factor GATA3.

[0676] 49. A TREG or TREG / Th2 cell population having at least 5% of CD4+ or CD8+ T cells expressing GATA3.

[0677] 50. A TREG or TREG / Th2 cell population having at least 5% CD4+ or CD8+ T cells expressing FoxP3.

[0678] 51. A TREG or TREG / Th2 cell population having at least 10% of CD4+ or CD8+ T cells expressing CD73.

[0679] 52. A TREG or TREG / Th2 cell population having at least 10% of CD4+ or CD8+ T cells expressing CD103.

[0680] 53. A TREG or TREG / Th2 cell population having at least 20% of CD4+ or CD8+ T cells expressing CD150.

[0681] 54. A TREG or TREG / Th2 cell population that expresses at least 5 pg / mL / 1 x 106 cells / day of IL-4 following co-stimulation using anti-CD3 / anti-CD28 beads at a 3:1 beads:T cell ratio.

[0682] 55. A TREG or TREG / Th2 cell population that expresses at least 100 pg / mL / 1 x 106 cells / day of IL-2 following co-stimulation using anti-CD3 / anti-CD28 beads at a 3:1 beads:T cell ratio.

[0683] 56. A TREG or TREG / Th2 cell population that expresses less than 100 pg / mL / 1 x 106 cells / day of IFN-γ or GM-CSF following co-stimulation using anti-CD3 / anti-CD28 beads at a 3:1 beads:T cell ratio.

[0684] 57. A TREG or TREG / Th2 cell population that expresses less than 100 pg / mL / 1 x 106 cells / day of TNF-α or IL-17F following co-stimulation using anti-CD3 / anti-CD28 beads at a 3:1 beads:T cell ratio.

[0685] 58. A TREG or TREG / Th2 cell population having one or more of the following properties:

[0686] expression of one or more of CD25, CD27, 2B4, BTLA, CTLA4, TIGIT, TIM3, ICOS, LAIR1, OXO40, and combinations thereof is increased by at least 10% relative to control Th1 / Tc1 cells as measured by flow cytometry;

[0687] IFN-γ secretion was reduced by at least 10% relative to control Th1 / Tc1 cells;

[0688] The secretion of TNF-α was reduced by at least 10% relative to control Th1 / Tc1 cells;

[0689] The expression of TBET was reduced by at least 10% relative to control Th1 / Tc1 cells;

[0690] The expression of FOXP3 increased by at least 10% relative to control Th1 / Tc1 cells;

[0691] At least 5% of CD4 + or CD8+ T cells express GATA3;

[0692] At least 5% of CD4 + or CD8 + T cells express FOXP3;

[0693] At least 5% of CD4 + or CD8 + T cells express CD73;

[0694] At least 5% of CD4 + or CD8 + T cells express CD103;

[0695] At least 5% of CD4 + or CD8 + T cells express both FOXP3 and GATA3;

[0696] At least 20% of the CD4 + or CD8 + T cells express CD150;

[0697] Relative to the T REG or T REG / a T cell population characterized by T cells of the Th2 cell population, wherein the expression of one or more of GATA3, FoxP3, CD73, CD103 and CD150 is increased by at least 50%;

[0698] After costimulation with anti-CD3 / anti-CD28 beads at a 3:1 beads:T cell ratio, at least 5 pg / mL / 1×10 6 cells / day of IL-4;

[0699] After costimulation with anti-CD3 / anti-CD28 beads at a 3:1 beads:T cell ratio, at least 100 pg / mL / 1×10 6 cells / day of IL-2;

[0700] After costimulation with anti-CD3 / anti-CD28 beads at a 3:1 beads:T cell ratio, less than 100 pg / mL / 1×10 6 cells / day IFN-γ;

[0701] After costimulation with anti-CD3 / anti-CD28 beads at a 3:1 beads:T cell ratio, less than 100 pg / mL / 1×10 6 cells / day GM-CSF;

[0702] After costimulation with anti-CD3 / anti-CD28 beads at a 3:1 beads:T cell ratio, less than 10 pg / mL / 1×10 6 cells / day TNF-α;

[0703] After costimulation with anti-CD3 / anti-CD28 beads at a 3:1 beads:T cell ratio, less than 10 pg / mL / 1×10 6 cells / day of IL-17; and

[0704] Its combination.

[0705] 59. A TREG or TREG / Th2 cell having one or more of the following properties:

[0706] expression of one or more of CD25, CD27, 2B4, BTLA, CTLA4, TIGIT, TIM3, ICOS, LAIR1, OXO40, and combinations thereof is increased by at least 10% relative to control Th1 / Tc1 cells as measured by flow cytometry;

[0707] IFN-γ secretion was reduced by at least 10% relative to control Th1 / Tc1 cells;

[0708] The secretion of TNF-α was reduced by at least 10% relative to control Th1 / Tc1 cells;

[0709] The expression of TBET was reduced by at least 10% relative to control Th1 / Tc1 cells;

[0710] The expression of FOXP3 increased by at least 10% relative to control Th1 / Tc1 cells;

[0711] After costimulation with anti-CD3 / anti-CD28 beads at a 3:1 beads:T cell ratio, at least 5 pg / mL / 1×10 6 cells / day of IL-4;

[0712] After costimulation with anti-CD3 / anti-CD28 beads at a 3:1 beads:T cell ratio, at least 100 pg / mL / 1×10 6 cells / day of IL-2;

[0713] After costimulation with anti-CD3 / anti-CD28 beads at a 3:1 beads:T cell ratio, less than 100 pg / mL / 1×10 6 cells / day IFN-γ;

[0714] After costimulation with anti-CD3 / anti-CD28 beads at a 3:1 beads:T cell ratio, less than 100 pg / mL / 1×10 6 cells / day GM-CSF;

[0715] After costimulation with anti-CD3 / anti-CD28 beads at a 3:1 beads:T cell ratio, less than 10 pg / mL / 1×10 6 cells / day TNF-α;

[0716] After costimulation with anti-CD3 / anti-CD28 beads at a 3:1 beads:T cell ratio, less than 10 pg / mL / 1×10 6 cells / day of IL-17;

[0717] Expression of GATA3, FOXP3, CD73, and CD103; and

[0718] Its combination.

[0719] 60. The method of any one of claims 1 to 41, wherein the dedifferentiated T cells have one or more of the following properties:

[0720] The mRNA expression of one or more of granzyme B, IL-10 and IFN-γ is reduced by at least 10%, and more preferably by 50%, relative to a control T cell population incubated under the same conditions in the absence of temsirolimus, vitamin D and an IL-2 signaling inhibitor;

[0721] mRNA expression of one or more of Nanog, KLF4, KLF10 and CD127 is increased by at least 10%, and more preferably by 50%, relative to a control T cell population incubated under the same conditions in the absence of temsirolimus, vitamin D and an IL-2 signaling inhibitor;

[0722] mRNA expression of one or more of T-Bet and STAT1 is reduced by at least 10%, and more preferably by 50%, relative to a control T cell population incubated under the same conditions in the absence of temsirolimus, vitamin D, and an IL-2 signaling inhibitor;

[0723] HIF-1-α expression was within approximately 20% of control T cell populations incubated under the same conditions without temsirolimus, vitamin D, and IL-2 signaling inhibitors;

[0724] p62 expression is increased by at least 10%, and more preferably by 50%, relative to a control T cell population incubated under identical conditions in the absence of temsirolimus, vitamin D, and an inhibitor of IL-2 signaling;

[0725] The expression level of RAPTOR or RICTOR is reduced by at least 50%, and more preferably by 90%, relative to a control T cell population made under the same conditions as the culture input cell population in the absence of temsirolimus, an IL-2 signaling inhibitor, and vitamin D;

[0726] The 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 population made under the same conditions as the culture input cell population in the absence of temsirolimus, an IL-2 signaling inhibitor, and vitamin D; and

[0727] Its combination.

[0728] 61. The method of any one of claims 1 to 41, wherein the dedifferentiated T cells have one or more of the following properties:

[0729] The mRNA expression of one or more of granzyme B, IL-10 and IFN-γ is reduced by at least 10%, and more preferably by 50%, relative to control T cells incubated under the same conditions in the absence of temsirolimus, vitamin D and an IL-2 signaling inhibitor;

[0730] mRNA expression of one or more of Nanog, KLF4, KLF10 and CD127 is increased by at least 10%, and more preferably by 50%, relative to control T cells incubated under the same conditions in the absence of temsirolimus, vitamin D and an IL-2 signaling inhibitor;

[0731] mRNA expression of one or more of T-Bet and STAT1 is reduced by at least 10%, and more preferably by 50%, relative to control T cells incubated under the same conditions in the absence of temsirolimus, vitamin D, and an IL-2 signaling inhibitor;

[0732] HIF-1-α expression was within approximately 20% of control T cells incubated under the same conditions without temsirolimus, vitamin D, and IL-2 signaling inhibitors;

[0733] p62 expression is increased by at least 10%, and more preferably by 50%, relative to a control T cell population incubated under identical conditions in the absence of temsirolimus, vitamin D, and an inhibitor of IL-2 signaling;

[0734] The expression level of RAPTOR or RICTOR is reduced by at least 50%, and more preferably by 90%, relative to a control T cell produced under the same conditions as the culture input cell population in the absence of temsirolimus, an IL-2 signaling inhibitor, and vitamin D;

[0735] The expression level of RAPTOR or RICTOR normalized by a housekeeping protein is reduced by at least 50%, and more preferably by 90%, relative to control T cells made under the same conditions as the culture input cell population in the absence of temsirolimus, an IL-2 signaling inhibitor, and vitamin D; and

[0736] Its combination.

Claims

1. Pentostatin and / or cyclophosphamide and T REG Use of a combination of a composition of cells in the preparation of a medicament for treating amyotrophic lateral sclerosis in a subject in need thereof, wherein: The subject undergoes one or more primary treatment cycles, each of the one or more primary treatment cycles comprising: administering pentostatin to the subject; and / or administering cyclophosphamide to the subject; and The subject undergoes one or more immunotherapy treatment cycles comprising: Administering to the subject a therapeutically effective amount of a manufactured T REG The method comprises culturing T cells obtained from a subject in a dedifferentiation medium to produce dedifferentiated T cells; and culturing the dedifferentiated T cells in a redifferentiation medium comprising IL-2, TGF-β, IL-4, and anti-CD3 / anti-CD28 coated magnetic beads at a bead:T cell ratio of 3:1 to produce the manufactured T cells. REG Cells, wherein the dedifferentiation medium comprises vitamin D, temsirolimus and an anti-IL-2 receptor antibody, and wherein the expression levels of RAPTOR and RICTOR in the dedifferentiated T cells are reduced by at least 50% relative to a control T cell population cultured in the absence of temsirolimus, an anti-IL-2 receptor antibody and vitamin D.

2. The use according to claim 1, wherein the manufactured T REG The cells include a ratio of central memory cells to effector memory cells selected from 1:1, 3:1, 10:1, 1:3 and 1:

10.

3. The use according to any one of claims 1 and 2, wherein the IL-2 is present in the redifferentiation medium at a concentration of 10000 IU / ml.

4. The use according to any one of claims 1 and 2, wherein the TGF-β is present in the redifferentiation medium at a concentration of 10 ng / mL.

5. The use according to any one of claims 1 and 2, wherein the step of culturing the dedifferentiated T cells in a redifferentiation medium is performed for 3 to 40 days.

6. The use according to any one of claims 1 and 2, wherein IL-4 is present in the redifferentiation medium at a concentration of 1000 IU / mL.

7. Made in T REG Use of a cell in the preparation of a medicament for treating amyotrophic lateral sclerosis in a subject in need thereof, wherein: The subject undergoes one or more treatment cycles comprising: Administering a therapeutically effective amount of the manufactured T to the subject REG The manufactured T cells are produced by culturing T cells obtained from a subject in a dedifferentiation medium to produce dedifferentiated T cells; and culturing the dedifferentiated T cells in a redifferentiation medium comprising IL-2, TGF-β, IL-4, and anti-CD3 / anti-CD28 coated magnetic beads at a bead:T cell ratio of 3:

1. REG Cells, wherein the dedifferentiation medium comprises vitamin D, temsirolimus and an anti-IL-2 receptor antibody, and wherein the expression levels of RAPTOR and RICTOR in the dedifferentiated T cells are reduced by at least 50% relative to a control T cell population cultured in the absence of temsirolimus, an anti-IL-2 receptor antibody and vitamin D.

8. The use according to claim 7, wherein the manufactured T REG The cells include a ratio of central memory cells to effector memory cells selected from 1:1, 3:1, 10:1, 1:3 and 1:

10.

9. The use according to any one of claims 7 to 8, wherein the IL-2 is present in the redifferentiation medium at a concentration of 10000 IU / ml.

10. The use according to any one of claims 7 and 8, wherein the TGF-β is present in the redifferentiation medium at a concentration of 10 ng / mL.

11. The use according to any one of claims 7 and 8, wherein the step of culturing the dedifferentiated T cells in a redifferentiation medium is performed for 3 to 40 days.

12. The use according to any one of claims 7 and 8, wherein IL-4 is present in the redifferentiation medium at a concentration of 1000 IU / mL.

13. The use according to claim 1 or 7, wherein the manufactured T REG Cells have one or more of the following properties: At least 5% of CD4 + or CD8 + T cells express GATA3; At least 5% of CD4 + or CD8 + T cells express FOXP3; At least 5% of CD4 + or CD8 + T cells express CD73; At least 5% of CD4 + or CD8 + T cells express CD103; At least 5% of CD4 + or CD8 + T cells express both FOXP3 and GATA3; At least 20% of CD4 + or CD8 + T cells express CD150; and Its combination.

14. A method of manufacturing T REG / Th2 cell method, the method comprising: culturing the T cells in a dedifferentiation medium to produce dedifferentiated T cells; and culturing the dedifferentiated T cells in a redifferentiation medium comprising IL-2, TGF-β, IL-4, and anti-CD3 / anti-CD28 coated magnetic beads at a bead:T cell ratio of 3:1 to generate T cells. REG / Th2 cells, wherein the dedifferentiation medium comprises vitamin D, temsirolimus and anti-IL-2 receptor antibody, and wherein the expression levels of RAPTOR and RICTOR in the dedifferentiated T cells are reduced by at least 50% relative to a control T cell population cultured in the absence of temsirolimus, anti-IL-2 receptor antibody and vitamin D.

15. The method of claim 14, wherein the IL-2 is present in the redifferentiation medium at a concentration of 10000 IU / mL.

16. The method of claim 14 or 15, wherein the IL-4 is present in the redifferentiation medium at a concentration of 1000 IU / mL.

17. The method according to any one of claims 14 and 15, wherein the TGF-β is present in the redifferentiation medium at a concentration of 10 ng / mL.

18. The method of any one of claims 14 and 15, wherein the dedifferentiation medium comprises vitamin D, temsirolimus, anti-IL-2 receptor antibody, and X-Vivo 20 supplemented with 5% human AB serum, and the redifferentiation medium comprises IL-2, TGF-β, IL-4, anti-CD3 / anti-CD28 coated magnetic beads at a bead:T cell ratio of 3:1, and X-Vivo 20 supplemented with 5% human AB serum.

19. The method according to any one of claims 14 and 15, wherein the step of culturing the dedifferentiated T cells in a redifferentiation medium is performed for 3 to 40 days.

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